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Contains fulltext : 172250.pdf (Publisher’s version ) (Open Access) BACKGROUND: Ectopic adrenocorticotropic hormone-producing primary liver tumors are rare, especially in children. We report the case of an adolescent boy of mixed Dutch and Moroccan descent with an adrenocorticotropic hormone-producing calcifying nested stromal-epithelial tumor with long-term follow-up. Thus far, only two such cases have been reported. CASE PRESENTATION: A 16-year-old boy of mixed Dutch and Moroccan descent presented with Cushing syndrome and a palpable abdominal mass. A calcifying nested stromal-epithelial tumor was diagnosed. Postoperatively, his plasma adrenocorticotropic hormone concentration normalized. He remains in complete remission 13 years after tumor resection. CONCLUSIONS: Calcifying nested stromal-epithelial tumor should be in the differential diagnosis of liver tumors, especially if associated with Cushing syndrome as significant morbidity and mortality may be associated. Literature on the topics involved is comprehensively reviewed.

In the first part of our review of cardiac development,1 we discussed the initial changes involved in transformation of the heart forming regions of the embryo into the great veins, the atrial and ventricular chambers, and the arterial trunks. We showed that this first part of cardiac development could be divided into phases of formation of the primary myocardial tube, looping of the tube, during which additional parts are added that give the future compartments their definitive topography, and the assembly of these components into the cardiac chambers and arterial trunks. In this second review, we discuss the processes that complete the separation of the two sides of the definitive heart, for the most part involving septation of the parts of the primary tube not themselves directly involved in formation of the chamber-specific compartments of the atriums and ventricles. In this respect, when concluding our first review, we explained how the arterial trunks were also formed by septation within the solitary outflow tract of the primary heart tube. We also showed, however, that subsequent to formation of the two arterial trunks, there was disappearance of the cushions that initially divided them. Thus, in the definitive heart, the proximal parts of the aorta and pulmonary trunk, along with the sinuses of the arterial roots and the subpulmonary infundibulum, possess their own discrete walls, separated by extra-cardiac space. Although septation by fusion of endocardial cushions will be a topic included in this second review, septation and separation of the outflow tracts is sufficiently complicated to require special treatment. Because of this, we will devote a third review specifically to the mechanisms underscoring the remodelling of the outflow tracts. In this review, therefore, we will confine our considerations to the formation of the atrial, atrioventricular, and ventricular septal structures. As will become …

Through the 20th century, knowledge of the events occurring during cardiac development was clouded by conflicting descriptions, coupled with use of notably different terminologies. Furthermore, not all accounts were based on direct study of embryonic material, instead being constructed on the basis of interpretations of previous reports, supported by inferences made from the structure of the congenitally malformed heart. Such processes, in themselves, are understandable, since it is axiomatic that proper appreciation of the events occurring during formation of the heart will aid in the analysis of the morphogenesis of cardiac malformations, this being a desirable prerequisite in the search for optimal treatment. Over the past decade, this has all changed. There has been an explosion of work, both anatomical and molecular, devoted to cardiac development. Advances in technology, coupled with the use of suitable animal models, now enable us to provide a more accurate account of the steps involved in formation and septation of the cardiac chambers. Not all of this new information is concordant with the “classical” accounts. In these reviews, therefore, we will describe, first, the steps involved in formation of the primary heart tube, and its conversion to the four cardiac chambers and the paired arterial trunks. We will then look in greater detail at the events occurring during the separation of the initial solitary heart tube into discrete systemic and pulmonary circulations. The mesodermal tissues that give rise to the heart first become evident when the embryo is undergoing the process known as gastrulation. In the human, this occurs during the third week of development, while for the mouse, at a comparable stage of development, around seven days will have elapsed from fertilisation, and the embryo will be in the presomitic stage. The embryonic plate in humans, initially possessing two layers, is ovoid, and is …

Internal ribosome entry site-mediated translation of a mammalian mRNA is regulated by amino acid availability.Fernandez J, Yaman I, Mishra R, Merrick WC, Snider MD, Lamers WH, Hatzoglou M.Departments of Nutrition and Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio, 44106, USA.The cationic amino acid transporter, Cat-1, facilitates the uptake of the essential amino acids arginine and lysine. Amino acid starvation causes accumulation and increased translation of cat-1 mRNA, resulting in a 58-fold increase in protein levels and increased arginine uptake. A bicistronic mRNA expression system was used to demonstrate the presence of an internal ribosomal entry sequence (IRES) within the 5'-untranslated region of the cat-1 mRNA. This study shows that IRES-mediated translation of the cat-1 mRNA is regulated by amino acid availability. This IRES causes an increase in translation under conditions of amino acid starvation. In contrast, cap-dependent protein synthesis is inhibited during amino acid starvation, which is well correlated with decreased phosphorylation of the cap-binding protein, eIF4E. These findings reveal a new aspect of mammalian gene expression and regulation that provides a cellular stress response; when the nutrient supply is limited, the activation of IRES-mediated translation of mammalian mRNAs results in the synthesis of proteins essential for cell survival

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.

