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Biomedical subjects

H C Pitot

Publications and source records attributed to H C Pitot.

At least 343 records · Page 19Linked to original sources
PubMed1976

The effect of various treatments in vitro and in vivo on the binding of 125I-labeled anti-rat serum albumin Fab' to rat tissue polyribosomes.

A quantitative assay was developed to permit estimation of the relative amounts of albumin-synthesizing polyribosomes in rat liver. The polyribosomes synthesizing albumin were identified by their capacity to bind anti-RSA Fab' radiolabeled with 125I. The anti-RSA Fab'-binding sites occur on the nascent peptide chains attached to liver polyribosomes. These binding sites can be saturated by preincubation of the polyribosomes with large quantities of unlabeled anti-RSA Fab'. The iodinated antibody did not react with polyribosomes isolated from a tissue which does not synthesize rat serum albumin. Pretreatment of hepatic polyribosomes with bovine pancreatic ribonuclease resulted in a 42% enhancement of binding of anti-RSA Fab'. Pretreatment of these polyribosomes with detergents or various levels of Mg2+ did not significantly affect the specific binding of the iodinated antibody. Anti-RSA Fab' associated preferentially with membrane-bound polyribosomes when compared with free polyribosomes following their isolation from animals maintained either on a 90% or a 0% protein diet fed ad libitum. Binding of anti-RSA Fab' to each A260 unit of membrane-bound polyribosomes is from 2.4 to 27 times greater than to each A260 unit of free polyribosomes. However, each A260 unit of free polyribosomes was found to associate with 1.8 times more anti-RSA Fab' when compared with the "loosely bound" subclass of membrane-bound polyribosomes. Each A260 unit of the "tightly bound" subclass of membrane-bound polyribosomes reacted with 4.3 times as much antibody as compared with free polyribosomes. Polyribosomes isolated from the livers of rats sacrificed 6 h after treatment with actinomycin D showed a 42% reduction in their capacity to bind anti-RSA Fab'. Polyribosomes from rats sacrificed 2 h after treatment with actinomycin D showed no reduction in binding capacity. Free polyribosomes from three Morris hepatomas were capable of binding anti-RSA Fab' whereas the antibody would not associate with the membrane-bound polyribosomes of the same hepatomas. Thus the binding of 125I-labeled Fab' antibody molecules to polyribosomes is a useful technique for the subcellular localization of polyribosomes synthesizing specific proteins and for the estimation of the relative proportions of such polyribosomes.

Animals↗
PubMed1976

Hormonal and nutritional effects on the binding of 125I-labeled anti-serine dehydratase Fab' to rat tissue polysomes.

With 125I-labeled Fab' specific for rat liver serine dehydratase it has been possible to localize polyribosomes synthesizing the enzyme under several different environmental conditions. Evidence is presented to show that, following the administration of amino acids in vivo, the relative synthetic capabilities of free and membrane-bound polyribosomes synthesizing serine dehydratase vary with time. Early during the period of induction of the enzyme by administration of amino acids or by feeding a high protein diet the majority of the newly synthesized enzyme is derived from membrane-bound polyribosomes. Later in the induction process an increasing proportion of the enzyme is synthesized by the free polyribosomes. Subcellular localization studies clearly show that serine dehydratase is synthesized by both subclasses of hepatic membrane-bound polyribosomes, the loose and tight membrane-bound polyribosomes, as well as by the free polyribosomes. It was found that the membrane-bound polyribosomes are the preferential sites of synthesis of the majority of serine dehydratase molecules in the Morris hepatomas 5123C and 7800. It is concluded that the synthesis of the enzyme, serine dehydratase, in rat liver is not discretely compartmentalized in either class of free or membrane-bound polyribosomes. Rather, the relative proportions of the serine dehydratase synthesizing polyribosomes within these two classes of polyribosomes can vary depending on the metabolic and physiologic state of the liver cell.

Animals↗
PubMed1976

Protein phosphorylation of the smooth and rough endoplasmic reticulum in normal and neoplastic liver of the rat.

