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Biomedical subjects

A Ichihara

Publications and source records attributed to A Ichihara.

At least 181 records · Page 10Linked to original sources
PubMed1986

Immunohistochemically demonstrated suppressed expression of tryptophan oxygenase, a marker for liver differentiation, within putative preneoplastic rat liver lesions.

The behaviour of rat liver putative preneoplastic lesions with respect to the enzyme tryptophan oxygenase (TO), a liver-specific differentiation marker, and a possible growth-related marker, glucose-6-phosphate dehydrogenase (G6PD) was investigated during and after their induction by diethylnitrosamine initiation and subsequent 'selection pressure'. Using specific antibodies to rat liver TO and G6PD and the avidin-biotin complex method for immunohistochemical staining it was demonstrated that all of the nodular lesions showing increased expression of G6PD during the induction phase were also negative or deficient in TO enzyme protein. With the onset of 'phenotypic instability' or loss of marker enzymes, a gradual return to normal expression of TO activity was evident. Administration of dexamethasone and L-tryptophan 11 weeks after cessation of carcinogen treatment allowed differentiation between morphologically altered, apparently persisting lesions in which no, or little, enzyme induction was apparent and instable lesions showing a strong increase in levels of TO protein. Thus, persisting nodular lesions share a common lack of response to normal homeostatic physiological control.

Animals↗
PubMed1986

Immunohistochemical demonstration of increased glucose-6-phosphate dehydrogenase in preneoplastic and neoplastic lesions induced by propylnitrosamines in F344 rats and Syrian hamsters.

Changes in the level of expression of glucose-6-phosphate dehydrogenase (G6PD) within propyl nitrosamine-induced preneoplastic and neoplastic lesions in F344 rats and Syrian golden hamsters were investigated using an immunohistochemical approach. Previously demonstrated increases in G6PD activity in rat liver and hamster pancreatic foci of altered cells were revealed as being due to elevation in the quantity of enzyme protein, suggesting an underlying change in gene expression. Furthermore, strong positive binding of G6PD antibody in thyroid, lung, urinary bladder and kidney lesions indicated that increase in this enzyme protein might be a common marker for neoplastic alteration, regardless of organ. While the function of elevated G6PD may be related to growth requirements, the finding that preneoplastic lesions in some cases bind more strongly than more malignant populations suggests additional involvement of the enzyme in other biochemical pathway(s) relevant to tumorigenesis.

Animals↗
PubMed1986

Distinct effects of transforming growth factor-beta on EGF receptors and EGF-induced DNA synthesis in primary cultured rat hepatocytes.

Previous studies showed that transforming growth factor-beta (TGF-beta, 1 ng/ml) strongly inhibited DNA synthesis induced by epidermal growth factor (EGF) in primary cultures of adult rat hepatocytes. This paper reports that TGF-beta (4 ng/ml) caused marked increase of EGF-binding to cultured rat hepatocytes. The binding increased biphasically with time to a maximum after treatment with TGF-beta for 12 h. Scatchard analysis showed that adult rat hepatocytes had a single class of non-cooperative binding sites with a Kd of 1.5 nM, that there were 1.4 X 10(5) binding sites/cell, and that TGF-beta increased the number of binding sites without changing the Kd value. The increase in EGF binding sites by TGF-beta was dose-dependent and the dose that elicited the maximum increase was about 10 times that which inhibited DNA synthesis stimulated by EGF. These findings suggest that the effect of TGF-beta in modulating the EGF receptor is not directly related to that in inhibiting DNA synthesis in adult rat hepatocytes.

Animals↗
PubMed1985

Inhibitory effect of transforming growth factor-beta on DNA synthesis of adult rat hepatocytes in primary culture.

A transforming growth factor-beta (TGF-beta) found in platelets strongly inhibited DNA synthesis of adult rat hepatocytes in primary culture stimulated by insulin plus EGF or by hepatocyte growth factor (HGF) from rat platelets, but not the syntheses of secretory and intracellular proteins by the cells. TGF-beta had no cytotoxic effect, as judged by phase-contrast microscopic examination of the cell morphology. The inhibition of DNA synthesis by TGF-beta was correlated with marked decrease in the labeling index. TGF-beta did not inhibit growth of hepatoma cell line. These findings indicate that TGF-beta is a strong growth inhibitor of adult rat hepatocytes and may block their shift from the G1 phase to the S phase. The physiological role of TGF-beta in inhibiting growth of adult hepatocytes during liver regeneration is discussed.

