Overexpression of carbamoyl-phosphate synthase 1 significantly improves ureagenesis of human liver HepaRG cells only when cultured under shaking conditions.

Hyperammonemia is an important contributing factor to hepatic encephalopathy in end-stage liver failure patients. Therefore reducing hyperammonemia is a requisite of bioartificial liver support (BAL). Ammonia elimination by human liver HepaRG cells occurs predominantly through reversible fixation into amino acids, whereas the irreversible conversion into urea is limited. Compared to human liver, the expression and activity of the three urea cycle (UC) enzymes carbamoyl-phosphate synthase1 (CPS1), ornithine transcarbamoylase (OTC) and arginase1, are low. To improve HepaRG cells as BAL biocomponent, its rate limiting factor of the UC was determined under two culture conditions: static and dynamic medium flow (DMF) achieved by shaking. HepaRG cells increasingly converted escalating arginine doses into urea, indicating that arginase activity is not limiting ureagenesis. Neither was OTC activity, as a stable HepaRG line overexpressing OTC exhibited a 90- and 15.7-fold upregulation of OTC transcript and activity levels, without improvement in ureagenesis. However, a stable HepaRG line overexpressing CPS1 showed increased mitochondrial stress and reduced hepatic differentiation without promotion of the CPS1 transcript level or ureagenesis under static-culturing conditions, yet, it exhibited a 4.3-fold increased ureagenesis under DMF. This was associated with increased CPS1 transcript and activity levels amounting to >2-fold, increased mitochondrial abundance and hepatic differentiation. Unexpectedly, the transcript levels of several other UC genes increased up to 6.8-fold. We conclude that ureagenesis can be improved in HepaRG cells by CPS1 overexpression, however, only in combination with DMF-culturing, suggesting that both the low CPS1 level and static-culturing, possibly due to insufficient mitochondria, are limiting UC.

Hepatocyte Antigen Expression in Barrett Esophagus and Associated Neoplasia.

Hepatocyte antigen or hepatocyte paraffin 1 (Hep Par 1) is widely used as a diagnostic immunomarker for hepatocellular carcinoma. It has also been identified as a rate-limiting enzyme of the urea cycle, carbamoyl phosphate synthetase 1. Hep Par 1 has been detected in non-neoplastic small intestinal epithelium, but its expression in Barrett esophagus and its related neoplasia has not been well investigated. We immunohistochemically evaluated expression of Hep Par 1 on 75 cases of Barrett esophagus (25 cases without dysplasia, 16 cases with low-grade dysplasia, 25 cases with high-grade dysplasia, and 9 cases with intramucosal adenocarcinoma) on endoscopic biopsies and endoscopic mucosal resections. All 25 cases without dysplasia (100%) showed granular cytoplasmic Hep Par 1 staining (24 diffuse and 1 focal). Of the 16 cases with low-grade dysplasia, 12 (75%) were positive (5 diffuse and 7 focal), whereas 4 (25%) were negative (P=0.018). Of the 25 cases with high-grade dysplasia, 9 (36%) showed focal positivity, whereas 16 (64%) were negative (P=0.0001). Similarly of the 9 cases of intramucosal adenocarcinomas 3 (33%) were focally positive, whereas 6 (67%) were negative (P=0.0001). Hep Par 1 is diffusely expressed in non-neoplastic Barrett esophagus while it is frequently lost in related dysplasia and adenocarcinoma, suggesting decreased level of HepPar1 may represent an early event in Barrett-related tumor genesis. This warrants additional investigation to look for the possible role of carbamoyl phosphate synthetase 1 in the pathogenesis of Barrett-related neoplasia.

Transcription start site sequence and spacing between the -10 region and the start site affect reiterative transcription-mediated regulation of gene expression in Escherichia coli.

