The recessive phenotype displayed by a dominant negative microphthalmia-associated transcription factor mutant is a result of impaired nucleation potential.

In the DNA binding domain of microphthalmia-associated transcription factor (MITF), four mutations are reported: mi, Mi wh, mi ew, and mi or. MITFs encoded by the mi, Mi wh, mi ew, and Mi or mutant alleles (mi-MITF, Mi wh-MITF, Mi ew-MITF, and Mi or-MITF, respectively) interfered with the DNA binding of wild-type MITF, TFE3, and another basic helix-loop-helix leucine zipper protein in vitro. Polyclonal antibody against MITF was produced and used for investigating the subcellular localization of mutant MITFs. Immunocytochemistry and immunoblotting revealed that more than 99% of wild-type MITF and Mi wh-MITF located in nuclei of transfected NIH 3T3 and 293T cells. In contrast, mi-MITF predominantly located in the cytoplasm of cells transfected with the corresponding plasmid. When the immunoglobulin G (IgG)-conjugated peptides representing a part of the DNA binding domain containing mi and Mi wh mutations were microinjected into the cytoplasm of NRK49F cells, wild-type peptide and Mi wh-type peptide-IgG conjugate localized in nuclei but mi-type peptide-IgG conjugate was detectable only in the cytoplasm. It was also demonstrated that the nuclear translocation potential of Mi or-MITF was normal but that Mi ew-MITF was impaired as well as mi-MITF. In cotransfection assay, a strong dominant negative effect of Mi wh-MITF against wild-type MITF-dependent transactivation system on tyrosinase promoter was observed, but mi-MITF had a small effect. However, by the conjugation of simian virus 40 large-T-antigen-derived nuclear localization signal to mi-MITF, the dominant negative effect was enhanced. Furthermore, we demonstrated that the interaction between wild-type MITF and mi-MITF occurred in the cytoplasm and that mi-MITF had an inhibitory effect on nuclear localization potential of wild-type MITF.

The effects of fluorouracil on thymidylate synthase and thymidine kinase in regenerating rat liver after partial hepatectomy.

The injection of 5-fluorouracil to partial hepatectomized rat prevented the rise of activities of thymidylate synthase and thymidine kinase as well as DNA synthesis in 24-h regenerating liver in a dose-dependent manner. The extent of the decrease of thymidine kinase activity was more than that of thymidylate synthase level. The immunoblotting assay showed the existence of fluorodeoxyuridine monophosphate bound thymidylate synthase in 5-fluorouracil injected rat liver. These results indicate that the reduction of DNA synthesis in regenerating liver by 5-fluorouracil result from the inhibitions of the rise of activities of both thymidylate synthase and thymidine kinase, while the mechanisms in their inhibitions may be different.

Effect of folic acid on thymidylate synthase and thymidine kinase in regenerating rat liver after partial hepatectomy.

The effects of folic acid on liver regeneration after partial hepatectomy were investigated. The injection of folic acid inhibited the increases in the activities of thymidylate synthase and thymidine kinase in regenerating rat liver at 24 h after partial hepatectomy, with a concomitant reduction in DNA content. Northern blot analysis showed that this inhibition was due to the delay of the elevation of the mRNA levels of thymidylate synthase and thymidine kinase after partial hepatectomy. At 48 and 72 h, after partial hepatectomy, the thymidylate synthase activities in the folic acid injected rats increased to about 1.9- and 1.7-fold the corresponding control level, respectively, while thymidine kinase activities were similar to the control. Immunoblotting assay indicated that the increases in the thymidylate synthase activity at 48 and 72 h after partial hepatectomy were caused by a three fold increase in its protein level. Folic acid suppressed chymotryptic hydrolysis of thymidylate synthase. These suggest that folic acid increases the protein level of thymidylate synthase, at least in part, through protection against proteolysis.

Retinoic acid repressed the expression of c-fos and c-jun and induced apoptosis in regenerating rat liver after partial hepatectomy.