Animals↗
PubMed1985

Molecular cloning of DNA complementary to mRNA of rat liver serine dehydratase.

A cDNA clone containing sequences complementary to the mRNA cording for rat hepatic serine dehydratase was isolated to study the multihormonal regulation of this enzyme. Serine dehydratase mRNA was partially purified (50-fold enrichment, 8.2% of the total mRNA activity) from the liver of rats fed high protein diet by polysome immunoadsorption followed by oligo(dT)-cellulose column chromatography. This preparation was used as template for synthesis of cDNA. Double-stranded cDNA sequences were inserted into the plasmid pBR322 and cloned in Escherichia coli DH1. Of 860 transformants screened, 6 clones containing DNA complementary to serine dehydratase mRNA were identified by differential colony hybridization and hybrid-selected translation. The length of serine dehydratase mRNA was estimated to be 1,500 bases by Northern blot analysis. One cloned cDNA comprised about 1,000 base pairs, or 65% of the length of the mRNA. The amount of the mRNA was greatly increased in the liver of rats given high protein diet.

Animals↗
PubMed1985

Enzyme leakage due to change of membrane permeability of primary cultured rat hepatocytes treated with various hepatotoxins and its prevention by glycyrrhizin.

Various hepatotoxins were added to the medium of primary cultures of adult rat hepatocytes and the release of the cytosolic enzymes lactic dehydrogenase, glutamic-oxaloacetic and glutamic-pyruvic aminotransferases were measured 24 h later. CCl4 at low concentrations caused dose-dependent release of soluble enzymes into medium without appreciable cytolysis of the hepatocytes. Mitochondrial enzymes were not released under these conditions. At 5 mM CCl4, both soluble and mitochondrial glutamic-oxaloacetic aminotransferase were found in the culture medium. Glycyrrhizin, a triterpenoid glycoside of licorice roots, prevented the enzyme release caused by CCl4.

Animals↗
PubMed1985

Control of growth and expression of differentiated functions of mature hepatocytes in primary culture.

Since methods to disperse and culture hepatocytes were developed 15 years ago, numerous investigations have shown that primary cultures of mature hepatocytes retain most liver functions and respond as well to various hormones as those in vivo. Thus they are the most suitable system in vitro for studies on the liver. Moreover, recently it was found that differentiated hepatocytes in culture can grow under certain conditions and that this growth is regulated not only by several hormones, such as insulin, epidermal growth factor and serum growth factor, but also by a cell membrane factor and proteins in the environmental matrix through cell contact. This article describes the biochemical characterization of regulatory factors for hepatocyte growth and functions and their reciprocal expression. The mechanisms of liver regeneration, differentiation and carcinogenesis and the importance of the tissue architecture for these events are discussed mainly on the basis of our findings.

Animals↗
PubMed1985

Comparison of the various forms of glutathione S-transferase with glucose-6-phosphate dehydrogenase and gamma-glutamyltranspeptidase as markers of preneoplastic and neoplastic lesions in rat kidney induced by N-ethyl-N-hydroxyethylnitrosamine.

Alterations in gamma-glutamyl transpeptidase (gamma-GT) and alkaline phosphatase (ALP) activities and binding of specific antibodies to glucose-6-phosphate dehydrogenase (G6PDH), gamma-GT and the A, B, C, D, and P forms of glutathione S-transferase (GST-A, -B, -C, -D, and -P, respectively) in preneoplastic and neoplastic lesions induced by N-ethyl-N-hydroxyethylnitrosamine (EHEN) in the rat kidney were investigated. Morphologically the lesions were of basophilic type and were classified either as altered tubules or adenomas, the latter being further subdivided into microadenomas and adenomas depending on size and the presence of compression. The fact that all lesions demonstrated only weak (or negative) gamma-GT, ALP and GST-B stainings, all of which are normally evident in proximal tubules, and also the occasional finding of cells bearing a periodic acid-Schiff-positive apical brush border within altered tubules strongly positive for GST-A, indicated their histogenesis from the proximal segment of the nephron. Thus a directed shift to a phenotype opposite to that observed in the cells of origin was demonstrated. Comparison of phenotype within the range of EHEN-induced lesions strongly suggested putative preneoplastic character for the altered tubules. Transition to adenomas appeared to be correlated with progressive loss of GST-A and moderate to slight increase of G6PDH.