Reiterative transcription is a reaction catalyzed by RNA polymerase, in which nucleotides are repetitively added to the 3' end of a nascent transcript due to upstream slippage of the transcript without movement of the DNA template. In Escherichia coli, the expression of several operons is regulated through mechanisms in which high intracellular levels of UTP promote reiterative transcription that adds extra U residues to the 3' end of a nascent transcript during transcription initiation. Immediately following the addition of one or more extra U residues, the nascent transcripts are released from the transcription initiation complex, thereby reducing the level of gene expression. Therefore, gene expression can be regulated by internal UTP levels, which reflect the availability of external pyrimidine sources. The magnitude of gene regulation by these mechanisms varies considerably, even when control mechanisms are analogous. These variations apparently are due to differences in promoter sequences. One of the operons regulated (in part) by UTP-sensitive reiterative transcription in E. coli is the carAB operon, which encodes the first enzyme in the pyrimidine nucleotide biosynthetic pathway. In this study, we used the carAB operon to examine the effects of nucleotide sequence at and near the transcription start site and spacing between the start site and -10 region of the promoter on reiterative transcription and gene regulation. Our results indicate that these variables are important determinants in establishing the extent of reiterative transcription, levels of productive transcription, and range of gene regulation.

Epithelial VEGF signaling is required in the mouse liver for proper sinusoid endothelial cell identity and hepatocyte zonation in vivo.

Vascular endothelial growth factor (VEGF) is crucial for vascular development in several organs. However, the specific contribution of epithelial-VEGF signaling in the liver has not been tested. We used a mouse model to specifically delete Vegf from the liver epithelial lineages during midgestational development and assessed the cell identities and architectures of epithelial and endothelial tissues. We find that without epithelial-derived VEGF, the zonal endothelial and hepatocyte cell identities are altered. We also find decreased portal vein and hepatic artery branching coincident with an increase in hepatic hypoxia postnatally. Together, these data indicate that VEGF secreted from the hepatic epithelium is required for normal differentiation of cells and establishment of three-dimensional vascular branching and zonal architectures in both epithelial and endothelial hepatic tissues.

Transcriptional regulation of N-acetylglutamate synthase.

The urea cycle converts toxic ammonia to urea within the liver of mammals. At least 6 enzymes are required for ureagenesis, which correlates with dietary protein intake. The transcription of urea cycle genes is, at least in part, regulated by glucocorticoid and glucagon hormone signaling pathways. N-acetylglutamate synthase (NAGS) produces a unique cofactor, N-acetylglutamate (NAG), that is essential for the catalytic function of the first and rate-limiting enzyme of ureagenesis, carbamyl phosphate synthetase 1 (CPS1). However, despite the important role of NAGS in ammonia removal, little is known about the mechanisms of its regulation. We identified two regions of high conservation upstream of the translation start of the NAGS gene. Reporter assays confirmed that these regions represent promoter and enhancer and that the enhancer is tissue specific. Within the promoter, we identified multiple transcription start sites that differed between liver and small intestine. Several transcription factor binding motifs were conserved within the promoter and enhancer regions while a TATA-box motif was absent. DNA-protein pull-down assays and chromatin immunoprecipitation confirmed binding of Sp1 and CREB, but not C/EBP in the promoter and HNF-1 and NF-Y, but not SMAD3 or AP-2 in the enhancer. The functional importance of these motifs was demonstrated by decreased transcription of reporter constructs following mutagenesis of each motif. The presented data strongly suggest that Sp1, CREB, HNF-1, and NF-Y, that are known to be responsive to hormones and diet, regulate NAGS transcription. This provides molecular mechanism of regulation of ureagenesis in response to hormonal and dietary changes.

Expression of glutamine synthetase and carbamoylphosphate synthetase i in a bioartificial liver: markers for the development of zonation in vitro.