Smooth and rough endoplasmic reticulum from rat liver and hepatomas exhibited endogenous protein kinase activity independent of adenosine 3':5'-monophosphate. The phosphorylation of smooth membranes by this process was consistently higher than that of rough membranes. When histone was added along with the smooth endoplasmic reticulum, cyclic AMP stimulated protein phosphorylation. Analysis of membrane-phosphorylated proteins by gel electrophoresis showed 5 major phosphorylated bands with estimated molecular weights of 155 000, 62 000, 50 000, 46 000 and 43 000, whereas major bands having estimated molecular weights of 62 000, 50 000 and 43 000 were found in membranes of the smooth endoplasmic reticulum of the Morris hepatoma 5123 C. Since previous studies in this and other laboratories have demonstrated the similarity of the protein components of membranes of the endoplasmic reticulum of normal liver and hepatoma, our findings indicate an inability of the protein kinase of hepatoma intracellular membranes to phosphorylate protein species that are found in membranes of both liver and the neoplasm.

Animals↗
PubMed1976

Direct association of messenger RNA labeled in the presence of fluoroorotate with membranes of the endoplasmic reticulum in rat liver.

Liver rough endoplasmic reticulum (RER) membranes were isolated from rats given [3H]orotic acid for 48 h (ribosomal RNA [rRNA] label) or for 3 h along with 5-fluoroorotate; this latter procedure permits the labeling of cytoplasmic messenger RNAs (mRNAs) in the absence of rRNA labeling. More than 50% of the labeled mRNA remained attached to membranes of the RER after complete removal of ribosomes with a buffer of high ionic strength in the presence of puromycin. Under similar conditions, membranes retained 40% of their polyadenylate as determined by a [3H]-polyuridylate hybridization assay. Treatment of mRNA-labeled endoplasmic reticulum membranes with pancreatic RNase indicates that the polyadenylate and possibly nonpolyadenylate-pyrimidine portions of the messenger are involved in the binding of mRNA to the membranes. The implication of these results in furthering our understanding of the mechanisms of the translational regulation of genetic expression is discussed.

Animals↗
PubMed1976

Hepatocarcinogenicity of estragole (1-allyl-4-methoxybenzene) and 1'-hydroxyestragole in the mouse and mutagenicity of 1'-acetoxyestragole in bacteria.

Approximately 20% of a dose of estragole, a naturally occurring flavoring agent, was excreted in the urine of outbred male CD -1 mice as a conjuage (presumably the glucuronide) of 1'-hydroxyestragole, Estragole and its 1'-hydroxy metabolite caused significant increases in the incidences of hepatocellular carcinomas in male CD-1 mice that received the compounds by sc injection at 1-22 days of age. Estragole induced hepatocellular carcinomas by 15 months in 23 and 39% of the mice that received total doses of 4.4 and 5.2 mumoles, respectively, and lived to an age of 12 months or more. Of the 12-month survivors given a total dose of 4.4 mumoles of 1'-hydroxyestragole, 70% developed hepatocellular carcinomas; the incidence in mice that received only the vehicle (trioctanoin) was 12%. Multiple tumors ocurred in 5, 28, 64, and 0%, respectively, of the mice in each of these 4 groups. Of the mice given a total dose of 4.4 mumoles of 1'-hydroxysafrole, 59developed hepatocellular carcinomas; 39% of the mice bore multiple liver tumors. As previsously demonstrated for 1'-acetoxysafrole, 1'-acetoxyestragole and 1'-acetoxy-1-allyl-4-methoxynaphthalene reacted nonenzymatically with guanosine and inosine to form adducts. These electrophilic esters were strongly mutagenic for the Salmonella typhimurium missense mutant TA100. 1'-Acetoxyallybenzene had little or no activity in either of these tests. Attempts to demonstrate liver-mediated mutagenicty for 1'-hydroxysafrole and 1'-hydroxyestragole in the bacterial test system were unsuccessful.

Allylbenzene Derivatives↗
PubMed1976

The effect of spermidine and spermine on proliferation in vitro of fibroblasts from normal and cystic fibrosis patients.

The effects of spermidine and spermine at varying concentrations upon the replicative ability of human fibroblasts in cell culture have been studied. The average concentrations of spermidine causing a 50% inhibition of prolifertion (ID50) after 3 days of growth for three normal cell strains and three strains derived from patients with cystic fibrosis (CF) were 4.4 X 10(-6) +/- 1.2 M and 6.2 X 10(-6) +/- 2.1 M, respectively. The values for spermine were 2.0 X 10(-6) +/- 0.5 M for normal and 2.2 X 10(-6) +/- 0.1 M for fibroblasts from cystic fibrosis patients. No significant difference between the replicative ability of normal and CF cell strains was seen over a wide range of polyamine concentrations employed for a period of up to 3 days.