Retinoic acid (RA), which was injected within 4 h after partial hepatectomy (PH), inhibited DNA synthesis in regenerating liver. The inhibition was accompanied by apoptosis, evidenced by in situ end labeling and gel electrophoresis of DNA fragmentation. Characteristic DNA fragmentation was obvious at 4 h and reached a maximum at 8 h after injection. Northern blot analysis revealed that RA repressed the expression of c-fos and c-jun at 15 and 30 min with the up-regulation of retinoic acid receptor gamma (RARgamma) and RARbeta at 2 h after PH. The transglutaminase II mRNA level and activity were increased by RA injection at 4 h and 8 h after PH, respectively. The mRNA levels of thymidylate synthase and thymidine kinase, which are rate determining enzymes of DNA synthesis, decreased in RA injected rats. No change was seen in the expression of p53 and p21WAF1/CIP1 which have been suggested to participate in the apoptosis process. These results suggest that RA exerts the antiproliferative activity only on the early stage of liver regeneration accompanied by the repression of c-fos and c-jun expression and induction of apoptosis.

Quercetin inhibited DNA synthesis and induced apoptosis associated with increase in c-fos mRNA level and the upregulation of p21WAF1CIP1 mRNA and protein expression during liver regeneration after partial hepatectomy.

Quercetin, a widely distributed bioflavonoid, inhibited DNA synthesis in regenerating liver after partial hepatectomy. This inhibition was accompanied by apoptosis, evidenced by in situ end-labeling and gel electrophoresis of DNA fragmentation. Characteristic DNA fragmentation was detected as early as 2 h after injection. Northern blot analysis revealed that quercetin induced the increases in c-fos and p21WAF1CIP1 mRNA levels within 2 h. The expression of p21 protein was also enhanced, while p53 mRNA and protein levels were not affected by quercetin. These results suggest that quercetin-induced apoptosis is associated with the increase in c-fos mRNA level and the upregulation of p21 mRNA and protein expression, probably in a p53-independent pathway.

Effect of colchicine and vincristine on DNA synthesis in regenerating rat liver.

The increases in the activities of hepatic thymidylate synthetase and thymidine kinase were significantly suppressed at 24 h after 70% partial hepatectomy in rats which had been administered a microtubule disrupter, colchicine or vincristine. The decrease of these enzymic activities was accompanied by a reduction of DNA content in 24 h regenerating liver. The immunoblotting assay showed that the depression of the thymidylate synthetase activity by the injection of colchicine or vincristine was due to the decrease of the enzyme protein. These results indicate that colchicine and vincristine inhibit the DNA synthesis during liver regeneration by inhibiting the induction of the key enzyme in DNA synthesis.

Prolonged Jun N-terminal kinase (JNK) activation and the upregulation of p53 and p21(WAF1/CIP1) preceded apoptosis in hepatocytes after partial hepatectomy and cisplatin.

Cisplatin induced apoptosis in regenerating liver after partial hepatectomy (PH). Apoptosis was determined by in situ end-labeling and gel electrophoresis of DNA fragmentation. Characteristic DNA fragmentation was obvious at 4 h and peaked at 8 h after PH. The activity of Jun N-terminal kinase (JNK) transiently increased at 1 h after PH. However, in cisplatin-injected rats, the JNK activity increased at 30 min and the increased level was maintained up to 4 h after PH. The in vivo activation of JNK was confirmed by the increased level of the phosphorylated c-Jun protein. Western blot analysis showed that the phosphorylated c-Jun level increased at 1 h and reached more than 30-fold the control level at 2 h after PH with cisplatin. The c-jun mRNA levels also markedly increased at 1 h after PH with cisplatin. The protein level of p53 increased after 1 h on cisplatin injection, but no significant change in the mRNA level was observed. The rise in the p53 protein level was followed by the upregulation of p21(WAF1/CIP1) mRNA and protein levels. These results suggested that the enhanced and sustained JNK activation and the upregulation of p53 and p21(WAF1/CIP1) were involved in hepatocyte apoptosis induced by PH with cisplatin.

The sex difference in the regulation of liver regeneration after partial hepatectomy in the rat.