BACKGROUND: Mechanisms underlying hepatic zonation are not completely elucidated. In vitro test systems may provide new insights into current hypotheses. In this study, zonally expressed proteins, i.e. glutamine synthetase (GS; pericentral) and carbamoylphosphate synthetase (CPS; periportal), were tested for their expression patterns in the bioartificial liver of the Academic Medical Center (AMC-BAL). METHODS: Distribution and organization of porcine hepatocytes inside the AMC-BAL as well as GS and CPS expression were analyzed (immuno-)histochemically in time. Ten zonally expressed proteins were analyzed by RT-PCR on cell isolate and bioreactor samples. General metabolic and hepatocyte-specific functions were determined as well. RESULTS: Viable hepatocyte layers of approximately 150 microm were observed around gas capillaries, whereas inside the matrix, single cells or small aggregates were present. GS protein and mRNA levels were upregulated in time. GS protein was preferentially expressed in hepatocytes adjacent to oxygen-supplying capillaries and in previously CPS-positive hepatocytes. No shift towards a periportal or pericentral phenotype was observed from RT-PCR analysis. CONCLUSION: Induction of GS expression inside the AMC-BAL is not dependent of (low) oxygen tensions and hepatic nuclear factor 4alpha transcript levels. GS expression might be related to (1) low substrate levels and/or autocrine soluble factors, or (2) to cytoskeleton interactions, putatively associated with the beta-catenin signaling pathway.

Changes of activity and mRNA expression of urea cycle enzymes in the liver of developing Holstein calves.

Urea is an important reutilizable nitrogen source for the ruminant and is mainly synthesized through the urea cycle in the liver. The cycle is undertaken by 5 enzymes: carbamoyl phosphate synthetase (CPS), ornithine transcarbamoylase (OTC), arginino-succinate synthetase (AS), argininosuccinate lyase (AL), and arginase. The purpose of this study was to investigate changes in the activity of the enzymes and mRNA expression, given that previous observations have indicated an increase in plasma urea concentrations with age in Holstein calves. First, plasma concentrations of metabolites and hormones were determined in calves at 1, 3, 8, 13, and 19 wk of age (n = 4, weaned at 6 wk of age). The plasma concentration of urea drastically increased after weaning (P < 0.001). The plasma concentration of glucose was lowest at 8 wk. The plasma concentration of IGF-I gradually increased with age, although those of NEFA, glucagon, and cortisol decreased (P < 0.001). Concentrations of triglyceride, alpha-amino nitrogen, growth hormone, and insulin did not change significantly with age of the calf. Next, using the liver tissues taken from calves at 2, 13, and 19 wk of age (n = 4 to 6 at each time point, weaned at 6 wk of age), we measured the activity and mRNA expression of the enzymes by biochemical methods and quantitative reverse transcription-PCR, respectively. The activities of CPS (P < 0.001), OTC (P = 0.001), and AS (P = 0.015) increased with age, whereas AL (P = 0.003) decreased. Although mRNA expression was decreased with age for AL (P = 0.002) and arginase (P = 0.007), no significant change was observed for CPS, OTC, or AS mRNA expression. We conclude that the increased urea production in the liver may be explained not only by an increase in the activities of the urea cycle enzymes, but also by increased ammonia production by rumen fermentation and gluconeogenesis from amino acids around weaning time.

Air-breathing catfish, Clarias batrachus upregulates glutamine synthetase and carbamyl phosphate synthetase III during exposure to high external ammonia.

We assessed the possible upregulation of glutamine synthetase (GS) and typical 'fish type' carbamyl phosphate synthetase III (CPS III) in detoxification of ammonia in different tissues of the walking catfish (Clarias batrachus) during exposure to 25 mM NH(4)Cl for 7 days. Exogenous ammonia led to an increase in ammonia and urea concentrations in different tissues. The results revealed the presence of relatively high levels of GS activity in the brain, liver and kidney, unexpectedly, also in the muscle, and even higher levels in the intestine and stomach. Exposure to high external ammonia (HEA) caused significant increase of activities of GS, CPS III and CPS I-like enzymes, accompanied with the upregulation of GS and CPS III enzyme proteins in different tissues. Exposure to HEA also led to a sharp rise of plasma cortisol level, suggesting being one of the primary causes of upregulation of GS and CPS III enzymes activity. Liver perfusion experiments further revealed that exposure to HEA enhances the capacity of trapping ammonia to glutamine and urea by the liver of walking catfish. These results suggest that the upregulation of GS and CPS III activity in walking catfish during exposure to HEA plays critical roles to ameliorate the toxic ammonia to glutamine, and also to urea via the induced ornithine-urea cycle possibly through the involvement of cortisol.

