Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor

Amino acid sequences of four peptide fragments of human hepatocyte growth factor purified from the plasma of patients with fulminant hepatic failure were determined. Based on the amino acid sequence of one of the fragments, two oligodeoxyribonucleotide mixtures were synthesized and used to screen a human placenta cDNA library. On the screening, two overlapping cDNA clones for human hepatocyte growth factor were isolated and the nucleotide sequence of the cDNA was determined. The entire primary structure of the protein was deduced from the sequence. The protein consists of 728 amino acid residues, including a possible signal peptide at the N-terminus. The sequence revealed that the heavy and light chains which comprise the protein are encoded by the same mRNA and are produced from a common translation product by proteolytic processing.     

Changes in whole body concentrations of thyroid hormones and cortisol in metamorphosing conger eel

Summary To clarify the hormonal regulation of metamorphosis of the conger eel (Conger myriaster), changes in whole body concentrations of thyroid hormones, thyroxine (T₄) and triiodothyronine (T₃), and cortisol during metamorphosis were examined, as well as the changes in the histological activity of the thyroid gland. In larvae before metamorphosis, T₄ and T₃ levels were less than 5 and 0.15 ng·g^-1 respectively. Levels of T₄ increased to about 30 ng·g^-1 during early metamorphosis, and decreased subsequently. Levels of T₃ increased gradually in early metamorphosis, and then increased abruptly to about 2.0 ng·g^-1 in late metamorphosis. Before metamorphosis, cortisol levels of the leptocephali less than 11 cm in total length were greater than 200 ng·g^-1. Cortisol levels decreased rapidly in larger premetamorphic leptocephali, and low levels were maintained throughout the metamorphic period. Histological observation revealed an activation of the thyroid gland in early metamorphosis; thyroid follicle epithelial cells became columnar and their nuclei larger. Active uptake of colloid by these cells and intensive vascularization of the gland were also observed. By the end of metamorphosis, follicle epithelial cells became squamous, indicating a low level of glandular activity. These results suggest that thyroid hormone plays an important role in regulation of conger eel metamorphosis.Abbreviations AL anal length - TL total length - T ₃ triiodothyronine - T ₄ thyroxine     

Aminoacyl-tRNA exclusively in the 3'-isomeric form is bound to polypeptide chain elongation factor Tu

2' and 3'-O-(N-acetyl-L-phenylalanyl)adenosine (Ac-Phe-Ado) were chemically synthesized. These two isomers were clearly separated from each other by high-performance liquid chromatography (HPLC). From the two isomers of [3H]Phe-tRNA in equilibrium, Ac-[3H]Phe-Ado was prepared, without any change in the 2'/3'-isomer ratio, by acetylation of the phenylalanyl residue with acetic anhydride followed by digestion with pancreatic RNase A. By HPLC analysis of this preparation of Ac-[3H]Phe-Ado, the abundance ratio of the 2'-isomer and the 3'-isomer of [3H]Phe-tRNA was found to be 0.20:0.80. Further, [3H]Phe-tRNA was bound to Escherichia coli polypeptide chain elongation factor Tu (EF-Tu) with the ligand of GTP or guanosine 5'-[beta, gamma-imido]triphosphate (GMP-P(NH)P). The ternary complex was treated with phenol and acetic anhydride, and then digested with pancreatic RNase A. By HPLC analysis of Ac-[3H]Phe-Ado, the abundance ratio of the 2'-isomer and the 3'-isomer of [3H]Phe-tRNA was determined to be 0.07:0.93 in the complex with EF-Tu.GTP and 0.04:0.96 in the complex with EF-Tu.GMP-P(NH)P. These results clearly indicate that the 3'-isomer, rather than the 2'-isomer, of aminoacyl-tRNA is exclusively involved in the ternary complex.     

Induction of peroxisomal beta-oxidation enzymes in primary cultured rat hepatocytes by clofibric acid

Rat hepatocytes were cultured for 72 h with or without the addition of 0.5 mM clofibric acid. The activities of individual enzymes of the peroxisomal beta-oxidation pathway (acyl-CoA oxidase, enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and 3-ketoacyl-CoA thiolase) decreased in the control culture, but markedly increased synchronously in the clofibric acid-treated culture. The levels of mRNAs coding for these enzymes and the rates of synthesis of the enzymes were also elevated in the clofibric acid-treated culture, although no proportional relationship was observed between the time-dependent changes of these parameters. The increase in mRNAs was much higher than the increase in the rate of synthesis of the enzymes. The activity of catalase, its mRNA level and the rate of its synthesis were slightly affected. The effects of clofibric acid on the peroxisomal beta-oxidation enzymes and catalase in primary cultured hepatocytes were very similar to those observed in vivo. These results, therefore, suggest that primary culture of hepatocytes should provide a useful means for investigating the mechanism of induction of peroxisomal enzymes and the mechanism of action of peroxisome proliferators.     