The increases in the activity of hepatic thymidylate synthetase and thymidine kinase, which catalyzes the formation of thymidylate via the de novo and salvage pathways, respectively, were significantly suppressed 24 h after 70% partial hepatectomy in female rats administered either alpha- or beta-adrenoreceptor antagonists. The injection of beta-antagonist to male or ovariectomized female rats had no effect on the activities of these enzymes. Only alpha-adrenoceptor antagonist depressed these enzymatic activities of 24-h-regenerating liver in male and ovariectomized female rats. The decrease of the activities of thymidylate synthetase and thymidine kinase was accompanied by a concomitant reduction of DNA content in 24-h-regenerating liver. It is concluded that catecholamine regulates the female rat liver regeneration through both alpha- and beta-adrenergic pathways by the inductions of thymidylate synthase and thymidine kinase, while in adult male and ovariectomized female rats, only the alpha-mediated pathway is involved.

The role of zinc with special reference to the essential thiol groups in delta-aminolevulinic acid dehydratase of bovine liver.

delta-Aminolevulinic acid dehydratase (5-aminolevulinic acid hydro-lyase (adding 5-aminolevulinic acid and cyclizing), EC 4.2.1.24 purified from bovine liver in the presence of both SH-reducing reagent and zinc during the purification contained one zinc atom and eight SH groups/subunit. This preparation showed the full enzymatic activity even in the absence of thiol activator. It was found that two cysteine residues, one zinc atom and two histidine residues were involved in the active site. The enzyme was fullly active as long as two SH groups in the active site remained in the reduced form even in the absence of zinc. However, the enzymatic activity was completely lost, with a concomitant loss of bound zinc, upon oxidation of the SH groups to a disulfide bond, modification of SH groups with chemical reagents, or mercaptide formation by heavy metals. Thus, it is apparent that the activity depends on the essential SH groups. The zinc is not absolutely essential for the activity but may be required to prevent the essential SH groups from autooxidation by coordination. Binding experiments indicated that there was one binding site of zinc/subunit. Photooxidation of histidine residues diminished both enzymatic activity and bound zinc, suggesting that the histidine residues not only constituted the active site but also served as a possible ligand to zinc.

Purification and characterization of thymidine kinase from regenerating rat liver.

Thymidine kinase (EC 2.7.1.21) from regenerating rat liver has been purified 70,000-fold to apparent homogeneity by affinity chromatography. Molecular weight of the native enzyme was found to be about 54,000, as determined by gel filtration. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate yielded a single band with a molecular weight of 26,000, suggesting that thymidine kinase is a dimer of very similar or identical subunits. The Michaelis constant for thymidine is 2.2 microM. ATP acts as a sigmoidal substrate with a 'Km' of 0.2 mM. Reaction kinetics and product inhibition studies reveal the enzymatic mechanism to be sequential.

Control of thymidine kinase during liver regeneration after partial hepatectomy.

To elucidate the molecular basis for the periodic change of thymidine kinase (TK) activity, the expressions of TK protein and TK mRNA were examined during liver regeneration. TK protein level, quantified by immunoblotting assay using the polyclonal antiserum against rat TK polypeptide produced in Escherichia coli, increased 13-fold compared with the normal at 24 h after partial hepatectomy. This was closely correlated with a 11-fold increase in TK activity. Northern blot analysis showed that the partial hepatectomy caused 12- and 8-fold increase in 2.6 kb and 1.1 kb TK mRNA at 24 h after surgery, respectively. During next 12 h the levels of both TK mRNA species reduced to 5-fold of the normal base level. This reduction was coupled with a similar decrease in the activity as well as in the amount of TK protein. The TK mRNA levels were strictly proportional to the levels of TK activity and TK protein at 48 h and 72 h after partial hepatectomy. These results demonstrate that the change in TK activity is controlled at the mRNA level during liver regeneration. The injection of alpha-adrenoceptor antagonist, phenoxybenzamine, calcium channel blocker, nifedipine or calmodulin inhibitor, trifluoperazine was also found to inhibit TK activity by the repression of its mRNA level.

Purification and characterization of thymidylate synthetase from rat regenerating liver.