We have studied the expression patterns of ammonia-metabolising enzymes and serum proteins in intraspleni-cally transplanted embryonic rat hepatocytes by in situ hybridisation and immunohistochemical analysis. The enzymic phenotype of individually settled hepatocytes was compared with that of hepatocytes being organised into a three-dimensional hepatic structure. Our results demon-strate that development towards the terminally differenti-ated state with zonal differences in enzyme content requires the incorporation of hepatocytes into lobular structures. Outside such an architectural context, phenotypic matura-tion becomes arrested and hepatocytes linger in the pro-todifferentiated state. These features identify the foetal period as a crucial time for normal liver development and show that the establishment of the terminally differentiated hepatocellular phenotype, beginning with the differen-tiation of hepatocytes from the embryonic foregut, is realised via a multistep process.

With respect to hepatocyte heterogeneity in ammonia and amino acid metabolism, two different patterns of sublobular gene expression are distinguished: 'gradient-type' and 'strict- or compartment-type' zonation. An example for strict-type zonation is the reciprocal distribution of carbamoylphosphate synthase and glutamine synthase in the liver lobule. The mechanisms underlying the different sublobular gene expressions are not yet settled but may involve the development of hepatic architecture, innervation, blood-borne hormonal and metabolic factors. The periportal zone is characterized by a high capacity for uptake and catabolism of amino acids (except glutamate and aspartate) as well as for urea synthesis and gluconeogenesis. On the other hand, glutamine synthesis, ornithine transamination and the uptake of vascular glutamate, aspartate, malate and alpha-ketoglutarate are restricted to a small perivenous hepatocyte population. Accordingly, in the intact liver lobule the major pathways for ammonia detoxication, urea and glutamine synthesis, are anatomically switched behind each other and represent in functional terms the sequence of the periportal low affinity system (urea synthesis) and a previous high affinity system (glutamine synthesis) for ammonia detoxication. Perivenous glutamine synthase-containing hepatocytes ('scavenger cells') act as a high affinity scavenger for the ammonia, which escapes the more upstream urea-synthesizing compartment. Periportal glutaminase acts as a pH- and hormone-modulated ammonia-amplifying system in the mitochondria of periportal hepatocytes. The activity of this amplifying system is one crucial determinant for flux through the urea cycle in view of the high Km (ammonia) of carbamoylphosphate synthase, the rate-controlling enzyme of the urea cycle. The structural and functional organization of glutamine and ammonia-metabolizing pathways in the liver lobule provides one basis for the understanding of a hepatic role in systemic acid base homeostasis. Urea synthesis is a major pathway for irreversible removal of metabolically generated bicarbonate. The lobular organization enables the adjustment of the urea cycle flux and accordingly the rate of irreversible hepatic bicarbonate elimination to the needs of the systemic acid base situation, without the threat of hyperammonemia.

Transgenic mice were used to investigate sequences within the promoter of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the rat (EC 4.1.1.32) (PEPCK) which are involved in tissue-specific and developmental regulation of gene expression. Segments of the PEPCK promoter between -2000 and -109 were linked to the structural gene for bovine growth hormone (bGH) and introduced into the germ line of mice by microinjection. Bovine growth hormone mRNA was found in tissues that express the endogenous PEPCK gene, mainly in the liver but to a lesser extent in the kidney, adipose tissue, small intestine, and mammary gland. In the liver the chimeric PEPCK/bGH(460) gene was expressed in periportal cells, which is consistent with the zonation of endogenous PEPCK. The PEPCK/bGH gene was not transcribed in the livers of fetal mice until immediately before birth; at birth the concentration of bGH mRNA increased 200-fold. Our results indicate that the region of the PEPCK promoter from -460 to +73 base pairs contains regulatory sequences required for tissue-specific and developmental regulation of PEPCK gene expression. Mice transgenic for PEPCK/bGH(460) were not hyperglycemic or hyperinsulinemic in response to elevated bGH, as were transgenic mice with the MT/bGH gene. The number of insulin receptors in skeletal muscle was no different in mice transgenic for MT/bGH when compared with mice transgenic for PEPCK/bGH(460) and control animals. However, mRNA abundance for the insulin-sensitive glucose transporter in skeletal muscle was decreased in mice transgenic for the MT/bGH gene. The differences in glucose homeostasis noted with the two types of transgenic mice may be the result of the relative site of expression, the different developmental pattern, or hormonal regulation of expression of the bGH gene.