A case of hyperinsulinism/hyperammonaemia syndrome with reduced carbamoyl-phosphate synthetase-1 activity in liver: a pitfall in enzymatic diagnosis for hyperammonaemia.

We report a patient who was first diagnosed as having congenital carbamoyl-phosphate synthetase-1 (CPS-1) deficiency on the basis of significantly low CPS-1 activity in the liver at 1 year of age. We then started therapy against hyperammonaemia with little effect and, at the age of 15 years, we analysed the GLUD1 gene and found a previously reported gain-of-function mutation in the gene, resulting in a change of her diagnosis to hyperinsulinism/hyperammonaemia (HI/HA) syndrome. This case demonstrates that low CPS-1 activity in liver, however significant it might be, does not always come from a primary CPS-1 deficiency and that we have to take into consideration the possibility of a secondary CPS-1 deficiency, such as HI/HA syndrome.

Modulation of intestinal urea cycle by dietary spermine in suckling rat.

Argininosuccinate synthetase, an ubiquitous enzyme in mammals, catalyses the formation of argininosuccinate, the precursor of arginine. Arginine is recognised as an essential amino acid in foetuses and neonates, but also as a conditionally essential amino acid in adults. Argininosuccinate synthetase is initially expressed in enterocytes during the developmental period, it disappeared from this organ then appeared in the kidneys. Although the importance of both intestinal and renal argininosuccinate synthetases has been recognised for a long time, nutrients have not yet been identified as inducers of the gene expression. In the context of a proteomic screening of intestinal modifications induced by dietary spermine in suckling rats, we showed that argininosuccinate synthetase and carbamoyl phosphate synthase disappeared from enterocytes after this treatment. The disappearance of argininosuccinate synthetase in small intestine was confirmed by immunodetection. Expression of carbamoyl phosphate synthase and argininosuccinate synthetase coding genes decreased also after spermine administration. Expression of other urea cycle enzyme coding genes was modulated by spermine administration: argininosuccinate lyase decreased and arginase increased. Our results fit with the developmental variation of argininosuccinate synthetase and carbamoyl phosphate synthase. Modulation of the gene expression for several urea cycle enzymes suggests a coordination between all the pathway steps and switch toward polyamine (or proline and glutamate) biosynthesis from ornithine.

The role of proximal-enhancer elements in the glucocorticoid regulation of carbamoylphosphate synthetase gene transcription from the upstream response unit.

As part of the urea cycle, carbamoylphosphate synthetase (CPS) converts toxic ammonia resulting from amino-acid catabolism into urea. Liver-specific and glucocorticoid-dependent expression of the gene involves a distal enhancer, a promoter-proximal enhancer, and the minimal promoter itself. When challenged with glucocorticoids, the glucocorticoid-responsive unit (GRU) in the distal enhancer of the carbamoylphosphate-synthetase gene can only activate gene expression if, in addition to the minimal promoter, the proximal enhancer is present. Here, we identify and characterise two elements in the proximal CPS enhancer that are involved in glucocorticoid-dependent gene activation mediated by the GRU. A purine-rich stretch forming a so-called GAGA-box and a glucocorticoid-response element (GRE) are both crucial for the efficacy of the GRU and appear to constitute a promoter-proximal response unit that activates the promoter. The glucocorticoid response of the CPS gene is, therefore, dependent on the combined action of a distal and a promoter-proximal response unit.

The crab-eating frog, Rana cancrivora, up-regulates hepatic carbamoyl phosphate synthetase I activity and tissue osmolyte levels in response to increased salinity.