Thymidylate synthetase (EC 2.1.1.45) from rat regenerating liver has been purified over 5000-fold to apparent homogeneity by a procedure involving two affinity methods. Molecular weight of the native enzyme was found to be about 68,000, as determined by gel filtration. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate yielded a single band of molecular weight of 35,000, suggesting that thymidylate synthetase is a dimer of very similar or identical subunits. The Michaelis constants for deoxyuridylate (dUMP) and (+/-)L-5,10-methylenetetrahydrofolate are 6.8 microM and 65 microM, respectively. Reaction kinetics and product inhibition studies reveal the enzymatic mechanism to be ordered sequential. 5-Fluoro-dUMP, halogenated analog of the nucleotide substrate is a competitive inhibitor of the enzyme, with an apparent Ki value of 5 nM. Amethopterin, analog of the cofactor is also a competitive inhibitor with an apparent Ki value of 23 microM.

Ethanol represses thymidylate synthase and thymidine kinase at mRNA level in regenerating rat liver after partial hepatectomy.

The effects of ethanol on liver regeneration after partial hepatectomy were investigated. The injection of ethanol inhibited the increases in the activities of thymidylate synthase and thymidine kinase in regenerating rat liver at 24 h after partial hepatectomy in a dose-dependent manner, with a concomitant reduction in DNA content. Northern blot analysis showed that the inhibition of thymidylate synthase and thymidine kinase activities was caused by comparable decreases in their mRNA levels. The immunoblotting assay confirmed the protein levels of thymidylate synthase and thymidine kinase as proportional to the activity and mRNA levels. These findings suggest that ethanol inhibits DNA synthesis by the repression of mRNA levels of dTMP-synthesizing enzymes during liver regeneration.

A new immunoblotting assay for thymidylate synthetase and its application to the regulation of enzyme activity in regenerating rat liver.

A highly sensitive and specific immunoblot assay has been developed to quantitate the content of rat liver thymidylate synthetase (EC 2.1.1.45). Applying the method, it is demonstrated that the increase of the activity of thymidylate synthetase in liver regeneration after partial hepatectomy is due to the de novo synthesis of the enzyme protein. Administration of cycloheximide, phenoxybenzamine, phorbol 12-myristate 13-acetate, nifedipine, dexamethasone or indomethacin to partially hepatectomized rats prevented the synthesis of thymidylate synthetase in regenerating liver. Thyroparathyroidectomy also inhibited the increase of the enzyme in liver regeneration. These observations are discussed in relation to the signal transduction concerning the alpha 1-receptor, which was shown to regulate liver regeneration in our previous papers.

Cellular localization of thrombopoietin mRNA in the liver by in situ hybridization.

The expression of thrombopoietin (TPO) mRNA is observed in several tissues, including liver, kidney, brain, skeletal muscle, intestine, spleen, and bone marrow. Among these organs, the highest expression of TPO mRNA is detected in the liver. We identified cells producing TPO by means of in situ hybridization of adult rat liver using digoxigenin-11-UTP-labeled cRNA probes. We found that the cells expressing TPO mRNA also expressed serum albumin mRNA. TPO mRNA was detected in parenchymal cells (hepatocytes) but not in non-parenchymal cells (including endothelial cells, epithelial cells, and so forth). To determine the location of TPO expression in embryogenesis, sections of fetal mice were further analyzed by in situ hybridization. TPO mRNA was detected only in hepatocytes of fetal liver, which was also the major site of hematopoiesis. The expression of TPO mRNA in fetal liver was observed from 12.5 days postcoitus. Northern blot analysis showed that mouse liver transcribed the same size of TPO mRNA in the fetus and in the adult. These results clearly demonstrate that hepatocytes are the primary site of TPO production in the liver from fetus to adult.

Impaired expression of noncollagenous bone matrix protein mRNAs during fracture healing in ascorbic acid-deficient rats.

