Glucosamine metabolism in regenerating rat liver

1. Glycoprotein synthesis was investigated with [1-14C]glucosamine in vivo. [14C]Glucosamine was administered intravenously 24h after hepatectomy to rats. 2. Incorporation into the acid-soluble fraction was maximum at 15 min after injection both in sham-operated and hepatectomized rats. 3. Enhancement of incorporation into UDP-N-acetylhexosamine in regenerating liver was observed. However, its specific activity was lower, because of a greater enhancement of synthesis de novo of the amino sugar. 4. In the liver acid-insoluble fraction, maximum incorporation of [14C]glucosamine was at 30 min in sham-operated rats and 2 h in hepatectomized rats respectively. 5. In sham-operated rats, incorporation into the plasma acid-insoluble fraction followed that of the liver acid-insoluble fraction, but hepatectomy resulted in a rapid enchancement of incorporation into plasma. 6. It is concluded that synthesis of liver glycoproteins is stimulated after partial hepatectomy and that glycoproteins synthesized are released rapidly into the plasma.     

Inhibition of DNA methylation by S-adenosylethionine with the production of methyl-deficient DNA in regenerating rat liver

Ethionine, a liver carcinogen, was administered p.o. (300 mg/kg) to rats 17 hr after partial hepatectomy. At 6 hr after administration of the ethionine, hepatic S-adenosylethionine levels were 30- to 40-fold greater than the hepatic level of S-adenosylmethionine. A 10-fold ratio of S-adenosylethionine to S-adenosylmethionine still persited at 24 hr after ethionine administration. When given at 17 hr after partial hepatectomy, ethionine produced a 30% inhibition of DNA synthesis, measured by the incorporation of [methyl-3H]thymidine at 23 to 24 hr after partial hepatectomy (6 to 7 hr after ethionine administration). DNA synthesized during this interval was methyl deficient as judged by the reduced incorporation of radioactivity from L-[methyl-3H]methionine into 5-methylcytosine residues of DNA. In an assay for DNA methylation in vitro using whole nuclei, the methyl-deficient DNA was methylated by S-adenosylmethionine 8 times more than was control DNA; the DNA methylation was competitively inhibited by S-adenosylethionine. These data suggest that S-adenosylethionine, formed in vivo from ethionine, competitively inhibits the methylation of DNA in vivo by S-adenosylmethionine, resulting in the production of methyl-deficient DNA.     

Effects of 9-aminocamptothecin on newly synthesized DNA in patient bone marrow samples

9-Aminocamptothecin (9-AC) inhibited cell growth and DNA synthesis in HCT 116 human colon cancer cells in a concentration- and time-dependent manner. Interference with nascent DNA chain elongation was monitored using pH step alkaline elution. After a 3-day 9-AC exposure, 38% (10 nM) and 53% (50 nM) of the total [3H]DNA eluted with pH steps 11.3-11.7, compared to 9% in control cells. Effects on nascent DNA integrity were also evaluated by fixed elution with pH 12.1 buffer. After a 3-day exposure to 9-AC, 27% (10 nM) and 82.5% (50 nM) of the total [3H]DNA eluted relative to control. Paired bone marrow samples were then obtained in 10 patients before treatment and between 42 and 72 h of a continuous i. v. infusion of 9-AC (35-74 microgram/m2/h for 72 h). The mononuclear cells were incubated with [3H]dThd for 2 or 4 h, and then analyzed using either pH step or fixed pH alkaline elution, respectively. In seven patients receiving >/=47 microgram/m2/h 9-AC, 4% +/- 1.5% (mean +/- SE) of the total [3H]DNA eluted with pH steps /=59 microgram/m2/h 9-AC (n = 7). Since hematological toxicity is dose limiting on this 9-AC schedule, these cellular pharmacodynamic studies provide evidence of a DNA-directed cytotoxic effect of 9-AC in a sensitive host target tissue.     

