The ornithine-urea cycle is the major pathway for detoxication of ammonia formed in amino acid metabolism and is responsible for nitrogen balance of the body. The cycle is also involved in biosynthesis of arginine. Ornithine transcarbamylase (OTC) catalyzes the second among the five enzymatic steps of the cycle. The enzyme is present in the liver and to a lesser extent in the small intestine. In the fetal liver, the level of the enzyme increases late in gestation, coordinately with other urea cycle enzymes. OTC deficiency is known in humans and mice, as an inborn error of metabolism that results in protein intolerance and hyperammonemia. The rat and human OTC genes span a region of 70-75 kb on the X chromosome. Transient expression analysis showed that the promoter of this gene is much more active in a hepatoma cell line (HepG2) than in a non-hepatic cell line (CHO) . Two sites of the promoter region are recognized by both HNF-4 (hepa-tocyte nuclear factor-4) and COUP-TF (chicken ovalbumin upstream promoter-transcription factor) that are members of steroid receptor superfamily. In cotransfection analysis, HNF-4 activated expression from the OTC promoter, whereas COUP-TF repressed it. In transgenic mice, the promoter of the rat OTC gene directed liver-and small intestine-specific expression, but the expression of the transgene in the liver was very low. We found a hepatoma-specific enhancer region of -110 by located 11 kb upstream of the transcription start site. There are four protein binding sites in this region; two were recognized by HNF-4 and the other two by a factor (s) related to C/EBP (CCAAT/enhancer binding protein) . Both HNF-4 and C/EBP sites were required for the enhancer activity. In transgenic mice, an OTC enhancer/OTC promoter/OTC cDNA transgene was expressed more strongly in the liver than in the small intestine, thus resembling the expression of the endogenous gene. Introduction of the OTC promoter/OTC cDNA transgene into the OTC-deficient spf-ash mouse apparently corrected the OTC deficiency under normal dietary conditions. However, this transgenic spf-ash mouse was not completely healthy under stressed conditions. Introduction of the OTC enhancer-containing transgene into the OTC deficiency mouse is underway.

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We report the apparent therapeutic effects of the introduction of rat ornithine transcarb-amylase (OTC) gene composed of 1.3 kb of the 5' flanking region fused onto rat OTC cDNA into OTC-deficient spf ash mice as a fundamental approach to gene therapy. The OTC transgene caused an increase in hepatic and intestinal OTC activities of spf ash mice from 5 % to about 10 and 30 %, respectively, of controls, an increase in serum citrulline, and a decrease in urinary orotic acid, to each control level under fed conditions. The transgene was still effective under fasting conditions, but was not sufficient under nitrogen-load (oral tryptone administration at 5.6 mmol/ kg body weight) conditions, judged from urinary orotic acid excretion. Accidental tick infection caused a suppression of the OTC transgene expression which resulted in a decrease in OTC activities to those in spf-ash mice and an increase in urinary orotic acid under fasting and nitrogenload conditions. Relation between OTC activities in the liver and small intestine and urinary orotic acid under various conditions including data under conditions at tick extirmination sug-gests more important role of intestinal OTC than hepatic OTC.

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Recently, mutations in the glucokinase gene have been found in patients with maturity-onset diabetes of the young (MODY), which has raised the possibility that a decrease in glucokinase activity may impair the glucose-stimulated insulin secretion in β cells, leading to diabetes. To test the effect of a reduced glucokinase activity in β cells on β-cell function and their ability to maintain glucose homeostasis, we generated transgenic mice expressing an anti-sense glucokinase mRNA in β cells. The transgene construct contains the human insulin promoter linked to a 5' part of glucokinase cDNA of -280 by long including the start codon and is designed to produce an RNA that hybridizes specifically to glucokinase mRNA molecules, resulting in reduced levels of glucokinase mRNA in cells expressing the transgene. Fourteen mouse lineages were generated. Their fasting blood glucose levels were significantly higher than those of negative littermates. Studies are underway to determine the glucokinase activity in islets isolated from these mice. These mice are likely to provide insights into the role of glucokinase in glucose sensing in β cells under various physiological conditions. These results also demonstrate the potential of applying anti-sense RNA for cell-specific reduction in gene expression in vivo.

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Interleukin-6 (IL-6) was originally identified as a B cell differentiation factor that induces final maturation of activated B cells into antibody-producing plasma cells. It is now known that IL-6 is a multifunctional cytokine that is produced by a wide range of cells and suggested to play important roles in host defense mechanisms.
Recently, it has been suggested that deregulated expression of IL-6 is associated with the pathogenesis of plasma cell neoplasias. The growth factor for murine hybridoma/plasmacytoma was found to be identical to IL-6. Furthermore, IL-6 was shown to be a growth factor for human myeloma cells.
To clarify how IL-6 plays a role in the generation of plasma cell neoplasias, transgenic mice were produced by introducing the human IL-6 gene.
IL-6 transgenic mice of C57BL/6 origin developed massive polyclonal IgGi plasmacytosis, but not plasmacytomas. However, introduction of BALB/c genetic background into IL-6 transgenic mice was found to induce malignant transformation of plasma cells in about 1/3 of transgenic mice. These plasmacytoma cells were shown to be IgA plasmacytoma cells and contain the chromosomal translocation t (12; 15), which is common in pristane-induced plasmacytomas in BALB/c mice and is thought to induce the c-myc gene rearrangement.
These data demonstrated that overexpression of the IL-6 gene could induce plasma cell neo-plasias through a polyclonal plasmacytosis in concert with a certain BALB/c genetic background.

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Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant genetic disease, characterized by the extracellular deposition of amyloid fibrils and by prominent peripheral nerve involvement. The amyloid fibrils derived from FAP consist of variant transthyretin (TTR) with single amino acid substitutions. We demonstrated that the main cause of the Japanese FAP is the presence of a point mutation in the ttr gene. To investigate the molecular basis of FAP, we constructed transgenic mice carrying the human mutant ttr gene. In the transgenic mice, human TTR was deposited as amyloid fibrils in various tissues. Thus the transgenic mouse model of FAP can be used to elucidate the mechanism of amyloid deposition in FAP.
These transgenic mice, however, do not develop peripheral neuropathy. TTR is a serum protein which is involved in the transport of thyroid hormones and retinol. TTR is synthesized in the choroid plexus of brain and the retina of eye as well as in the liver. These observations suggest that TTR may play important roles in the nervous system. To elucidate the physiological function of TTR and the etiology of peripheral neuropathy in FAP, we have generated a unique strain of mice carrying a null mutation at the ttr locus by gene targeting.
Unexpectedly, the TTR-deficient mice are phenotypically normal. However, the levels of serum retinol, retinol-binding protein, and thyroid hormone (T_4I) are significantly depressed in the mutant animals. These observations provide evidence that TTR is a major plasma carrier of retinol and T_4I in mice. The TTR-deficient mice will be useful for investigating the transport and metabolism of retinoid and thyroid hormones.
In an attempt to generate a mouse model of FAP homozygous for the mutant ttr gene and to elucidate the function of the human variant TTR, we introduced the human mutant ttr gene into the TTR-deficient mice. In the TTR-deficient mice carrying the human mutant ttr gene, amyloid began to deposit at the age of 11 months. They appear to develop amyloid deposition earlier than wild type mice carrying the human mutant ttr gene.

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