In scorbutic patients, fractures are slow to heal because of impaired collagen synthesis. To investigate the influence of impaired collagen synthesis on the differentiation and proliferation of osteogenic and chondrogenic cells, we examined the expression of genes encoding bone matrix proteins, including osteonectin (ON), osteopontin (OPN), osteocalcin (OC), and matrix Gla protein (MGP), as differentiation markers for osteogenic and chondrogenic cells during fracture healing in Osteogenic Disorder Shionogi (ODS) rats, which have a hereditary defect in the ability to synthesize ascorbic acid (Asc). In ODS rats without Asc supplementation, intramembranous ossification was completely inhibited. Although a few fibroblast-like cells expressing ON mRNA were observed, no OPN mRNA-expressing cells were detected. During endochondral ossification, a small amount of metachromatic staining cartilage appeared at the fracture site, but there was no provisional calcification zone in the cartilage. Chondrocytes expressed ON and MGP mRNAs, but not OPN mRNA. When Asc was given to these rats, callus formation was soon detected around the fracture site, while OPN mRNA was expressed by differentiated osteoblasts and hypertrophic chondrocytes. Our data indicate that impaired collagen synthesis due to Asc deficiency inhibited the increase of ON and MGP mRNA-expressing cells as well as the appearance of OPN mRNA-expressing cells. Since OPN is considered to play an important role in normal and pathological mineralization, lack of OPN mRNA expression accompanying impaired collagen synthesis may have a role in defective mineralization and delayed fracture healing in scurvy.

Molecular cloning and expression of a rat cDNA encoding 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase.

The cDNA of a 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (AICARFT/IMPCHase) was isolated from rat liver RNA by reverse transcription and the polymerase chain reaction (PCR). The rat AICARFT/IMPCHase cDNA included 1928 bp containing a coding region of 1779 bp for a 592-amino acid polypeptide (Mr = 64 200). Rat and human AICARFT/IMPCHase cDNAs show 84 and 91% homology at the nucleotide and amino acid sequence level, respectively. The protein produced by the rat cDNA using pET-expression system catalysed the penultimate and final steps of de novo purine biosynthesis. Northern analysis identified a 2.8-kb AICARFT/IMPCHase mRNA and the level of the AICARFT/IMPCHase transcripts increased markedly at 24 h after partial (70%) hepatectomy.

Inhibition of thymidylate synthase and thymidine kinase by okadaic acid in regenerating rat liver after partial hepatectomy.

The effects of okadaic acid, a potent and specific inhibitor of protein phosphatases 1 and 2A, on liver regeneration after partial (70%) hepatectomy were investigated. The injection of okadaic acid (25 micrograms/kg body weight) inhibited the increases in the activities of thymidylate synthase and thymidine kinase in regenerating rat liver at 24 hr after partial hepatectomy, with a concomitant reduction in DNA content. Northern blot analysis showed that the suppression of thymidylate synthase and thymidine kinase activities was caused by comparable decreases in their mRNA levels. The protein levels of thymidylate synthase and thymidine kinase were confirmed by immunoblotting assay to be proportional to the activity and mRNA levels. These findings suggest that okadaic acid-sensitive protein phosphatases are involved in transcriptional control of the dTMP-synthesizing enzymes during liver regeneration.

Effect of calcium channel blockers and trifluoperazine on rat liver regeneration.

The activity of hepatic thymidylate synthetase and thymidine kinase at 24 h after 70% partial hepatectomy of rats was suppressed significantly compared with that in the control group by the administration of calcium channel blockers (verapamil, diltiazem and nifedipine) 8 h after partial hepatectomy. The decrease of thymidylate synthetase and thymidine kinase activities was accompanied by a reduction of DNA content in 24 h regenerating liver. Trifluoperazine showed an effect similar to that of the calcium channel blockers on DNA synthesis during liver regeneration. These results suggest that calcium entry into the hepatic cell is an essential event in liver regeneration.

Alpha-adrenergic regulation of the activity of thymidylate synthetase and thymidine kinase during liver regeneration after partial hepatectomy.

The increases in activity of hepatic thymidylate synthetase and of thymidine kinase, which catalyze the formation of thymidylate via the de novo and salvage pathways, respectively, were significantly suppressed during liver regeneration in rats which had been given alpha-adrenoceptor antagonists (phenoxybenzamine and phentolamine) or adrenergic neuron blockers (guanethidine and reserpine). These suppressions were not observed with a beta-adrenoceptor antagonist (propranolol), or an anticholinergic agent (atropine methyl nitrate). The rise in the activity of the thymidylate-synthesizing enzymes was closely correlated with the increase in the DNA content of the liver. It is concluded that catecholamine regulates the increase in the activity of thymidylate synthetase and thymidine kinase, which are key enzymes in DNA synthesis in regenerating liver. It is also suggested that sympathetic nerves play an important role in liver regeneration.