Enrichment and characterization of clonogenic epithelial cells from adult rat liver and initiation of epithelial cell strains

Summary A highly efficient method is described for obtaining prolifertive epithelial cells from adult rat livers for the reproducible establishment of liver epithelial cell strains. When cells were isolated from livers of 10-to 15-wk-old male Fischer 344 rats by a collagenase-perfusion method, collected by centrifugation at 50×g for 5 min, and cultured in Williams' medium E containing fetal bovine serum and dexamethasone, colonies of epithelial cells different in size and morphology from hepatocytes were obtained. Sequential perfusion with collagenase and dispase yielded numerous epithelial cell colonies. When isolated cells were fractionated by differential centrifugation, the great majority of hepatocytes were sedimented at 50 ×g for 1 min, whereas many non-hepatocytic cells remiined in the supernatant and could be sedimented by a second centrifugation at 50×g for 5 min. Culture of the two fractions revealed that almost all the epithelial cell colonies were derived from cells in the non-hepatocytic cell fraction. The epithelial cells were cytochemically negative for γ-glutamyl transpeptidase activity, whereas an increase in the activity was detected in hepatocytes with duration in culture. Ultrastructural characteristics of hepatocytes were not found in the cells of newly established cell strains. These results suggest that adult rat liver epithelial cells propagable in culture were derived from a cell type other than the hepatocyte.     

Differential Localization of the γ3 and γ12 Subunits of G Proteins in the Mammalian Brain

Abstract: The localization of two forms of the γ subunit of G proteins, γ₃ and γ₁₂, was examined in the mammalian brain. Concentrations of these two γ subunits increased markedly, as did those of glial fibrillary acidic protein, during postnatal development in the rat cerebral cortex. In aged human brains, by contrast, the concentration of γ₃ tended to decrease with age, whereas that of γ₁₂ in the temporal cortex increased slightly. An immunohistochemical study of human brains revealed that γ₃ was abundant in the neuropil, whereas γ₁₂ was localized in glial cells. In the hippocampal formation of aged human brains, levels of γ₁₂-positive cells, as well as levels of glial fibrillary acidic protein- and vimentin-positive astrocytes, increased, in particular in the CA1 subfield and the prosubiculum, in which there was a decrease in the number of pyramidal cells. The appearance of γ₁₂-positive cells associated with the loss of pyramidal cells was also observed in the hippocampus of rats that had been treated with kainic acid. These results indicate that γ₁₂ is strongly expressed in reactive astrocytes. In a study of cultured neural cells, we found that γ₁₂ was predominant in glioma cells, such as C6 and GA-1 cells, in contrast with the specific localization of γ₃ in PC12 pheochromocytoma cells, which are neuron-like cells. Taken together, the results indicate that γ₃ and γ₁₂ are selectively expressed in neuronal and glial cells, respectively, and that concentrations of γ₃ and γ₁₂ in the brain are related to the numbers and/or extent of maturation of these cells.     

In Vitro Culture of a Root Parasite, Aeginetia indica L. II. The Plane of Cell Division in the Tendril

Factors affecting septation (cell division) of the tendril whichfacilitates the organic connection with the host were studiedin a root parasite Aeginetia indica L. Transverse cell division,which occurs perpendicular to the long axis of the tendril,was promoted by additions of sucrose, glucose and cytokininsto the basal medium. Longitudinal cell division of the tendril,which takes place parallel or obliquely to the long axis, wasstimulated by cytokinins, but not by sucrose. The latter typeof cell division was frequent in basal and sub-basal cells ofthe tendril but was extremely rare in apical cells. The orientationof the planes of these cell divisions was closely related tocell shape. Abnormal growth of the tendril was seen in germinatingseeds grown for six weeks or more in media containing both Miscanthus(a host) root extract and cytokinin. (Received February 23, 1984; Accepted June 12, 1984)     

Two Forms of GO Type G Proteins: Identification and Distribution in Various Rat Tissues and Cloned Cells

