Hiroshi Terayama

Preparation and chemical composition of rat liver cell membranes.

Abstract 1. 1. Rat liver cell membranes were prepared from the rat liver homogenate under mild and controlled conditions. The phase contrast micrograph as well as electronmicrographs were presented. 2. 2. Analysis of the chemical components of the cell membrane preparation revealed the following composition: Proteins 41–54 per cent, phospholipids 26–32 per cent, total cholesterol 4–7 per cent, RNA 3–4 per cent, hexosamine about 1 per cent, sialic acids 0.1 per cent. Fatty acids and neutral fat seemed to be present, but their contents were not measured accurately. 3. 3. Among phospholipids, phosphatidyl compounds (72 per cent), sphingo-myeline (ca 8 per cent) and plasmalogens (3 per cent) were detected. Phosphatidylcholine and phosphatidylethanolamine were two major components. Cholesterolester was about 1 10 − 1 20 of the total cholesterol. 4. 4. Amino acid composition of proteins from the cell membrane preparation did not show any specific feature or any resemblance to collagen. Hydroxyproline was not detected in the membrane preparation.

Isolation and identification of glycosaminoglycans associated with purified nuclei from rat liver.

Nuclei isolated from rat liver were purified extensively and then subjected to extraction of glycosaminoglycans by the conventional method with a slight modification including the treatments with amylase, nucleases (DNAase and RNAase), and sialidase in addition to the pronase treatment. The nuclear glycosaminoglycan fraction thus prepared was subjected to chromatography on Dowes 1-X2 (Cl-) and electrophoresis before or after digestion with specific enzymes such as Streptomyces hyaluronidase, chondroitinase ABC and AC. These results together with the results of chemical analyses have revealed that the purified nuclei from rat liver contain glycosaminoglycans equivalent to 0.2-0.3 microgram hexuronic acid per mg DNA. A major component of the nuclear glycosaminoglycans has been identified as hyaluronic acid, while a minor component as chondroitin sulfate A (OR C). Preliminary investigations have shown that most of the nuclear glycosaminoglycans are associated with the chromatin fraction.

Liver plasma membranes and proteoglycan prepared therefrom inhibit the growth of hepatoma cells in vitro

1. Plasma membranes from rat liver or kidney inhibited the growth of hepatoma (AH-130) cells in vitro. AH-130 plasma membranes or erythrocyte ghosts inhibited the growth of AH-130 cells less effectively. The inhibitory activity of liver plasma membranes was lost by heat treatment, or mild protease (papain or bromelin, but not trypsin or pronase) treatment, whereas it was retained after sialidase treatment of delipidation by ethanol/ether. 2. Proteoglycan (proteoheparan sulfate) prepared from liver plasma membranes inhibited the growth of AH-130 cells, but heparan sulfate was less active. The inhibitory activity of liver plasma membranes seemed, however, not to be ascribable solely to proteoheparan sulfate associated with plasma membranes. 3. Preliminary investigation suggested that the molecular weight 40 000 component may be a major inhibitory principle in liver plasma membranes.

Prostaglandin receptor-adenylate cytase system in plasma membranes of rat liver and ascites hepatomas, and the effect of GTP upon it

1. Adenylate cyclase in plasma membranes from rat liver was stimulated by prostaglandin E1, and to a lesser extent by prostaglandin E2. Prostaglandin F1alpha and A1 did not stimulate the cyclase. The prostaglandin E1-mediated activation was found to require GTP when the substrate ATP concentration was reduced from 3 mM to 0.3 mM in the reaction mixture. Adenylate cyclase of the plasma membranes from rat ascites hepatomas AH-130 and AH-7974 was not stimulated by prostaglandin E1 in the presence or the absence of GTP, although the basal activity of adenylate cyclase as well as its stimulation by GTP alone were similar to normal liver plasma membranes. 2. Liver plasma membranes were found to have two specific binders for [3H] prostaglandin E1 with dissociation constants of 17.6-10(-9) M and 13.6-10(8) M (37 degrees C) and one specific binder for [3H]prostaglandin F2alpha with a dissociation constant of 2.31-10(8) M (37 degrees C). The specific binders for prostaglandin E1 could not be detected in the hepatoma plasma membranes. 3. Binding of [3H] prostaglandin E1 to the liver plasma membranes was exchange by, GTP dGPT, GDP, ATP and GMP-P(N)P, but not by GMP, CGMP, DTTP, UTP or CTP. The increase in the binding of [3H] prostaglandin E1 was found to be due to the increased affinity of the specific binders to prostaglandin F2alpha was not affected by GTP. 4. GTP alone was found to increase V of adenylate cyclase of liver plasma membranes, while GTP plus prostaglandin E1 was found to decrease Km of adenylate cyclase in addition to the increase of V to a further extent.