Two methods are described for directing the expression of genes to the livers of animals using retroviral vectors containing the predominantly liver-specific promoter from the gene for phosphoenolpyruvate carboxykinase (PEPCK)-linked to the structural gene for either amino 3'-glycosyl phosphotransferase (neo) or bovine growth hormone (bGH). Replication-incompetent retrovirus was used to infect the livers of fetal rats by intraperitoneal injection of animals in utero or to infect adult rats by direct injection into the portal vein after partial hepatectomy. The proviruses were integrated into the hepatic DNA, and the chimeric genes were expressed from the PEPCK promoter for as long as 8 months after infection. The expression of the PEPCK-bGH gene was regulated by diet and hormones in a manner similar to the regulation of the endogenous PEPCK gene in the liver. The potential of this method for targeting genes to the liver is discussed.

Article de synthese sur le metabolisme de l'azote et la synthese d'uree chez les mammiferes. Degradation des proteines. Transport des acides-amines entre les organes et a travers la membrane plasmique des hepatocytes. Cycle de l'ornithine et distribution des enzymes de ce cycle dans le foie. Ontogenese de la synthese d'uree. Pathophysiologie de l'hyperammonemie

MRI is becoming important in the management of rectal cancer patients. Knowledge of relevant MR anatomy of the rectum and mesorectum is mandatory for radiologists, surgeons, and radiation oncologists involved in the multidisciplinary approach of rectal cancer. This chapter aimed at providing detailed information on the MR anatomy of clinically relevant structures.

In recent years, the rat genes encoding glutamate dehydrogenase (GLUD; Das et al. 1993), glutamine synthetase (glutamateammonia ligase, GLUL; van de Zande et al. 1990) and carbamoylphosphate synthetase 1 (CPS; Van den Hoff et al. 1995) have been isolated. These enzymes have important functions in ammonia metabolism, and each of them is encoded by a single gene. GLUD (E.C. 1.4.1.3) catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate and ammonia, using NAD+ or NADP+ as cofactor. GLUL (E.C. 6.3.1.2) catalyzes the synthesis of glutamine from glutamate, thereby hydrolyzing ATP to ADP. CPS (E.C. 6.3.4.16) is the first and rate-determining enzyme of the ornithine cycle and catalyzes the production of carbamoylphosphate from ammonia, bicarbonate and ATP. In the present communication, we mapped the position of these three genes in the rat by FISH and by somatic cell hybrids. In addition, we determined the chromosomal location of the human GLUL gene and of the mouse, Cps1 gene by FISH. For the FISH mapping of the human glutamine synthetase gene (also called glutamate-ammonia ligase, approved human gene symbol GLUL), a human cDNA (total 2738 bp in pBluescript; Van den Hoff et al. 1991) was used. As shown in Fig.1a, the gene could be unequivocally mapped to Chromosome (Chr) (HSA) 1, band q25. The three genes were mapped in the rat both with a rat-mouse somatic cell hybrid panel (Szpirer et al. 1984; Klinga Levan et al. 1993) and with FISH. For the mappings with the cell hybrid panel, cDNA probes of approximately 1000 bp from the 38 ends of the genes were used (Glul 1111 bp, van de Zande et al. 1990; Glud1 957 bp, Das et al. 1989; Cps1 883 bp, De Groot et al. 1986). The probes were labeled with radioactivity by use of a-P-CTP and the random priming method. They were subsequently hybridized to filters containing 15 mg of genomic DNA from each hybrid restricted with EcoRI or BamHI. In each case the rat hybridizing fragments could be distinguished from the mouse bands, and the genes were assigned as follows: Glud1 to rat Chr (RNO) 16, Cps1 to RNO9, and Glul to RNO13. In the rat, the FISH results corroborated and refined the findings from the somatic cell hybrid panel. Longer probes are preferred in FISH analysis, and for the regional mapping of the Glul gene with FISH, two genomic clones were used (pgGS2, 5000 bp including exons 2–6, and pgGS4, 4500 bp including exon 1; van de Zande et al. 1990). Both probes gave very clear signals at the same chromosomal location, and rat Glul could be sublocalized to RNO13q22 (Fig. 1b). The rat G-band nomenclature is according to Levan (1974); for an updated recent version of the idiogram, see RATMAP database (URL http://ratmap.gen.gu.se/ratmap/WWW Nomen/RNOIdiogrRev96new.GIF). For the mapping of rat Glud1, two clones of genomic DNA (pgGDH6d, 6700 bp containing exon 1, and pgGDH2u, 5800 bp containing exons 8–12; Das et al. 1993) were used. The results from each of the probes were the same, and Glud1 could be sublocalized to RNO16p16 (Fig. 1c). Since RNO16 is a metacentric chromosome in which both chromosome arms have very similar stainability and banding pattern except in optimal metaphases, we wanted to check our conclusion with respect to which chromosome arm carried the Glud1 gene. Sasaki and associates (1994) have published excellent pictures of FISH mapping of the Atp7b gene (Wilson Disease gene homolog), and convincingly assigned this gene to RNO16q12.3. We used the same probe (designated pWD4) in simultaneous hybridizations with the Glud1 probe and could show that the two genes were located on opposite chromosome arms (Fig. 1d), thus verifying our conclusion that Glud1 is at RNO16p16. For the FISH mapping of the rat carbamoyl-phosphate synthetase gene (Cps1), a full-length cDNA probe (cCPSf.1, 5500 bp; De Groot et al. 1986) was used. The findings corroborated the previous hybrid panel mapping, and the Cps1 gene could be sublocalized to 9q34 (Fig. 1e). Since the Cps1 gene had not been mapped in the mouse, we attempted to map it by FISH with the rat cDNA probe. This worked out well, and the mouse Cps1 gene could be assigned to mouse Chr (MMU) 1, band C3 (Fig. 1f). Comparative mapping shows that the human GLUL gene is comprised in a region spanning bands HSA 1q22–1q42 and containing 11 human genes for which there are homologous rat genes on RNO13 (Table 1). Only seven of these genes have been mapped also in the mouse, but they are all situated distally in MMU1 (spanning about 26 cM from C4bp at map position 68 to Atp1a2 at map position 94; mouse data from Mouse Genome Database, MGD). The human homolog of the rat Glud1 gene is located at HSA 10q23.3, and the mouse homolog is on MMU14. Glud1 is included in a group of three genes (also comprising Rbp3 and Sftp1) that is conserved on these chromosomes. In contrast, the human and mouse genes homologous to the Atp7b gene on the long arm of RNO16 are on HSA13 and MMU8, respectively, as is the Atp4b gene, which is also located on RNO16. Thus, it looks as if RNO16 resulted from the fusion of two chromosome segments that are on separate chromosomes in both humans and mice, and, therefore, occurred after the separation of the rat lineage from human and mouse lineages. The human homolog of Cps1 is on HSA2q33–36. In total, there are nine genes on HSA2q (spanning the segment 2q32–2q37) that have their homologs on RNO9 (Table 1). A corresponding segment in the mouse is located on MMU1 (spanning 31 cM from Slc9a2 at map position 21 to Ugt1a1 and Akp3 at position 52). We have pointed out earlier that Correspondence to: G. Levan Mammalian Genome 8, 362–364 (1997).

