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H C Pitot

Publications and source records attributed to H C Pitot.

At least 289 records · Page 16Linked to original sources
PubMed1981

Structure and function of rat liver polysome populations. I. Complexity, frequency distribution, and degree of uniqueness of free and membrane-bound polysomal polyadenylate-containing RNA populations.

Free and membrane-bound polysomes were isolated from rat liver in high yields with minimal degradation, cross-contamination, or contamination by nuclear or nonpolysomal cytoplasmic ribonucleoprotein. Poly(A)+ RNA fractions isolated from free and bound polysomal RNA (poly(A)+ RNAfree and poly(A)+ RNAbound) by oligo(dT) cellulose chromatography exhibited number-average lengths of 1,600 and 1,200 nucleotides, respectively, on formamide sucrose gradients. Poly(A)+ RNAfree and poly(A)+ RNAbound contain 9.1 +/- 0.55 and 10.7 +/- 0.50% poly(A) as measured by hybridization to [3H]poly(U) and comprise 2.37 and 1.22% of their respective polysomal RNA populations. Homologous poly(A)+ RNA-cDNA hybridizations revealed that greater than 95% of the mass of poly(A)+ RNAfree and poly(A)+ RNAbound contain nucleotide complexities of about 3.4 x 10(7) and 6.0 x 10(6), respectively. This represents about 20,000 and 5,000 poly(A)+ RNA species of average sizes. Heterologous hybridizations suggested that considerable overlap exists between poly(A)+ RNAfree and poly(A)+ RNAbound sequences that cannot be attributed to cross-contamination. This was confirmed by conducting heterologous reactions using kinetically enriched cDNA populations. Heterologous hybridizations involving poly(A)+ RNA derived from tightly bound polysomes and cDNAfree indicated tha most of the overlapping sequences are not contributed by loosely bound (high-salt releasable) polysomes. The ramifications of these findings are discussed.

Animals↗
PubMed1981

Quantitative analysis of the time-dependent development of glucose-6-phosphatase-deficient foci in the livers of mice treated neonatally with diethylnitrosamine.

The development of glucose-6-phosphatase (G-6-Pase-)-deficient hyperbasophilic foci was analyzed at 4-week intervals in the livers of CD-1 and C57BL/6J x C3H/HeJ F1 (hereafter called B6C3F1) mice given a single i.p. injection of diethylnitrosamine (DEN) (0.1, 0.2, or 0.4 mumol/g body weight) within 24 hr after birth. Transections of G-6-Pase-deficient foci of hepatocytes were readily discernible in liver sections of DEN-treated mice of either sex at 8 weeks of age. The size and number of these foci per liver increased with time. The occurrence of G-6-Pase-deficient focus transections with diameters as large as 1 mm coincided with the gross appearance of 1-mm gray-white nodules in the livers of male B6C3F1 mice at 16 weeks of age and in females at 32 weeks of age. Transections of all grossly visible hepatic nodules from male and female mice were G-6-Pase deficient and hyperbasophilic; the great majority were diagnosed as mouse hepatomas type A. After a single neonatal dose of DEN, the number and rate of growth of the G-6-Pase-deficient foci and the incidence and rate of appearance of gross hepatomas were greater in the liver of male than in those of female mice. In contrast, the average numbers of G-6-Pase-deficient foci in the livers of male and androgenized female B6C3F1 mice at 36 weeks of age were approximately equal and about twice that observed for the livers of DEN-treated female controls. Quantitation of carcinogen-induced histochemically detectable foci and hepatomas as a function of time provides a useful tool for the analysis of initiation and promotion in the mouse liver.

Age Factors↗
PubMed1980

Characterization of protein kinase activity associated with rat liver polysomal messenger ribonucleoprotein particles.