Effects of protease inhibitors on liver regeneration

Summary Effects of microbial protease inhibitors, leupeptin and pepstatin, on rat liver regeneration were studied biochemically and histochemically. Leupeptin plus pepstatin (1 mg each) or leupeptin only (2 mg) given to rats by intraperitoneal injections four times every three hours after partial hepatectomy inhibited or retarded the surges of initial RNA synthesis as well as of subsequent DNA synthesis and mitosis in the regenerating livers. The results were discussed in relation to the possible involvement of lysosomal proteases (cathepsins) in initiating the liver cell proliferation.

Inhibition of DNA synthesis in ascites hepatoma cells by normal liver extract

Abstract 1. 1. The kinetics of the incorporation of [ 14 C]thymidine or [ 14 C]orotate into the DNA or RNA of ascites hepatoma cells, AH-414, incubated in vitro in the presence, or absence, of cell sap from the normal rat liver, have been studied. 2. 2. Remarkable inhibition of DNA synthesis was observed in the presence of the cell sap. However, RNA synthesis was not seriously affected. 3. 3. The inhibitory effect of the cell sap on DNA synthesis depended upon the concentration of the cell sap added, and its effect was removed on washing the cells. 4. 4. A similar effect was also observed with brain, spleen or kidney cell sap. Regenerating liver cell sap showed the same inhibitory activity as normal liver cell sap, but a cell extract or cell sap from ascites hepatoma cells, or from 3′-methyl-4-dimethylaminoazobenzene-induced primary hepatomas, failed to exhibit inhibitory activity. Normal rat liver nuclear sap showed the same activity as the cell sap. 5. 5. The active substance, which was present abundantly in the normal rat-liver cell sap, was thermolabile and displayed protein-like features. Its behaviour during DEAE-cellulose chromatography, zone-electrophoresis, precipitation by change in pH, and precipitation by ethanol, ammonium sulphate and heat, were investigated. The fact that the activity was not found to be associated with the pH 4.5 precipitate suggested that the active substance is not related to deoxyribonuclease, which was reported by Burdon, Smellie and Davidson 10 to be an active principle in the rat-liver extract inhibiting cell-free DNA synthesis. 6. 6. The nature and significance of the active substance, which is present in normal rat-liver cells, but absent in malignant cells, is discussed with respect to the control mechanism of cellular growth.

Inhibitory effects of hydrocortisone upon the phytohemagglutinin-induced RNA-synthesis in human lymphocytes.

Abstract The early increase in the incorporation of [ 3 H]uridine into ribonucleic acids of human lymphocytes in vitro , occurring after an addition of phytohemagglutinin, was inhibited by the presence of hydrocortisone in concentrations of 1–100 μg/ml. Similar inhibition was observed with prednisolone in lower concentrations, whereas deoxycholate in concentrations of 10–100 μg/ml had no inhibitory effect. The inhibitory effect of hydrocortisone was considered to be specific for phytohemagglutinin-stimulated RNA synthesis, since hydrocortisone in concentrations as high as 100 μg/ml did not show a significant effect upon the basal RNA synthesis in lymphocytes. Neither phytohemagglutinin nor hydrocortisone showed any significant effect upon the decomposition rate of lymphocyte RNA. Exposure of lymphocytes to hydrocortisone for various periods of time prior to the phytohemagglutinin stimulation showed variable suppressing effects upon the RNA synthesis which followed, depending upon the length of the previous exposure to hydrocortisone.