To provide an embryological explanation for the presence of ovarian stroma in cystadenomas of the liver and pancreas.Investigation of patients and embryos.From 1997 to 2001 in the Academic Medical Centre, Amsterdam, the Netherlands, nine women were treated for a cystadenoma with ovarian stroma, six of which were situated in the liver and three in the tail of pancreas. In one patient with a cystadenoma in the liver, malignant changes had taken place. In embryos at 5-8 weeks development, the regional differences in the morphology of the epithelium of the peritoneal cavity and the position of the gonads in relation to the embryonic liver, pancreas and spleen were examined.In the foetal period before the gonads begin to descend, they are situated directly dorsal to the liver, tail of pancreas and spleen, but are separated from these by the peritoneal cavity. The cells that cover the urogenital folds distinguish themselves from those elsewhere in the peritoneal cavity as they are bulging in shape as opposed to flattened. This activated morphology suggests that on physical contact with a neighbouring organ the cells covering the gonads may become detached and lodge in that organ.It is likely that cystadenomas of the liver and pancreas have their origin in the cells that cover the embryonic gonads. The anomalous morphology of these covering cells in fact suggests that they are relatively easily mobilized. They are probably comparable with inoculation metastasis in the coelomic cavity. Taking the chance of malignant transformation of a cystadenoma into account, the treatment of choice is radical resection of the abnormality.

Perennial ryegrass (Lolium perenne L.) is the most important temperate forage grass species. Unfortunately, the nutritional value of perennial ryegrass declines as maturity progresses, mainly because of a high concentration of poorly digestible compounds in inflorescences. Therefore, the development of forage-type ryegrass varieties with extended vegetative growth is of interest for agriculture. To delay floral transition in perennial ryegrass the Arabidopsis ATH1 gene driven by the maize ubiquitin promoter, the rice actin promoter or the rice OSH1 promoter, respectively was introduced. In ATH1-expressing plants heading was delayed, and in a number of cases the plants never flowered at all. Such non- or late-heading was accompanied by the outgrowth of normally quiescent lateral meristems into extra leaves, resulting in a leafy growth habit. When eventually heading, these plants generally produced a reduced number of inflorescences. These observations suggest that ATH1-mediated delay of heading may be useful to improve fodder quality of perennial ryegrass.

To identify the nephron segments expressing PEPCK in control and acidotic conditions, PEPCK mRNA was localized in rat kidney using the technique of reverse transcription and polymerase chain reaction (RT-PCR) in individual microdissected S1 S2, and S3 segments of the rat proximal tubule. In controls, the number of tubules expressing PEPCK mRNA was greatest in the S3 segment, moderate in the S2 segment, and least in the S1 segment of the proximal tubule. After NH4Cl feeding, strong signals for PEPCK mRNA were detected in all three proximal tubule segments. In situ hybridization demonstrated expression of PEPCK mRNA only in the medullary rays in controls. After NH4Cl, PEPCK mRNA was expressed throughout the cortex, confirming the RT-PCR results. These data demonstrate the ability of the rat kidney cortex to modulate the expression of PEPCK mRNA during metabolic acidosis by recruitment of additional cells in the proximal nephrons. Studies with cultured LLC-PK1-F+ cells indicated that increased PEPCK gene transcription at acid pH required a cis-acting element (enhancer) in the more distal 5' flanking region of the promoter.