Synthesis of secretory protein in regenerating liver of rat after partial hepatectomy

Albumin immunoreactivity in the liver was examined on days 2, 5 and 10 after two-thirds partial hepatectomy by light and ultrastructural immunoperoxidase methods and the ultrastructural area of the rough endoplasmic reticulum (ER) in hepatocytes was measured. Albumin immunoreactivity was seen in the rough ER and Golgi apparatus of all hepatocytes in the hepatectomized liver and ultrastructural analysis showed a significantly greater area of rough ER on day 5 than on days 2 or 10. Albumin mRNA was studied by the in situ hybridization technique using radioisotopes and their numbers were determined visually. Albumin mRNA was present as grains in all hepatocytes and the grains varied in number during regeneration of the liver, being more abundant on day 5 than on days 2 or 10. The activity of [3H]-leucine incorporated into albumin synthesis, an indicator of translational activity, was higher on days 5 and 10 than on day 2 and was highest on day 5. In conclusion, albumin synthesis varied during liver regeneration after partial hepatectomy, being reduced at the peak of cell proliferation on day 2 and being most active on day 5.     

Manipulation of metallothionein expression in the regenerating rat liver using antisense oligonucleotides

In order to gain some insight into the fate of fumarate synthesised in the cytosol in the purine nucleotide cycle and in amino acid catabolism, the capability of both rat kidney mitochondria and acidotic rat kidney mitochondria to take up either externally synthesised, via adenylsuccinate lyase, or added fumarate in exchange with intramitochondrial malate or aspartate was tested by means of both spectrophotometric and isotopic techniques. The appearance of either malate or aspartate caused by the presence of fumarate was revealed outside normal and acidotic mitochondria by using specific substrate detecting systems. Consistently, externally added fumarate was found to cause efflux of either [14C]-malate or [14C]-aspartate from loaded mitochondria. The occurrence in rat kidney mitochondria of two separate translocators, i.e., fumarate/malate and fumarate/aspartate carriers, is shown in the light of saturation kinetics and the different inhibitor sensitivity. The fumarate/aspartate antiporters found in normal and acidotic mitochondria appear to differ from each other.     

Trafficking of newly synthesized surfactant protein A in isolated rat alveolar type II cells

We examined the synthesis, transport, and localization of surfactant protein A (SP-A) in primary cultures of alveolar type II cells. In type II cells maintained in culture for 6 h, 39% of the SP-A pool detected with an enzyme-linked immunosorbent assay (ELISA) was found in lamellar bodies (LBs). After 24 h in culture, 53% of the cellular SP-A pool was found in LBs. The absolute amount of SP-A in the LB compartment was almost identical at 6 and 24 h of culture. In contrast to the results obtained with ELISA, 35S labeling of newly synthesized SP-A revealed that less than 7% of the cellular SP-A pool was in LBs at either 6 or 24 h of culture. In the 6-h cultures, 17% of the total (i.e., cells and media) [35S]SP-A pool was extracellular. In the 24-h cultures, 70% of the [35S]SP-A pool was extracellular. The secretion of [35S]SP-A was blocked by brefeldin A at all times. When medium containing newly secreted [35S]SP-A was incubated with alveolar type II cells maintained in culture for 24 h, the protein was taken up and incorporated into the LB fraction. More than 80% of the internalized SP-A was associated with the LB compartment after a 6 h incubation. The uptake of [35S]SP-A was blocked at 4 degrees C and was promoted by addition of unlabeled SP-A at 37 degrees C. These findings support a pathway of extracellular routing of SP-A prior to its accumulation in LBs in cultured type II cells.     

Formation and fate of tyrosine. Intracellular partitioning of newly synthesized tyrosine in mammalian liver

Tyrosine in an hepatocyte is transported from the plasma, synthesized from phenylalanine, or released during protein turnover. Effects of phenylalanine and tyrosine on the formation and fate (partitioning) of tyrosine from the different sources were examined in primary rat hepatocyte cultures. Rates of tyrosine degradation, transport, incorporation into and release from protein, and synthesis from phenylalanine were measured as well as the intracellular dilution of labeled tyrosine and phenylalanine incorporated into protein. We found tyrosine had little effect on phenylalanine hydroxylation over a wide range of conditions, that transported tyrosine and tyrosine from phenylalanine are in different metabolic pools, and that there appears to be channeling of newly synthesized tyrosine during degradation. In addition, under some conditions, intracellular partitioning of tyrosine is determined by tyrosine concentration. Specifically, if extracellular tyrosine is low and phenylalanine is at a normal plasma level, tyrosine use in protein synthesis takes precedence over tyrosine degradation or export. It is proposed that the mechanism controlling this is kinetic, based on relative rates of tyrosyl-tRNA formation and tyrosine degradation and export. A quantitative model of tyrosine and phenylalanine in-flow and out-flow in hepatocytes is given, incorporating tyrosine synthesis, degradation, plasma membrane transport, and tyrosine and phenylalanine use and release during protein turnover.     