Molecular cloning and nucleotide sequence of the cDNA for rat peroxisomal enoyl-CoA: hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme

For the studies on the mechanism of induction of peroxisomal beta-oxidation enzymes and biogenesis of the organelle, we have isolated cDNA clones for rat peroxisomal enoyl-CoA: hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme. On blotting experiments with liver RNA, the cDNAs hybridized to a 3.0-kilobase RNA which was increased 5-7-fold by the administration of di-(2-ethylhexyl)phthalate to rats. Nucleotide sequencing was carried out for four cloned cDNAs and one obtained by a primer extension method. By overlapping these sequences with each other, we identified 20 nucleotides of 5'-noncoding, 2,166 nucleotides of coding, and 910 nucleotides of 3'-noncoding regions. The deduced amino acid sequence of the enzyme is composed of 722 residues, and the composition agrees with that of the protein data. The sequence was confirmed by the amino acid compositions and sequence analyses of some of the tryptic peptides. The molecular weight of the mature enzyme is calculated to be 78,511 from the predicted amino acid sequence. The enzyme has no terminal peptide extension as a signal for translocation into peroxisomes.     

Characterization of a growth-inhibiting protein present in rat serum that exerts a differential effect onin vitro growth of nonmalignant rat liver cells when compared with Rous sarcoma virus-transformed rat liver cells

Summary We have previously reported the transformation by Rous sarcoma virus of a cloned epithelial cell line (BRL) established from Buffalo rat liver by H. Coon. The nontransformed (BRL) and transformed (RSV-BRL) cells grew at comparable rates in culture, whereas only the transformed cells were tumorigenic in vivo. We report here on the existence in rat and mouse sera of a growth inhibitor for the nontransformed BRL cells. The transformed BRL cells (RSV-BRL) were insensitive to this inhibitor. The inhibitory activity was not prominent in sera from other species of animals tested except for rabbit; this serum inhibited the growth of RSV-BRL cells more strongly than that of BRL cells. The growth inhibitor was partially purified from rat serum. It is a protein free of lipid and has a molecular weight of about 220 000. The inhibitor could be separated into three components of pI 4.6, 5.2 (major) and 5.6 by isoelectric electrophoresis. EDITOR'S STATEMENT Although compelling theoretical arguments sometimes can be made for the likely existence of growth-inhibitory substances of physical relevance in the control of cell proliferation, experiments aimed at identifying and studying such factors often are difficult to design and interpret, and little strong data exists to suggest that growth-inhibitory substances are important regulatorsin vivo. The information presented in this paper represents a start toward developing a useful system for studying growth-inhibitory factor. David W. Barnes     

Molecular cloning of cDNA for rat liver catalase

For the studies on the induction of peroxisomal enzymes by hypolipidemic agents, we have tried to isolate a cDNA clone for rat liver catalase. A recombinant clone, pMJ501, was isolated, of which cDNA insert specifically hybridized to catalase mRNA in hybridization-selected translation. On RNA blot hybridization, it hybridized to 2.4-kilobases RNA which was increased about 1.5-fold by the administration of di-(2-ethylhexyl)phthalate to the rats. The nucleotide sequence of the cDNA contains a reading frame for 109 amino acid residues which match the reported amino acid sequence of bovine liver catalase at the carboxyl end with 82% homology. It is concluded that pMJ501 contains a cDNA sequence for rat liver catalase.     

A simple method for preparing physiologically active mitochondria from plant leaves rich in oils and phenolics

Amberlite XAD-7, a nonionic polyacrylate adsorbent, was found to be a very effective protectant for isolating mitochondria from tissues rich in oils and phenolics. Physiologically active, well-coupled mitochondria were successfully prepared from young green leaf tissues of citrus, apple, pear and tobacco.Abbreviations DNP 2,4-dinitrophenol - CCCP carbonyl cyanide m-chlorophenylhydrazone - BSA bovine serum albumin - PVP polyvinylpyrrolidone     

Purification and properties of carnitine octanoyltransferase and carnitine palmitoyltransferase from rat liver

The activities of carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) in rat liver were markedly increased by administration of di(2-ethyl-hexyl)phthalate. COT and CPT were purified from the enzyme-induced rat liver. COT was a 66,000-dalton polypeptide. The molecular weight of native CPT was 280,000--320,000 daltons, and the enzyme consisted of 69,200-dalton polypeptides. CAT, COT, and CPT were immunologically different. COT exhibited activity with all of the substrates tested (acyl-CoA's and acylcarnitines of saturated fatty acids having carbon chain lengths of C2--C20), though maximum activity was observed with hexanoyl derivatives. CPT exhibited catalytic activity with medium- and long-chain acyl derivatives. 2-Bromo-palmitoyl-CoA inactivated COT but not CPT. Malonyl-CoA inhibited CPT but not COT. CPT was confined to mitochondria, whereas COT was found in peroxisomes and the soluble compartment but not in mitochondria.