A G(o) type G protein distinct from the major species of G(o) was recently isolated from bovine brain and designated G(o)*. The cDNAs encoding two forms of mammalian G(o) alpha were also isolated and designated GoA alpha and GoB alpha. To recognize two forms of G(o) type G proteins, we raised antibodies in rabbits against two peptides with sequences found only in the respective proteins of murine GoA alpha (SNTYEDAAAYIQTQF) and GoB alpha (TEAVAHIQGQYWSK). Purified anti-GoA alpha antibodies reacted with the major species of G(o) alpha purified from bovine and rat brain, whereas anti-GoB alpha antibodies reacted only with rat G(o)*alpha, but not with the major species of G(o) alpha or bovine G(o)*alpha. These results indicate that the major species of G(o) alpha is encoded by GoA alpha cDNA and G(o)*alpha is encoded by GoB alpha cDNA. Using these antibodies, the distribution of GoA and GoB was studied in various rat tissues and cloned cells. Both GoA and GoB were present in many tissues, but their distribution in peripheral tissues was distinct. GoA alpha seemed to associate mainly with neural tissues. On the other hand, relatively high concentrations of GoB alpha were present in the brain, pituitary gland, adipose tissue, lung, and testis. The concentrations of both GoA and GoB in the brain increased during ontogenic development, but the increase in GoB was observed at a later age. Both GoA and GoB were found in such cloned cells as PC12, NG108-15, C6, GA-1, G8, and 3T3-L1 cells. Treatment of PC12 cells with nerve growth factor caused the extension of neuron-like processes and the increase in the level of GoA, but not in the level of GoB.     

Studies on the Induced Synthesis of Maleate cis-trans Isomerase by Malonate

Maleate cis-trans isomerase in Alcaligenes faecalis IB–14 was induced by malonate and purified about 100-fold over the crude cell-free extract by treatments of ammonium sulfate fractionation, Sephadex G–100 gel filtration, DEAE-cellulose and DEAE-Sephadex A–50 column chromatography. The preparation was shown to be monodisperse on ultracentrifugal analysis and Svedberg value was found to be 3.84 S.

The enzyme was most active at pH value around 8.3 and was stable over the range of pH 5.0 to 7.0 in the presence of dithiothreitol (DTT) for a few weeks, but in the absence of it, the enzyme activity was markedly decreased, especially in the alkaline region. The enzyme activity was inhibited by various sulfhydryl reagents and oxidizing agents, whereas it was not affected by metal chelating agents. The inhibition by Hg²⁺ and PCMB was overcome by the addition of sulfhydryl compounds such as DTT, 2-mercaptoethanol, l-cysteine and glutathione. It was observed that the enzyme did not require co-factor for its function.

Kinetic studies showed that Michaelis constant for maleate was 2.8×10^?3 m and the enzyme did not catalyze the reverse reaction.     

Gliadain, a gibberellin-inducible cysteine proteinase occurring in germinating seeds of wheat, Triticum aestivum L., specifically digests gliadin and is regulated by intrinsic cystatins

We cloned a new cysteine proteinase of wheat seed origin, which hydrolyzed the storage protein gliadin almost specifically, and was named gliadain. Gliadain mRNA was expressed 1 day after the start of seed imbibition, and showed a gradual increase thereafter. Gliadain expression was suppressed when uniconazol, a gibberellin synthesis inhibitor, was added to germinating seeds. Histochemical detection with anti-gliadain serum indicated that gliadain was present in the aleurone layer and also that its expression intensity increased in sites nearer the embryo. The enzymological characteristics of gliadain were investigated using recombinant glutathione S-transferase (GST)-progliadain fusion protein produced in Escherichia coli. The GST-progliadain almost specifically digested gliadin into low molecular mass peptides. These results indicate that gliadain is produced via gibberellin-mediated gene activation in aleurone cells and secreted into the endosperm to digest its storage proteins. Enzymologically, the GST-progliadain hydrolyzed benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin (Z-Phe-Arg-NH(2)-Mec) at K(m) = 9.5 microm, which is equivalent to the K(m) value for hydrolysis of this substrate by cathepsin L. Hydrolysis was inhibited by two wheat cystatins, WC1 and WC4, with IC(50) values of 1.7 x 10(-8) and 5.0 x 10(-8) m, respectively. These values are comparable with those found for GST-progliadain inhibition by E-64 and egg-white cystatin, and are consistent with the possibility that, in germinating wheat seeds, gliadain is under the control of intrinsic cystatins.     