The accumulation of urine in the renal pelvis causes depolarisation of non-specific muscular pace-maker cells. The wave of depolarisation spreads distally in the ureteric smooth muscle cells via gap junctions. This wave of excitation causes a coordinated peristaltic contraction which transports the urine bolus distally to the bladder. The EMG activity in the distal porcine ureter was studied and analyzed to establish the characteristics of ureteric excitation.Ten female New Yorkshire pigs (50 to 60 kg.) were studied in two groups under light halothane anesthesia (5% at induction and 1% for maintenance anesthesia). In both groups each pig was studied in two separate sessions at a week's interval. In group I (n = 5), bipolar needle electrodes (o: 0. 09 mm.) were implanted through a lower mid-line abdominal incision in the posterior bladder wall, the trigone and the pelvic ureter at intervals of 3 and 8 cm., respectively, from the ureteric orifice. In group II (n = 5), EMG spike burst activity was studied using a twin bipolar ring-electrode attached to an endoluminal ureteric catheter. EMG complexes were recorded using 0 to 30 Hz filters. The duration of spike burst complexes and their intervals were analyzed using a Nicollet, Pathfinder II(R) machine and a Poly(R) 4.9 digital signal processing program.Two types of spike burst activity could be distinguished between the electrodes: A, the migrating type and B, the non-migrating type. Frequency distribution analysis of spike burst duration revealed two main classes in experimental group II, a short spike burst (96%) which lasted 4.5 +/- 1.8 seconds and a longer one lasting 13.4 +/- 1.5 seconds. The conduction velocity of the migrating spike bursts (n = 177, 42% of total) between the proximal and the distal electrode had an average of 2.3 +/- 1.3 cm./sec. No relationship was found between the duration of the proximal spike burst and the conduction velocity. Data from experimental group I correlated well with data from group II.The results of our EMG study in the distal ureter reveal an approximately 9 cm. long electrically active zone in/= 90% of EMG activity recordings. The duration of activity was approximately 5 seconds. Such an excited segment of ureter led to a contraction which occluded the ureter and could prevent retrograde leakage of intraluminal contents.

The development of the atrial chambers in the human heart was investigated immunohistochemically using a set of previously described antibodies. This set included the monoclonal antibody 249-9G9, which enabled us to discriminate the endocardial cushion-derived mesenchymal tissues from those derived from extracardiac splanchnic mesoderm, and a monoclonal antibody recognizing the B isoform of creatine kinase, which allowed us to distinguish the right atrial myocardium from the left. The expression patterns obtained with these antibodies, combined with additional histological information derived from the serial sections, permitted us to describe in detail the morphogenetic events involved in the development of the primary atrial septum (septum primum) and the pulmonary vein in human embryos from Carnegie stage 14 onward. The level of expression of creatine kinase B (CK-B) was found to be consistently higher in the left atrial myocardium than in the right, with a sharp boundary between high and low expression located between the primary septum and the left venous valve indicating that the primary septum is part of the left atrial gene-expression domain. This expression pattern of CK-B is reminiscent of that of the homeobox gene Pitx2, which has recently been shown to be important for atrial septation in the mouse. This study also demonstrates a poorly appreciated role of the dorsal mesocardium in cardiac development. From the earliest stage investigated onward, the mesenchyme of the dorsal mesocardium protrudes into the dorsal wall of the primary atrial segment. This dorsal mesenchymal protrusion is continuous with a mesenchymal cap on the leading edge of the primary atrial septum. Neither the mesenchymal tissues of the dorsal protrusion nor the mesenchymal cap on the edge of the primary septum expressed the endocardial tissue antigen recognized by 249-9G9 at any of the stages investigated. The developing pulmonary vein uses the dorsal mesocardium as a conduit to reach the primary atrial segment. Initially, the pulmonary pit, which will becomes the portal of entry for the pulmonary vein, is located along the midline, flanked by two myocardial ridges. As development progresses, tissue remodeling results in the incorporation of the portal of entry of the pulmonary vein in left atrial myocardium, which is recognized because of its high level of creatine. Closure of the primary atrial foramen by the primary atrial septum occurs as a consequence of the fusion of these mesenchymal structures.