Poly(adenylic acid)-containing rat liver polysomal messenger ribonucleoprotein particles (pmRNP) were isolated and found to contain protein kinase activity. The association of the enzyme(s) with the particles was confirmed by experiments showing that the protein kinase activity comigrated with the pmRNP on metrizamide gradients and bound to oligo-(dT)-cellulose columns only under conditions where the pmRNP bound. The following properties were determined for the pmRNP-associated kinase(s). Casein and phosvitin were preferred substrates over histone and protamine. The optimal MgCl2 and KCl concentrations were found to be 12.5 and 50 mM, respectively. MnCl2 and CaCl2 could not replace MgCl2 and were inhibitory at low concentrations. The optimum pH range was 7.7--9.0, and the enzyme activity was cAMP independent. A molecular weight of 55 000--60 000 was determined for the kinase(s) by sucrose gradient analysis. The enzyme(s) was capable of phosphorylating proteins endogenous to the pmRNP. Membrane-bound pmRNP contained much less kinase activity than free pmRNP while pmRNP from hepatoma 7777 contained an elevated level of the enzyme(s). The relationship between the protein kinase activity and one of the pmRNP proteins of molecular weight 66 000 is discussed.

Animals↗
PubMed1980

Separation of putrescine oxidase and spermidine oxidase in foetal bovine serum with the aid of a specific radioactive assay of spermidine oxidase.

1. A sensitive and specific assay for spermidine oxidase is described. The method involves the separation of [14C]spermidine (substrate) from [14C]putrescine (product) and other 14C-labelled products on a Dowex 50 cation-exchange column: 92% of the putrescine applied to the column was eluted by 2.3 M-HCl, but this treatment left 96% of the spermidine bound to the column. Unchanged spermidine could be removed from the column by elution with 6 M-HCl. 2. By means of this assay, foetal and adult bovine serum were each shown to contain spermidine oxidase activity, putrescine being a major product of the oxidation of spermidine by the serum enzymes. 3. In foetal bovine serum, spermidine oxidase activity is separable from putrescine oxidase activity by chromatography on a cadaverine-Sephadex column, by gel filtration and by ion-exchange column chromatography. Putrescine oxidase was purified 1900-fold and spermidine oxidase 130-fold by these procedures. The former oxidized putrescine but not spermidine, and spermidine oxidase exhibited no activity with putrescine as substrate.

Animals↗
PubMed1980

Regulation of gene expression in primary cultures of adult rat hepatocytes on collagen gels.

The studies described in this paper demonstrate rather conclusively the efficacy of the study of the regulation of gene expression in primary cultures of adult rat hepatocytes. The utilization of these cells in completely defined medium allows one to determine the exact environmental conditions for the regulation of the expression of specific genes. In the studies described in this work, we have demonstrated that the regulation of glucokinase involved three hormones, insulin, corticosteroids, and T3. In contrast, the regulation of an enzyme involved primarily in fatty acid metabolism, ATP-citrate lyase, required only insulin and T3 for its full expression. Cyclic GMP appeared to be involved in the regulation of glucokinase, but not ATP-citrate lyase, a fact that would be extremely difficult to demonstrate clearly in vivo. The regulation of the gluconeogenic enzyme, ornithine aminotransferase, in vitro involved only a single hormone, glucagon, the inhibition of induction by corticoid steroids demonstrable in vivo being absent in cell culture. However, the repressive effect of glucose on the induction of this enzyme was quite comparable to that seen in vivo and was not mediated through cyclic AMP or insulin, based on findings in cell culture. Thus, the requirements for and the mechanisms involved in enzyme induction and repression by hormones and glucose may be much more easily studied in primary cultures of rat hepatocytes than in vivo, or even in hepatoma cell lines, where relatively few genes are expressed as compared with adult liver. In addition to the regulation of enzyme levels, the characteristics of protein secretion may be investigated in primary cultures of rat hepatocytes and compared with the biochemical and physiological parameters in the whole organism. This was exemplified by the study of the synthesis and secretion of alpha 2u-globulin that was secreted into the culture medium in both glycosylated and nonglycosylated forms but was maintained in the circulation in vivo, principally as the glycosylated form. Furthermore, the function of glycosylation in this particular instance may be deduced from a combination of the in vivo and in vitro approaches. The advantages of the use of primary hepatocyte cultures for the study of the regulation of gene expression in mammalian tissue has only recently been explored. Future investigations of the regulation of a variety of enzymes in these cultures as well as a study of the regulation of the synthesis of their messenger RNA are now possible and should provide an exciting system in which to understand at a molecular level the regulation of the expression of a number of genes.

ATP Citrate (pro-S)-Lyase↗
PubMed1980

Diamine oxidase activity in fibroblasts from normal and cystic fibrosis patients.