Salicylic acid carboxyl methyltransferase induced in hairy root cultures of Atropa belladonna after treatment with exogeneously added salicylic acid

In Atropa belladonna hairy roots, exogeneously added salicylic acid (SA) is converted to methyl salicylate (MSA) through the reaction, which might be catalysed by S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase (SAMT). Here we cloned a cDNA for A. belladonna SAMT (AbSAMT1), which consisted of 357 aa residues. It was expressed in E. coli, and the recombinant AbSAMT1 showed SAMT activity. When A. belladonna hairy roots were exposed to a high concentration of SA, AbSAMT1 mRNA begins to be expressed 12 h after the exposure, and steady expression continued over 144 h.     

Diagnosis and monitoring of inborn errors of metabolism using urease-pretreatment of urine,isotope dilution,and gas chromatography-mass spectrometry

To diagnose inborn errors of metabolism, it would be desirable to simultaneously analyze and quantify organic acids, purines, pyrimidines, amino acids, sugars, polyols, and other compounds using a single-step fractionation; unfortunately, no such method currently exists. The present article will be concerned primarily with a practical yet comprehensive diagnostic procedure of inborn errors of metabolism (IEM). This procedure involves the use of urine or eluates from urine on filter paper, stable isotope dilution, and gas chromatography-mass spectrometry (GC-MS). This procedure not only offers reliable and quantitative evidence for diagnosing, understanding and monitoring the diseases, but also provides evidence for the diagnosis of new kinds of IEM. In this review, the differential diagnosis for hyperammonemia are described; deficiencies of ornithine carbamoyl transferase, argininosuccinate synthase (citrullinemia), argininosuccinate lyase and arginase, lysinuric protein intolerance, hyperammonemia-hyperornithinemia-homocitrullinemia syndrome, and citrullinemia type II. The diagnosis of IEM of purine and pyrimidine such as deficiencies of hypoxanthine-guanine phosphoribosyl transferase, adenine phosphoribosyl transferase, dihydropyrimidine dehydrogenase, dihydropyrimidinase and beta-ureidopropionase are described. During the pilot study for newborn screening, we found neonates with diseases at a rate of 1 per 1,400 including propionic acidemia, methylmalonic acidemia, orotic aciduria, beta-ureidopropionase deficiency, lactic aciduria and neuroblastoma. A rapid and reliable prenatal diagnosis for propionic acidemia is also described.     

Distribution and variable expression of secretory pathway protein reticulocalbin in normal human organs and non-neoplastic pathological conditions.

Reticulocalbin (RCN) is one member of the Ca(2+)-binding proteins in the secretory pathway and is localized in the endoplasmic reticulum. RCN may play a role in the normal behavior and life of cells, although its detailed role remains unknown. Overexpression of RCN may also play a role in tumorigenesis, tumor invasion, and drug resistance. The new antibody for human RCN is used in the distribution of RCN in normal human organs of fetuses and adults with or without inflammation. Immunohistochemical examination demonstrated a broad distribution of RCN in various organs of fetuses and adults, predominantly in the endocrine and exocrine organs. However, RCN expression was heterogeneous in each constituent cell of some organs. Among non-epithelial organs, vascular endothelial cells, testicular germ cells, neurons, and follicular dendritic cells showed strong staining. Plasma cells were the only RCN-positive cells among hematopoietic and lymphoid cells. In inflammatory conditions, RCN expression was enhanced in both epithelial and non-epithelial cells. Heterogeneous expression of RCN indicates that the amount of RCN needed for cell behavior and life may be variable, depending on each cell type and, therefore, RCN may be helpful in establishing the cell origin of neoplasms in some organs. However, further study is needed to establish the significance of RCN in tumorigenesis and in some peculiar features of neoplasms.     

Non-acidic compounds induce the intense sweet taste of neoculin, a taste-modifying protein

Neoculin, a sweet protein found in the fruit of Curculigo latifolia, has the ability to change sourness into sweetness. Neoculin turns drinking water sweet, indicating that non-acidic compounds may induce the sweetness. We report that ammonium chloride and certain amino acids elicit the intense sweetness of neoculin. Neoculin can thus sweeten amino acid-enriched foods.