Experiments were performed to quantify the duration and frequency of ureteric peristaltic activity in the laparotomized and non-laparotomized pig in its virgin and postinstrumented states.Pigs (n = 10) in a steady state of hydration were studied under halothane anesthesia in two groups. The study was undertaken in two separate sessions at a week's interval. In group I laparotomy and vesicotomy were undertaken to obtain ELUS images. In group II, peristalsis was studied using an ELUS probe introduced through the working channel of a 22F rigid cystoscope. Peristalsis was visualized as a periodic diameter-change of ureter and recorded (for approx. 30 minutes) on videotape after an initial period of adaptation of approx. 30 minutes.The ureter acts like a pump discharging urine into the bladder through peristaltic activity. ELUS imaging of ureteric peristalsis correlated well with "eyeballing" of the passage of peristalsis through a ureter (group I). The shortest peristaltic activity in group I was 6.0+/-2.0 seconds in the non-instrumented- and 5.1+/-1.4 seconds in the instrumented ureter. In group II it was 6.8+/-1.5 seconds in the non-instrumented- and 6.4+/-1.5 seconds in the instrumented ureter. Chronic dilatation of ureter led to decrease in peristalsis frequency. Interestingly, acute dilatation caused an increase in ureteric peristalsis frequency.Ureteric peristalsis acts as a pump discharging urinary boluses (intraluminal fluid load) unidirectionally into the bladder. ELUS provides us an opportunity to quantify and study ureteric peristalsis.

Background: Connexins are the protein subunits of intercellular gap junction channels. In mammals, they are encoded by a family of at least 15 genes, which show cell-type-specific but overlapping patterns of expression. Mice lacking connexin43 (Cx43) die postnatally from obstruction of the right ventricular outflow tract of the heart. To discriminate between the unique and shared functions of Cx43, Cx40 and Cx32, we generated two ‘knock-in' mouse lines, Cx43KI32 and Cx43KI40, in which the coding region of the Cx43 gene was replaced, respectively, by the coding regions of Cx32 or Cx40. Results: Heterozygous mutants were fertile and co-expressed the wild-type and the corresponding recombinant allele in all tissues analyzed. Heterozygous Cx43KI32, but not Cx43KI40, mutant mothers were unable to nourish their pups to weaning age, possibly reflecting a defect in milk ejection. Homozygous mutant males were sterile because of extensive germ-cell deficiency. The ovaries of homozygous Cx43KI32 neonates exhibited all stages of follicular development and ovulation. The hearts of homozygous Cx43KI32 neonates showed mild morphological defects, but the cardiac morphology of homozygous Cx43KI40 neonates was relatively normal. Spontaneous ventricular arrhythmias were observed in most Cx43KI40 and some Cx43KI32 mutant mice, suggesting increased ventricular vulnerability in these mice. Conclusions: The postnatal lethality of Cx43-deficient mice was rescued in Cx43KI32 or Cx43KI40 mice, indicating that Cx43, Cx40 and Cx32 share at least some vital functions. On the other hand, Cx43KI32 and Cx43KI40 mice differed functionally and morphologically from each other and from wild-type mice. Thus, these connexins also have unique functions.

In order to reveal the biological function(s) of the gap-junction protein connexin 45 (Cx45), we generated Cx45-deficient mice with targeted replacement of the Cx45-coding region with the lacZ reporter gene. Heterozygous Cx45+/− mice showed strong expression of the reporter gene in vascular and visceral smooth muscle cells. Cx45-deficient embryos exhibited striking abnormalities in vascular development and died between embryonic day (E) 9.5 and 10.5. Differentiation and positioning of endothelial cells appeared to be normal, but subsequent development of blood vessels revealed impaired formation of vascular trees in the yolk sac, impaired allantoic mesenchymal ingrowth and capillary formation in the labyrinthine part of the placenta, and arrest of arterial growth, including a failure to develop a smooth muscle layer surrounding the major arteries of the embryo proper. As a consequence, the hearts of most Cx45-deficient embryos were dilated. The abnormal development of the vasculature in the yolk sac of Cx45−/− embryos could be caused by defective TGFβ signalling, as the amount of TGF β1 protein in the epithelial layer of the yolk sac was largely decreased in the E9.5 Cx45−/− embryo, compared with the wild-type embryo. The defective vascular development was accompanied by massive apoptosis, which began in some embryos at E8.5 and was abundant in virtually all tissues of the embryos at E9.5. We conclude that in Cx45−/− embryos, vasculogenesis was normal, but subsequent transformation into mature vessels was interrupted. Development of different types of vessels was impaired to a varying extent, which possibly reflects the complementation by other connexin(s).