Amino acid incorporation into proteins in isolated rat liver nuclei

Abstract Rat liver nuclei, isolated in a concentrated sucrose solution, were able to incorporate [14C]leucine into proteins. Incorporation of the amino acid into nuclear proteins was insensitive to puromycin, cycloheximide, actinomycin D and ribonuclease, but was only slightly inhibited by deoxyribonuclease. It was, however, markedly inhibited by chloramphenicol and several reagents known to interfere with endogenous energy metabolism. Evidence was presented to show that [14C]leucine is incorporated into both intrapolypeptide and C-terminal residues but not into N-terminal residues. Distribution of the incorporated radioactivity among different nuclear protein fractions was investigated, and it was found that [14C]leucine is incorporated preferentially into the nuclear residual proteins. The subnuclear origin(s) of this protein fraction is not yet fully clarified, but it seems to be derived from some membranous and/or chromosomal component(s). Nucleoli were found not to be involved in the incorporation of [14C]leucine in the isolated nuclear system.

Pattern of glycosaminoglycans and glycoproteins associated with nuclei of regenerating liver of rat

1. Hyaluronic acid was detected as the largest glycosaminoglycan component in the glycosaminoglycan fraction from purified nuclei of regenerating livers as in the case of normal livers (Furukawa, K. and Tarayama, H. (1977) Biochim. Biophys. Acta 499, 278--289). However, the nuclear content of glycosaminoglycans tended to decrease after partial hepatectomy, reaching one-third of the normal liver level at 24--30 h after partial hepatectomy. On the other hand, two new polyanionic components were detected in the glycosaminoglycan fraction from regenerating liver nuclei. 2. One of these new components seems to be a sulfated glycopeptide. The 35SO4 incorporation into this component was stimulated biphasically after partial hepatectomy; the first stimulation occurring immediately after partial hepatectomy and the second stimulation occurring almost in parallel to the DNA synthesis. 3. Another polyacnionic component which also increases in the nuclear content after partial hepatectomy lacks hexuronic acid, sialic acid and 35SO4 and yet it is intensely stained by Alcian Blue. Preliminary investigations revealed the presence of hexose, ribose and phosphate as the major components. 4. In contrast to the primary localization of hyaluronic acid in the chromatin fraction and also in the nonhistone chromosomal protein fraction from it, these new polyanionic components were detected mainly in the karyosol fraction.

DNA synthesis in isolated nuclei from the brains of rats at different post-partal stages and the infant rat brain cytosol factor stimulating the DNA synthesis in infant rat brain nuclei.

Abstract [3H]TTP incorporation into DNA of isolated nuclei from the brains of rats at different post-partal stages, as well as the effects of the adult and the infant rat brain cytosols upon the DNA synthesis in the isolated nuclear system, have been investigated. The nuclear system requires the four deoxyribonucleoside triphosphates, ATP and Mg2+ for a maximum activity and DNA synthesis proceeds linearly within 30 min of incubation. A large portion of [3H]TTP incorporated into DNA is sensitive to actinomycin D inhibition and seems to be DNA polymerase dependent, while the remaining incorporation (approx. 20 % in the infant nuclear system and approx. 40 % in the adult nuclear system) is less sensitive to actinomycin D inhibition, proceeds without dATP, dCTP and dGTP and seems to be due to the terminal DNA nucleotidyltransferase activity. The nuclear activity of [3H]TTP incorporation into DNA increases after birth, reaches a maximum on the fifth day and then declines rapidly, reaching the adult level (i. e. one half the maximum level) on the 22nd day after birth. [3H]TTP incorporation into DNA of isolated infant rat brain nuclei is stimulated specifically by the infant rat brain cytosol. The active principle in the infant rat brain cytosol seems to be thermostable and low molecular, and differs from the stimulatory factor present in the serum.