The crab-eating frog Rana cancrivora is one of only a handful of amphibians worldwide that tolerate saline waters. They typically inhabit brackish water of mangrove forests of Southeast Asia, but live happily in freshwater and can be acclimated to 75% seawater (25 ppt) or higher. We report here that after transfer of juvenile R. cancrivora from freshwater (1 ppt) to brackish water (10 -->20 or 20 -->25 ppt; 4-8 d) there was a significant increase in the specific activity of the key hepatic ornithine urea cycle enzyme (OUC), carbamoyl phosphate synthetase I (CPSase I). At 20 ppt, plasma, liver and muscle urea levels increased by 22-, 21-, and 11-fold, respectively. As well, muscle total amino acid levels were significantly elevated by 6-fold, with the largest changes occurring in glycine and beta-alanine levels. In liver, taurine levels were 5-fold higher in frogs acclimated to 20 ppt. There were no significant changes in urea or ammonia excretion rates to the environment. As well, the rate of urea influx (J(in) (urea)) and efflux (J(out) (urea)) across the ventral pelvic skin did not differ between frogs acclimated to 1 versus 20 ppt. Taken together, these findings suggest that acclimation to saline water involves the up-regulation of hepatic urea synthesis, which in turn contributes to the dramatic rise in tissue urea levels. The lack of change in urea excretion rates, despite the large increase in tissue-to-water gradients further indicates that mechanisms must be in place to prevent excessive loss of urea in saline waters, but these mechanisms do not include cutaneous urea uptake. Also, amino acid accumulation may contribute to an overall rise in the osmolarity of the muscle tissue, but relative to urea, the contribution is small.

Expression of carbamoylphosphate synthetase I and glutamine synthetase in hepatic organoids reconstructed by rat small hepatocytes and hepatic nonparenchymal cells.

The objective of this study was to investigate the expression of carbamoylphosphate synthetase I (CPS) and glutamine synthetase (GS) in small hepatocyte colonies and whether the heterogeneous expression of the enzymes could be induced during the maturation of small hepatocytes. Small hepatocytes isolated from an adult rat liver were cultured and proliferated to form colonies. The expression of CPS and GS was examined using immunocytochemistry and immunoblotting. In this culture more than 99% of morphologically hepatic cells were positive for CPS and all small hepatocytes were negative for GS at day 5. CPS-positive cells dramatically decreased with time in culture, whereas GS-positive ones appeared and their number increased in the colonies. Two to 3 weeks after plating, colonies with rising and piled-up cells appeared and the number of such colonies reached about 25% of all colonies at day 30. In most rising and piled-up cells in colonies both proteins were strongly expressed, whereas many small hepatocytes in monolayer colonies did not express either protein. When small hepatocytes in monolayer colonies were overlayed with Matrigel, the cells gradually piled up and both CPS and GS proteins were dramatically induced. The expression of CPS and GS in small hepatocytes may interact with the extracellular matrix because the rising and piled-up cells appear to be induced by the extracellular matrix produced by hepatic nonparenchymal cells.

Expression of carbamoyl phosphate synthetase I and ornithine transcarbamoylase genes in Chinese hamster ovary dhfr-cells decreases accumulation of ammonium ion in culture media.

Ammonium ion accumulation in mammalian cell culture media causes toxicity which inhibits cell growth and productivity. To reduce the level of the accumulated ammonium ion, carbamoyl phosphate synthetase I (CPS I) and ornithine transcarbamoylase (OTC) were used, which catalyze the first and second steps of the urea cycle in the liver. To examine the effects of overexpressed CPS I and OTC genes on the concentration of the ammonium ion in culture media, the two genes were introduced into Chinese hamster ovary (CHO) dhfr-cells. The CPS I expressing cell lines (CPS I-CHO) and both CPS I and OTC expressing cell lines (CPS I/OTC-CHO) were confirmed at the mRNA level and analyzed in terms of the cell growth and the accumulation of ammonium ion in culture media. The accumulation of ammonium ion was approximately 25-33% less in CPS I/OTC-CHO than in either CPS I-CHO or the vector-control cell lines. Interestingly however, the cell growth was approximately 15-30% faster in both CPS I-CHO and CPS I/OTC-CHO than in the control cell lines. Forced expression of urea cycle enzymes in the CHO cells revealed that both the expression of CPS I and OTC can reduce the accumulation of ammonium ion in the culture media.