Adenoma↗
PubMed1984

L-proline is an essential amino acid for hepatocyte growth in culture.

For improvement of the culture conditions of adult rat hepatocytes in primary culture in collagen coated dishes, effects of various commercial culture media on the induction of replicative DNA synthesis of the cells stimulated by insulin plus epidermal growth factor were studied. Proline-deficient media, such as Leibovitz's L-15, Eagle's minimal essential medium and Dulbecco's modified minimal essential medium, did not induce DNA synthesis in hepatocytes, whereas proline-rich media, such as Williams medium E, McCoy's 5A and Ham's F-12, induced markedly hepatocyte proliferation. Moreover, when the proline-deficient media were supplemented with L-proline, the cells synthesized DNA in response to the two hormones. Cis-4-hydroxyl-L-proline strongly inhibited the induction of DNA synthesis, without affecting protein synthesis of the cells or showing any cytotoxicity. This inhibition was recovered completely by adding excess proline to the medium. Addition of other amino acids not present in the medium did not promote DNA synthesis. These findings indicate that L-proline is essential for induction of hepatocyte proliferation in culture, through its affect on synthesis of intracellular collagen.

Animals↗
PubMed1984

Partial purification and characterization of hepatocyte growth factor from serum of hepatectomized rats.

When rat serum was subjected to gel filtration on a Sephadex G-200 column, a factor, "hepatotropin", that promoted hepatocyte growth in primary culture was separated. Its Mr was about 150 KD and it was an anionic protein that was unstable on acid- and heat-treatments. Hepatotropin was purified 20-fold further by affinity chromatography on heparin-Sepharose CL-6B. The purified hepatotropin was effective at 20 micrograms/ml and maximally effective at 120 micrograms/ml, and its effect was additive with that of insulin plus epidermal growth factor. In rats after partial hepatectomy, the hepatotropin activity in the serum increased time-dependently reaching a maximum of about 5-fold the initial level 24 h after the operation. Various known growth factors, such as fibroblast growth factor, platelet derived growth factor, somatomedin, thrombin and transferrin, did not stimulate DNA synthesis in cultured hepatocytes. These results suggest that hepatotropin is a new growth factor.

Animals↗
PubMed1984

Reciprocal modulation of growth and liver functions of mature rat hepatocytes in primary culture by an extract of hepatic plasma membranes.

A cell-surface modulator with the ability to mimic the reciprocal effects of cell density on cell growth and liver-specific functions of mature rat hepatocytes in primary culture (Nakamura, T., Yoshimoto, K., Nakayama, Y., Tomita, Y., and Ichihara, A. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7229-7233) was solubilized with 4% octyl glucoside and 4 M guanidine HCl from plasma membranes purified from adult rat liver. The membrane-free modulator showed activities for inhibition of DNA synthesis stimulated by insulin with epidermal growth factor and stimulation of tyrosine aminotransferase induction by dexamethasone. The apparent Mr of the factor was estimated as 670,000 by gel filtration on a Sephacryl S-400 column equilibrated with 0.5% octyl glucoside and 4 M guanidine HCl. The modulator was heat-labile and sensitive to trypsin. These results suggest that the reciprocal regulations of cell growth and hepatocyte-specific functions are mediated via cell to cell contact by a cell-surface modulator(s) that is an integral membrane protein.

Animals↗
PubMed1984

Mechanism of heterologous desensitization of the adenylate cyclase system by glucagon in primary cultures of adult rat hepatocytes.