Diamine oxidase is the initial enzyme involved in the degradation of polyamines, whose metabolism has been reported as abnormal in cystic fibrosis (CF). Diamine oxidase activity, using putrescine as substrate, was measured in homogenates of human fibroblasts cultured in vitro from normal and cystic fibrosis patients. The pH optimum of the crude enzyme was 7.8. Diamine oxidase activity per microgram protein peaked during periods of greatest proliferative rate and reached its nadir when fibroblasts grew in static (confluent) phase. No difference was found between normal and CF fibroblasts in specific activity of diamine oxidase, but CF cells exhibited a slightly lower protein content during logarithmic growth when compared with normal cells.

Amine Oxidase (Copper-Containing)↗
PubMed1980

Quantitative evaluation of the promotion by 2,3,7,8-tetrachlorodibenzo-p-dioxin of hepatocarcinogenesis from diethylnitrosamine.

In order to test the potential of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as a promoter of hepatocarcinogenesis, rats which had received a single 10-mg/kg dose of diethylnitrosamine (DEN) following partial hepatectomy were given TCDD (0.14 and 1.4 micrograms/kg s.c. once every 2 weeks) for 7 months. Animals which received (a) only a single initiating dose of DEN after partial hepatectomy and no further treatment of (b) TCDD alone with no initiating dose of DEN exhibited relatively few enzyme-altered foci and no hepatocellular carcinomas. However, animals initiated with DEN and then given TCDD had a marked increase in enzyme-altered foci. At the higher dose of TCDD, hepatocellular carcinomas were present in five of seven rats. By means of three different enzyme markers used to evaluate the phenotypes of the enzyme-altered foci, a distinct phenotype heterogeneity of the foci was noted with a shift towards phenotypes exhibiting a greater deviation from normal liver when TCDD was given following DEN-partial hepatectomy. Quantitation of the numbers of enzyme-altered foci was performed by relating measurements made from two-dimensional tissue sections to the numbers of foci per unit volume of liver using relationships established in the field of stereology. The total volume of the liver occupied by the enzyme-altered foci, but not their number, increased with the dose of TCDD administered following DEN-partial hepatectomy. These studies demonstrate that TCDD is a potent promoting agent for hepatocarcinogenesis.

Adenosine Triphosphatases↗
PubMed1980

Accumulation of actin microfilaments in adult rat hepatocytes cultured on collagen gel/nylon mesh.

Adult rat hepatocytes cultured on a collagen gel/nylon mesh support were shown previously to exhibit fetal properties as a function of time in culture and to accumulate numerous microfilament structures as seen with electron microscopy. The present study characterizes the accumulation and properties of such microfilaments. Electron microscopy revealed that microfilaments, 40 to 60 A in diameter, accumulated as a compact network almost exclusively beneath the plasma membrane at the interface with the culture medium. Treatment with heavy meromyosin resulted in the formation of the characteristic arrowhead complexes on these microfilaments, indicating that they contain actin. Extraction of glycerinated hepatocytes with low-ionic-strength solutions removed most of the microfilaments, which could also be disrupted by treatment of the cells with cytochalasin B. The disruption was reversible, and reappearance of the network of microfilaments, though less compact, was observed as early as 30 min after removal of cytochalasin B. At later times in culture, the microfilaments developed into a more compact and extensive network, and their reappearance was not dependent on protein synthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins extracted from hepatocytes labeled with L-[35S]methionine or from acetone powders of the labeled hepatocytes showed a major band of a molecular weight of 42,000 that comigrated with skeletal muscle actin on the sodium dodecyl sulfate-polyacrylamide gels. The amount of radioactivity in this band increased with the age of the hepatocyte culture; this indicated an increase in the synthesis of the actin-like protein as well as a greater degree of polymerization into microfilaments. This finding was further confirmed by the demonstration of an increase in the proportion of the filamentous form of actin in the late cultures by means of a DNase I assay. The studies reported in this paper indicate that (a) the microfilaments in cultured hepatocytes contain actin, with properties similar to those found in other nonmuscle cells, and (b) the large accumulation of microfilaments is due in part to the synthesis of actin and the formation of new microfilaments.

Actins↗