Involvement of a cis-acting element in the suppression of carbamoyl phosphate synthetase I gene expression in the liver of carnitine-deficient mice.

The expression of carbamoyl phosphate synthetase I (CPS) gene is suppressed in the liver of carnitine-deficient juvenile visceral steatosis (JVS) mice at weaning and under starvation at adult age. To clarify the suppression mechanism, we produced CPSL transgenic JVS mice carrying a transgene composed of the chloramphenicol acetyltransferase (CAT) gene with the upstream region (-12 kb to +138) of the rat CPS gene and CPSE transgenic JVS mice carrying a transgene composed of the luciferase gene with minimal promoter (299 bp from -161 to +138) and enhancer (469 bp around -6.3 kb) fragments of the rat gene. The expression of the CAT gene as well as the endogenous CPS was suppressed in CPSL transgenic JVS mice, but luciferase gene expression was not suppressed in CPSE transgenic JVS mice. We isolated the 5'-upstream region of the mouse CPS gene and identified an activator protein-1 (AP-1) site downstream of the minimum enhancer region of both rat and mouse CPS genes. In conjunction with the 313-bp mouse promoter region, the 714-bp mouse enhancer fragment conferred a cell-type-dependent hormone responsiveness. In rat primary cultured hepatocytes, the addition of oleic acid suppressed reporter gene expression induced by dexamethasone in the construct containing the enhancer fragment of 714 bp with the AP-1 site, but not in its AP-1 site mutants or in 519 bp without the AP-1 site. These results strongly suggest that direct protein-protein interaction between AP-1 and glucocorticoid receptor is not involved in the suppression of the CPS gene in JVS mice and that the AP-1 element is the cis-element which is responsible for the suppression.

Effects of growth hormone on steroid-induced increase in ability of urea synthesis and urea enzyme mRNA levels.

Growth hormone (GH) reduces the catabolic side effects of steroid treatment due to its effects on tissue protein synthesis/degradation. Little attention is focused on hepatic amino acid degradation and urea synthesis. Five groups of rats were given 1) placebo, 2) prednisolone, 3) placebo, pair fed to the steroid group, 4) GH, and 5) prednisolone and GH. After 7 days, the in vivo capacity of urea N synthesis (CUNS) was determined by saturating alanine infusion, in parallel with measurements of liver mRNA levels of urea cycle enzymes, N contents of organs, N balance, and hormones. Prednisolone increased CUNS (micromol . min-1 . 100 g-1, mean +/- SE) from 9.1 +/- 1.0 (pair-fed controls) to 13.2 +/- 0.8 (P < 0.05), decreased basal blood alpha-amino N concentration from 4.2 +/- 0.5 to 3.1 +/- 0.3 mmol/l (P < 0.05), increased mRNA levels of the rate- and flux-limiting urea cycle enzymes by 20 and 65%, respectively (P < 0. 05), and decreased muscle N contents and N balance. In contrast, GH decreased CUNS from 6.1 +/- 0.9 (free-fed controls) to 4.2 +/- 0.5 (P < 0.05), decreased basal blood alpha-amino N concentration from 3. 8 +/- 0.3 to 3.2 +/- 0.2, decreased mRNA levels of the rate- and flux-limiting urea cycle enzymes to 60 and 40%, respectively (P < 0. 05), and increased organ N contents and N balance. Coadministration of GH abolished all steroid effects. We found that prednisolone increases the ability of amino N conversion into urea N and urea cycle gene expression. GH had the opposite effects and counteracted the N-wasting side effects of prednisolone.

Restoration of hepatic cytochrome c oxidase activity and expression with acetyl-L-carnitine treatment in spf mice with an ornithine transcarbamylase deficiency.