During treatment of primary cultured hepatocytes with either glucagon or isoproterenol for several hours, the stimulations of cAMP formation by these hormones decreased time dependently. Glucagon treatment also reduced the response to isoproterenol, but isoproterenol treatment did not decrease the response to glucagon. Treatment with isoproterenol caused more rapid desensitization than treatment with glucagon. Assays of glucagon and beta-adrenergic receptors showed that the receptor number of only the hormone with which the cells were treated decreased and that dissociation constants of the receptors did not change. Moreover, in glucagon-desensitized cells, the effect of GTP on competition of the bindings of antagonist and agonist for the beta-adrenergic receptor did not change. After treatment with isoproterenol, stimulation of adenylate cyclase activity by the agonist was decreased without any decrease in the stimulations of activity by other effectors. In contrast, glucagon treatment greatly decreased the stimulations of activity by glucagon, isoproterenol, and guanyl-5'-yl imidodiphosphate and slightly decreased that by fluoride. However, after glucagon treatment, the cells showed normal responses to cholera toxin of activation of adenylate cyclase and ADP-ribosylation of guanine nucleotide binding regulatory protein (Ns). The maximal response of glucagon-treated cells to forskolin was about two-thirds that of untreated cells and this treatment also impaired the shift toward a low Kact value for forskolin observed in the presence of either glucagon or isoproterenol. These results indicate that isoproterenol caused homologous desensitization consisting of only a "down regulation" of the beta-adrenergic receptor, whereas glucagon caused heterologous desensitization, mainly by alteration of the Ns component, as well as "down regulation" of glucagon receptor. This altered Ns seems to be coupled normally to the beta-adrenergic receptor, but to have impaired coupling to the catalytic component of adenylate cyclase.

Adenylyl Cyclases↗
PubMed1984

Purification and characterization of hemoglobin-hydrolyzing acidic thiol protease induced by leupeptin in rat liver.

Intraperitoneal administration of leupeptin to rats induced a hemoglobin-hydrolyzing protease which was most active at pH 3.5 and was insensitive to pepstatin in various tissues such as the liver, kidney, and muscle, as observed previously in adult rat hepatocytes in primary culture (Tanaka, K., Ikegaki, N., and Ichihara, A. (1979) Biochem. Biophys. Res. Commun. 91, 102-107). The induced acidic protease was purified about 600-fold in 30% yield from rat liver by conventional chromatographic techniques. The purified enzyme appeared homogeneous by polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate and was a monomeric protein of Mr = 20,000. The enzyme appeared to be a glycoprotein because its induction was blocked by the addition of tunicamycin to cultures of hepatocytes and because the induced protease was absorbed on concanavalin A-Sepharose and eluted with methylglucoside. It seemed to be present in lysosomes and was fairly stable at various pH values and temperatures. It showed endopeptidase activity on various protein substrates, but scarcely hydrolyzed N-substituted derivatives of arginine. It did not hydrolyze esters, showed no aminopeptidase or carboxypeptidase activity, and did not inactivate glucose-6-phosphate dehydrogenase or aldolase. The enzyme appeared to be a thiol protease, since it was strongly inhibited by sulfhydryl-reactive compounds and N-( [N-(1-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine and was not inhibited by reagents specific for carboxyl-, serine-, or metalloproteases. This induced protease could be separated from cathepsins B, D, and H by chromatography. The enzyme was similar to cathepsin L in chromatographic behavior, Mr and pI, but differed from the latter in stability and in its inability to inactivate some enzymes. These results suggest that it differs from any known proteases found previously in rat liver.

Animals↗
PubMed1984

Increased survival of rat hepatocytes in serum-free medium by inhibition of a trypsin-like protease associated with their plasma membranes.