Cell Division↗
PubMed1976

Environmental effects on glutathione-insulin transhydrogenase in rat liver.

The dietary and hormonal regulation of the level of glutathione-insulin tranhydrogenase in rat liver was investigated in these studies. In order to make valid comparisons, the assay of glutathione-insulin transhydrogenase was performed at near zero-order kinetics wherein enzyme rate was proportional to enzyme amount. Changing the protein content of the diet or administration of glucagon or cortisone did not significantly affect the specific activity of glutathione-insulin transhydrogenase in microsomes from rat liver. However, the Vmax and Km of this enzyme in the livers of adrenalectomized rats were increased three-and fourfold over these values in microsomes from normal liver. Administration of cortisone resulted in a return to the normal kinetic constants of microsomal GIT within 4 hr.

Animals↗
PubMed1976

Interaction of chemical carcinogens and drug-metabolizing enzymes in primary cultures of hepatic cells from the rat.

The experiments described in this paper have demonstrated that hepatocytes cultured on floating collagen membranes for periods of 10 days retain their ability to respond to the inducers of drug-metabolizing enzymes, phenobarbital and methylcholanthrene, by increases in cytochromes of the cytochrome P-450 complex. Since the regulation of these cytochromes is the rate-controlling factor in the metabolism of drugs and carcinogens in hepatocytes, such experiments indicate that hepatocytes cultured on floating collagen membranes retain those functions of the liver cell responsible for the metabolism and "activation" of carcinogenic substances. The data support this hypothesis and further indicate that this system may have potential application both in the investigation of hepatocarcinogenesis by chemicals in vitro and as a screening system for the detection of substances truly carcinogenic for the mammalian organism.

Animals↗
PubMed1976

Cystic fibrosis. A dilemma in the metabolic pathogenesis of genetic disease.

The fundamental pathological observations in cystic fibrosis (CF) were made over three decades ago. More recently, physiological findings have directed investigation toward a plethora of humoral substances and their possible role in disturbances of the secretory processes in CF. The application of the techniques of molecular biology and of tissue culture is beginning to increase our knowledge about the so-called CF factors and their diverse effects on epithelial and ciliated surfaces. Emphasis in current research is on the chemical characterization of the CF factors, the metabolism of cells cultured from patients with CF, and the biochemical events controlling the processes of secretion. Some current theories as to the metabolic pathogenesis of CF are considered in the context of existing findings.

Animals↗
PubMed1976

Translational regulatory mechanisms in neoplasia in vivo.

It is clear that regulation of genetic expression of cells need not involve alterations of the basic DNA structure. Between the production of messenger RNA and enzyme degradation there are a number of steps, any of which can be modulated by hormones and other factors to yield cellular abnormalities associated with malignancy. It is important to identify the exact step at which a carcinogen operates and to discover how this alteration is transmitted to the progeny of malignant cells.

Animals↗
PubMed1975

Acetylation of ribosome-associated proteins in vitro by an acetyltransferanse bound to rat liver ribosomes.

Incubation of rat liver ribosomes with [1-14-C]acetyl-coenzyme A results in the incorporation of [14-C]acetyl into a material insoluble in cold trichloroacetic acid. The acetyltransferase involved in the self-acetylation of ribosomes can be released by high salt washing of the ribosomes; the activity of the solubilized enzyme can be assayed using histones as acetyl acceptors. Electrophoretic analysis of acetylated risosomes or ribosomal proteins indicated that the acetyl radicals are associated with a group of relatively basic proteins, having molecular weights ranging from 10,000 to 45,000. Chromatographic analysis of the enzymatic hydrolsates of proteins extracted from acetylated ribosomes indicates that acetylation is mainly or exclusively NH2 terminal. Almost 80% of the acetyl proteins are released from the ribosomes by high salt treatment. Most of the acetyl radicals not solubilized by the high salt treatment were found in the 60S subunit, associated with a protein(s) having an apparent molecular weight of 43,000. This acetyl protein(s) was released from the 60S subunit by EDTA treatment and was found in a ribonucleoprotein complex having a bouyant density of 1.56.

Acetyltransferases↗