Ureteric peristalsis transports a urinary bolus from the renal pelvis to the bladder. We developed an intraluminal catheter with a pressure transducer on it to study intraluminal pressure changes and a twin bipolar electrode to record the ureteric EMG and impedance (Z) changes during a peristaltic wave. Five female New Yorkshire pigs (50-60 kg) were studied under light halothane anesthesia (5% at induction/1% for maintenance). A steady state of hydration was maintained using intravenous saline infusion. EMG spike burst activity was studied at a 10-cm interval using low (0-30) Hz filters. Impedance between the same electrodes is measured simultaneously in higher frequencies (1-5 KHz) as a function of ureteric motor activity. Pressure generation in the ureteric lumen was also measured simultaneously by a transducer on the same catheter. A digital signal processing program (Poly 4.9) was used for analysis. Parenteral furosemide was used to induce diuresis. Resting ureteric impedance (Z(R)) decreases to Z(B) (Z bolus) during the passage of the urinary bolus. Passage of a contractile zone during a peristaltic wave increases impedance from Z(B) to its Z(R) level and initiates a pressure rise. Bolus length (the length Z(B)) is not constant and decreases distally. EMG corresponds well in time to impedance. Z(R) disappears after infusion of furosemide because of increased urine load and changes of intraluminal ionic environment. The contractile segment of a ureteric peristaltic wave appears to be represented by an elevated Z segment (Z(C)). Pressure rise is recorded only at the beginning of a contractile zone. A specially adapted intraluminal catheter can be used to study peristalsis in the upper urinary tract. One can study all the three components of ureteric peristalsis (excitation, contraction, and intraluminal pressure rise) using such a catheter.

The embryonic heart consists of five segments comprising the fast-conducting atrial and ventricular segments flanked by slow-conducting segments, i.e. inflow tract, atrioventricular canal and outflow tract. Although the incorporation of the flanking segments into the definitive atrial and ventricular chambers with development is generally accepted now, the contribution of the outflow tract myocardium to the definitive ventricles remained controversial mainly due to the lack of appropriate markers. For that reason we performed a detailed study of the pattern of expression of myosin light chain (MLC) 2a and 2v by in situ hybridization and immunohistochemistry during rat and mouse heart development. Expression of MLC2a mRNA displays a postero-anterior gradient in the tubular heart. In the embryonic heart it is down-regulated in the ventricular compartment and remains high in the outflow tract, atrioventricular canal, atria and inflow tract myocardium. MLC2v is strongly expressed in the ventricular myocardium and distinctly lower in the outflow tract and atrioventricular canal. The co-expression of MLC2a and MLC2v in the outflow tract and atrioventricular canal, together with the single expression in the atrial (MLC2a) and ventricular (MLC2v) myocardium, permits the delineation of their boundaries. With development, myocardial cells are observed in the lower endocardial ridges that share MLC2a and MLC2v expression with the myocardial cells of the outflow tract. In neonates, MLC2a continues to be expressed around both right and left semilunar valves, the outlet septum and the non-trabeculated right ventricular outlet. These findings demonstrate the contribution of the outflow tract to the definitive ventricles and demonstrate that the outlet septum is derived from outflow tract myocardium.

This study was undertaken to investigate the effect of acute hyperglycemia on basal and bombesin-stimulated pancreaticobiliary secretion. Seven healthy subjects participated in two experiments performed in random order during normoglycemia and hyperglycemic clamping at 15 mM. Duodenal outputs of bilirubin, trypsin, amylase, and bicarbonate were measured by aspiration with a recovery marker under basal conditions for 60 min and during continuous infusion of bombesin (1 ng/kg x min) for 60 min. Plasma cholecystokinin (CCK) and pancreatic polypeptide (PP) levels were determined at regular intervals. Compared to normoglycemia, during hyperglycemia basal outputs of bilirubin (17 +/- 3 vs. 0.9 +/- 0.4 micromol/60 min), trypsin (24 +/- 4 vs. 4 +/- 1 U/60 min), amylase (12 +/- 1 vs. 3 +/- 1 kU/60 min), and bicarbonate (2.9 +/- 0.5 vs. 1.2 +/- 0.2 mmol/60 min) were significantly p < 0.05) reduced. Bombesin significantly (p < 0.05) increased pancreaticobiliary output during both normo- and hyperglycemia. During hyperglycemia bombesin-stimulated 60-min outputs of bilirubin, trypsin, amylase, and bicarbonate were not significantly different compared to those during normoglycemia. Basal and bombesin-stimulated plasma PP concentrations were significantly (p < 0.05) reduced during hyperglycemia, but plasma CCK levels were not significantly different. It is concluded that acute hyperglycemia reduces basal but does not affect bombesin-induced pancreaticobiliary secretion.