Bovine pancreatic trypsin-inhibitor (bPTI) is required for survival of adult rat hepatocytes for more than 2 days in primary cultures in serum-free medium. Of the various protease inhibitors tested, all trypsin inhibitors increased the survival of rat hepatocytes in serum-free medium, their potencies being in the order bPTI greater than alpha 2-plasmin inhibitor greater than leupeptin greater than soybean trypsin inhibitor greater than alpha 1-antitrypsin = alpha 2-macroglobulin. Elastatinal, a specific inhibitor of elastase, was also effective. bPTI did not inhibit the degradation of proteins with short or long lives, suggesting that it did not increase the survival of hepatocytes by inhibiting cellular protein degradation. alpha 2-Plasmin inhibitor immobilized on Sepharose 4B caused dose-dependent increase in survival. Plasma membranes purified from adult rat liver had significant protease activity, about 80% of which was sensitive to bPTI, alpha 2-plasmin inhibitor and leupeptin. From its specificity for substrates and sensitivity to inhibitors, the membrane-bound protease was characterized as a trypsin-like protease. The effects of various inhibitors on the membrane-bound protease correlated well with their abilities to increase survival of rat hepatocytes. Therefore, it seems that bPTI acts on the cell surface and increases hepatocyte survival in serum-free cultures by inhibiting a trypsin-like protease associated with the plasma membranes.

Animals↗
PubMed1984

Loss of reciprocal modulations of growth and liver function of hepatoma cells in culture by contact with cells or cell membranes.

In mature rat hepatocytes in primary culture, many metabolic functions and cell growth are controlled reciprocally by cell density, and this reciprocal regulation is mediated by a cell-surface modulator through cell-cell contact. Cultured RY-121B cells from Reuber hepatoma and MH1C1 cells from Morris hepatoma, which retain some liver-specific functions, did not show any cell-density dependency of either cell growth or hepatocyte-specific functions, such as induction of tyrosine aminotransferase by dexamethasone. However, when RY-121B cells were cocultured with a low density of rat hepatocytes as monolayers in direct contact, they exerted contact-dependent control of DNA synthesis and of differentiated function of the hepatocytes. Furthermore, plasma membranes from various tumor cells including these hepatoma cells had strong modulator activity on primary cultures of normal rat hepatocytes, and their activity mimicked the reciprocal effects of cell density on DNA synthesis and induction of tyrosine aminotransferase. On the contrary, addition of plasma membranes from normal adult rat liver to sparse cultures of RY-121B or MH1C1 cells did not cause any inhibition of active DNA synthesis or enhancement of induction of tyrosine aminotransferase in these cells. These results suggest that hepatoma cells have lost cell density-dependent regulations of many cellular activities and cell growth because they have lost the ability to respond to the cell surface modulator, although they retain modulator activity on their plasma membranes.

Animals↗
PubMed1984

Hormonal control of serine dehydratase mRNA in primary cultures of adult rat hepatocytes.

The mechanism of hormonal induction of serine dehydratase [EC 4.2.1.13, SDH] was studied in primary cultures of adult rat hepatocytes by measuring the rates of syntheses of the enzyme protein and its translatable mRNA. The rate of synthesis of enzyme protein, measured as incorporation of [3H]leucine into the enzyme protein in hepatocytes, was increased 4-5 times by dexamethasone (Dex) plus glucagon. Neither hormone alone increased the rate. The increased rate induced by the two hormones was suppressed by insulin and epinephrine. The decay curves of [3H]leucine-labeled SDH showed that these hormones did not affect the rate of enzyme degradation. The level of translatable mRNA was determined by measuring cell-free synthesis of SDH in a reticulocyte lysate system. Dex plus glucagon increased the level of mRNA of SDH in hepatocytes. Insulin and epinephrine suppressed this increase without changing the rate of mRNA degradation. The level of mRNA changed in parallel with that of the rate of synthesis of the enzyme protein. These results suggest that these hormones regulate transcription of SDH, rather than its translation. After pretreatment of hepatocytes with Dex, further addition of glucagon caused more rapid induction of mRNA of SDH than addition of both hormones together. The effect of glucagon after pretreatment with Dex was inhibited by actinomycin D and alpha-amanitin, suggesting that glucagon does not affect post-transcription, but transcription per se. The requirement for both Dex and glucagon for induction of this enzyme is discussed in comparison with the requirements for either hormone alone for inductions of other gluconeogenic enzymes.

Animals↗