Regenerating rat liver DNA polymerases: disimilitude or relationship between nuclear and cytoplasmic enzymes?

The possible relationship between the nuclear and cytoplasmic DNA polymerases of regenerating rat liver was studied by sucrose gradient analysis, salt dissociation, and with specific inhibitors. After aqueous subcellular fractionation and removal of the nuclear membranes, three species of DNA-dependent DNA polymerases were characterized: 1) a DNA polymerase-beta in the nuclei. 2) a DNA polymerase-alpha in the cytosol which was not dissociated at high salt concentrations; and 3) an intermediate form in the cytosol and in the Triton wash containing the nuclear membranes. The latter form behaved like DNA polymerase-alpha et low salt concentration but was dissociated at high salt concentrations to a low molecular weight species with properties like DNA polymerase-beta (resistance to inhibition by N-ethylmaleimide, heparin and KCL). In vitro reassociation experiments suggest that this intermediate form corresponds to the association of DNA polymerase-beta with a membrane component or cytoplasmic protein(s) which appear(s) in regenerating rat liver.     

Purification of rat liver nuclear protein kinase NII

Rat liver nuclear protein kinase activity (NII), which is eluted from DEAE-Sephadex columns, has been purified approximately 1500-fold from solubilized nuclear protein. The method of purification involved chromatography of protein eluted from DEAE-Sephadex successively on phosvitin-Sepharose, mixed histone-Sepharose, and histone H2b-Sepharose followed by gel filtration on Sephadex G-200. Resulting preparations are homogeneous by polyacrylamide gel electrophoresis. The enzyme consists of three polypeptides with molecular weights of 42,000 (alpha), 39,000 (alpha'), and 26,000 (beta) which are present in the ratio 1:1:2 indicating that the enzyme has a minimum tetrameric subunit composition of alphaalpha'beta2. The molecular weight and s20,w of the purified enzyme were 123,000 and 7.0, respectively, as determined by sucrose density gradient centrifugation in 0.4 M NaCl. The enzyme has maximal activity with phosvitin as substrate and is not stimulated by 10(-5) to 10(-4) M cAMP or cGMP using H2b as substrate.     

Mitochondrial DNA polymerase from rat liver

A DNA-dependent DNA polymerase from rat liver mitochondria was partially purified and characterized. Mitochondrial DNA polymerase has been found to be quite different from other DNA-dependent DNA polymerases alpha and beta present in the rat liver in the following points: elution patterns in a DEAE-cellulose column chromatography, sedimentation coefficients determined by the glycerol gradient centrifugation in the presence of high salt, and sensitivities to N-ethylmaleimide, ethidium bromide and KCl.     

Neuropharmacological actions of some newly synthesized Mannich bases

Benzamido (alkyl) methyl pyrrolidine Mannich bases were synthetized and subjected to certain neuropharmacological studies. All the bases reduced the pentobarbitone sleeping time and rota-rod grip of rats. The Mannich bases II, III and V raised the minimal electro-shock seizure threshold of rats. The TAB-induced pyrexia was not reduced by the bases I and III in rabbits. None of the bases showed any significant analgesic activity.     

Variation of H1(0) content throughout the cell cycle in regenerating rat liver

Histone H1(0), a differentiation-specific member of the histone H1 family, accumulates in cells during the terminal phase of cell differentiation, in tissues composed of arrested cells or cells exhibiting little proliferation. Moreover, the induction of cell proliferation in vivo, i.e., after partial hepatectomy, is accompanied by a decrease in H1(0) content. These observations suggest that H1(0) may be involved in the arrest of cell proliferation in vivo. In order to investigate this possibility, we took advantage of the fact that after partial hepatectomy the initiation of cell division is not synchronous. The strategy was to know, at the level of a single cell, whether H1(0) decreases prior to the initiation of the S phase or whether a cell can initiate DNA replication having a significant amount of H1(0) in the nucleus. We defined new protocols to analyze H1(0) content and cell proliferation at the level of a single cell, both in situ and by flow cytometry. The simultaneous determination of the relative amount of H1(0) and the position of cells in the cell cycle showed that no significant difference in H1(0) content was detected in cells actively replicating their DNA compared to nondividing cells. These observations have been confirmed by the successive immunodetections of H1(0) and BrdU in situ on the same cells. Therefore, we show here that in vivo, cells can initiate DNA replication with significant amounts of H1(0) and that the decrease of H1(0) is not a prerequisite of cell division. We propose that the accumulation of H1(0) is not related to the arrest of cell proliferation, but is controlled in such a manner that the protein accumulates in slowly dividing cells and decreases in rapidly growing cells.     