The sparse fur (spf) mutant mouse, with an X-linked ornithine transcarbamylase deficiency, is a model of congenital hyperammonemia in children. Our earlier studies indicated a deficiency of hepatic carnitine, CoA-SH, acetyl CoA, and ATP in spf mice. We have now studied the effects of a 7-day treatment with acetyl-L-carnitine (ALCAR) in the spf/Y mice on the activity and expression of the respiratory chain enzyme cytochrome c oxidase (COX; EC 1.9.3.1). We found decreased hepatic activity and expression of COX in the untreated hyperammonemic spf/Y mice, which was restored upon ALCAR treatment. Because COX is a mitochondrial membrane protein, we also carried out studies to explain the mechanism of ALCAR through its effect on membrane stability. Our results indicate a decrease of the mitochondrial membrane cholesterol/phospholipid molar ratio (CHOL/PL ratio) with the activity and expression of COX in untreated spf/Y mice. While ALCAR treatment normalized the ratios, it also restored the hepatic ATP production to normal. To study further if there was any effect of ALCAR on the mitochondrial matrix urea cycle enzymes, we measured the activity and expression of mutant ornithine transcarbamylase (OTC; EC 2.1.3.3) and normal carbamyl phosphate synthase-I (CPS-I; EC 6.3.4.16) in spf/Y mice. There was no general effect on the specific activities of the matrix enzymes upon ALCAR treatment, although their mRNA levels were enhanced. Our studies point towards the feasibility of an ALCAR treatment in conjunction with other treatment modalities, e.g. sodium benzoate and/or arginine, to improve the availability of cellular ATP and to counteract the effects of hereditary hyperammonemic syndromes in children.

Heterogeneous carbamoylphosphate synthetase I expression in testicular transplants of fetal mouse liver.

Expression of carbamoylphosphate synthetase I (CPSI; EC 6.3.4.16) was examined immunohistochemically in normal development of the mouse liver, and in testicular transplants of fetal liver fragments. CPSI started to be expressed in all hepatocytes around 15 days of gestation, and became heterogeneous (i.e. absent from pericentral hepatocytes) around 2 weeks after birth. Most hepatocytes in fetal liver fragments placed for 2 months under the testicular capsule expressed this enzyme except for the pericentral ones, most of which were positively stained with anti-glutamine synthetase (GS; EC 6.3.1.2) antiserum. This distribution resembled that in the adult liver. The steep change in CPSI immunostaining in liver lobules suggests that the microenvironment tightly connected to the central veins plays an important role in the suppression of CPSI expression in the pericentral hepatocytes. Some pericentral hepatocytes were also negative for both enzymes. Thus, control mechanisms of CPSI expression may be different from those of GS expression in pericentral hepatocytes.

Tissue-selective expression of enzymes of arginine synthesis.

Arginine is a non-essential amino acid in mammals as judged from nitrogen balance study. Citrulline is synthesized from glutamate in the small intestine, whilst kidneys and some other tissues convert citrulline to arginine. Ornithine transcarbamylase and carbamylphosphate synthetase are expressed in liver and small intestine. Tissue-selective expression depends on the regulatory elements in the promoter, or far 5', region of these genes to which tissue-selective transcription factors bind and activate transcription. Argininosuccinate synthetase and argininosuccinate lyase do not appear to have such elements, therefore their expression is more or less ubiquitous. The selective expression of pyrroline-5-carboxylate synthase activity in the intestine remains to be clarified.

Differentiation of biliary epithelial cells from the mouse hepatic endodermal cells cultured in vitro.

Differentiation of biliary epithelial cells from hepatic endodermal cells of the mouse embryo was examined with a special attention to the role of the connective tissue. When the whole liver primordium of the 9.5-day mouse embryo was cultured in vitro for 5 days, the endodermal cells differentiated into mature hepatocytes expressing carbamoylphosphate synthetase I (CPSI) and accumulating glycogen. Intrahepatic bile duct cells and connective tissue were poorly developed in this culture. However, when the hepatic endoderm was recombined with the 4-day embryonic chick lung mesenchyme and cultured in vitro, the endodermal cells differentiated into many ductal epithelial cells as well as mature hepatocytes with abundant connective tissue development. These results suggest that the ducts might be bile ducts, and that connective tissue is very important for bile duct development. In addition, this in vitro culture system might be useful for the study of mechanisms of bile duct differentiation and congenital biliary atresia.