Characteristics of rat pancreatic regenerating protein

BACKGROUND: The pancreatic regenerating (reg) gene is an acinar cell product involved in islet formation and maintenance. Human reg protein is mitogenic to pancreatic beta and ductular cells, and its amino acid sequence predicts it to be a calcium-dependent lectin. METHODS: We studied the biologic activity and cellular localization of rat reg I isolated from the acinar cell line AR42J and the lectin properties of reg from AR42J and pancreatic juice. Bioactivity was assayed by mitogenesis on the ductular cell line ARIP. Cellular localization was determined by differential centrifugation. Lectin properties were assessed by affinity chromatography. RESULTS: Reg protein from crude AR42J cellular lysate and purified reg protein from AR42J induced thymidine incorporation to ARIP. Conditioned medium from AR42J and co-culture of AR42J with morphologically distinguishable ARIP, however, failed to induce mitogenesis. Reg protein was localized within the vesicle fraction of the cell and was not membrane bound. Affinity chromatography revealed that reg protein did not bind to mannose or galactose in the presence or absence of calcium. In pancreatic juice a previously undescribed mannose-binding protein was discovered at 25,000 to 30,000 daltons. CONCLUSIONS: We conclude that reg produced in the acinar cell line AR42J is biologically active but not efficiently secreted, even though it localized within the cellular vesicles. Despite predictions based on its amino acid sequence, it does not appear to be a calcium-dependent lectin.     

Rapid genomic changes in newly synthesized amphiploids of Triticum and Aegilops. II. Changes in low-copy coding DNA sequences

Postischemic myocardium possesses considerable contractile and metabolic reserves, but their mobilization could result in increased cell death. We tested the hypothesis that beta-adrenergic stimulation of reperfused myocardium would increase segment work more than O2 consumption, thereby improving efficiency without increased cell death. In 16 open-chest anesthetized dogs, the left anterior descending coronary artery (LAD) was ligated for 2 h; during the reperfusion period, isoproterenol (ISO; 0.1 microg/kg/min, i.v.) was administered to nine of the animals. Regional myocardial segment length and force were measured in the anterior (LAD) and posterior circumflex coronary artery (CFX) regions of the left ventricular myocardium. Work was calculated as the integrated products of force and shortening for each region. Regional myocardial O2 consumption was obtained from LAD flow and arterial and local venous O2 saturations. Infarct size (tetrazolium) was measured in the treated and untreated hearts at the end of the experiment. In untreated hearts, the first derivative of left ventricular pressure, cardiac output, and external work were significantly depressed during reperfusion; ISO restored all values to preocclusion levels. Regional myocardial work in both LAD and CFX regions was significantly increased by ISO (from 564 +/- 207 to 1,635 +/- 543 g/mm/min in LAD, and from 753 +/- 90 to 1,426 +/- 245 g/mm/min in CFX). Efficiency (work/oxygen consumption) of the reperfused region was similarly increased. LAD flow was significantly increased by ISO, and O2 extraction was unchanged. Infarct size was 28.2 +/- 4.7% in untreated hearts and 29.0 +/- 3.5% in ISO hearts. Thus isoproterenol stimulation significantly improved both regional and global function without subsequent evidence of increased cell death.     

Effects of growth factors on the enzymes of purine metabolism in culture of regenerating rat liver cells

Hepatocytes are affected by many cytokines and growth factors during liver regeneration. In regenerating rat liver cells cultures, liver cell growth factor (LCGF), hepatic stimulator substance (HSS), interleukin-1 beta (IL-1 beta), as well as their combination, were tested for their ability to activate the enzymes involved in purine metabolism. The enzymes tested were 5' nucleotidase, AMP deaminase, adenosine deaminase and xanthine oxidase. The cytokines alone or in combination, activated 5' nucleotidase and adenosine deaminase. Activity of AMP deaminase was stimulated by IL-1 beta associated with LCGF, HSS and IL-1 beta. Xanthine oxidase was stimulated by IL-1 beta but not with HSS and LCGF. Associated with IL-1 beta these two substances decreased its activity. A novel approach to the understanding of the mechanisms involved in the regulation of purine metabolism during liver regeneration, is proposed.