Coexpression of alpha- and beta-myosin heavy chain (MHC) is a characteristic of the primary myocardial tube. To establish if the smooth-muscle proteins alpha-smooth-muscle actin (alpha-SMA) and calponin, and the intermediate filament protein, desmin, contribute to the specific functional properties of these early cardiomyocytes, we studied their spatiotemporal expression pattern.Sections of prenatal and neonatal Wistar rats were stained with antibodies against alpha- and beta-MHC, alpha-SMA, calponin, and desmin.The expression of alpha-SMA and calponin in embryonic cardiomyocytes increases to reach its highest level at ED14. Subsequently, these proteins gradually disappear, beginning in the interventricular septum (IVS) and followed successively by the compact myocardium of the left ventricle, the right ventricle, and the central atrium. Expression of alpha-SMA persists longer in the ventricular conduction system, making it a convenient marker for the ventricular conduction system of the fetal rat. Desmin becomes expressed one day later than alpha-SMA, but also reaches its peak at ED14, suggesting that a relatively high concentration is required to form mature sarcomeres.The results indicate that alpha-SMA, calponin, and desmin are involved in the myofibrillar development in rat heart. The presence of spatiotemporal differences in the expression of these proteins reveals regional differences in the developmental timing of cardiomyocyte maturation. The maturation process extends from the compact myocardium in the IVS to the left and right ventricular free walls, whereas the atrioventricular junction, the ventricular trabeculae, and developing ventricular conduction system show a relatively slow maturation. Smooth-muscle proteins may contribute to the slow shortening speed that is characteristic of the embryonic myocardium.

To resolve an apparent discrepancy in the developmental appearance of glutamine synthetase (GS) protein in rat [Gaasbeek Janzen et al. (1987) J. Histochem, Cytochem., 35:49-54] and mouse [Bennett et al. (1987) J. Cell Biol., 105:1073-1085] liver, we have investigated its expression during liver development in the mouse and compared it with that of carbamoylphosphate synthetase I (CPS). The expression of glutamate dehydrogenase was used as a marker to identify all hepatocytes in these strongly hematopoietic livers. GS protein accumulation starts in mouse hepatocytes at embryonic day (ED) 15. The first hepatocytes in which the enzyme accumulates were found around the major hepatic veins. CPS protein was found to accumulate in mouse hepatocytes from ED 13 onward: first, at the center of the median and lateral lobes, but temporarily not at the periphery of these lobes and not at the caudate lobe. The initial phase of accumulation of GS and CPS protein was characterized by a heterogeneity in enzyme content between hepatocytes. By ED 17, both enzymes were detectable in all hepatocytes at the center of the median and lateral lobes. This event marked the onset of the development of the complementary distribution of the enzymes typical of zonal heterogeneity in the adult mammalian liver. However, during the perinatal period, the pericentral hepatocytes temporarily accumulated CPS protein. We also observed heterochrony between species in the appearance of CPS protein in the small intestine.

The present review provides a starting point for setting up an image analysis system for quantitative densitometry and absorbance or fluorescence measurements in cell preparations, tissue sections or gels. Guidelines for instrumental settings that are essential for the valid application of image analysis in cytophotometry and cytofluorometry are described. The general principles of the working mechanism of CCD cameras in combination with general methods to improve the behaviour of the cameras are presented. Optimization of illumination of microscopical and macroscopical objects receives special attention because of its importance for valid cytometry. Sources of errors in quantitative measurements are listed and step-by-step charts for tuning the CCD camera, frame grabber and illumination for the optimal use of the systems are described. Suggestions are given for improvement of image arithmetic in difficult imaging situations, such as low fluorescence signals and high absorbance signals.

Previous studies showed that acetylcholinesterase (AChE) activity is present in the downstream (arterial) part of the embryonic chick and rat heart, but its functional significance was unclear. To establish whether other components of a cholinergic signal-transduction pathway are present in the embryonic heart, we localised the mRNAs encoding choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and the muscarinic receptor isoforms (mAChRs; m1-m5).Messenger RNA detection and localisation by in situ hybridisation and reverse transcriptase-polymerase chain reaction were employed.Expression of ChAT and AChE mRNAs was observed from 15 embryonic days onward in the neural tissue covering the dorsocranial wall of the atria. Muscarinic receptors (m1, m2, m4) were observed at the same localisation as AChE and ChAT mRNAs, both during embryogenesis and after birth. In addition, m1 and m4 mAChRs showed a low level of expression in the atrial myocardium during the fetal period. No expression of the m3 or the m5 mAChRs was observed in or near the embryonic hearts. ChAT, AChE, and mAChRs (m1, m2, m4) mRNAs always colocalised in the cardiac ganglia. However, none of these mRNAs was found at a detectable level in the outflow tract and/or the ventricular trabeculations.The AChE activity in the arterial part of the embryonic heart is probably synthesised elsewhere and subserves a function different from the hydrolysis of locally produced acetylcholine.

The valve function of the ureterovesical-junction (UVJ) is responsible for protection of the low pressure upper urinary tract from the refluxing of urine from the bladder. Controversy about the microanatomy of the human ureterovesical-junction persists.Ten (3 male and 7 female) fresh cadaveric bladders (mean age 70 years old) were studied. The bladders were fixed within 24 hours postmortem, frozen, and serially sectioned. Acetyl- and butyryl- (nonspecific) cholinesterase activity were visualised as described by Karnovsky and Roots. The three-dimensional distribution of the different muscle groups participating in the formation of the UVJ was reconstructed.Three different muscle groups were identified: (1) the detrusor muscle and the deep trigone were mainly acetylcholinesterase-positive, (2) the inner and outer layer of the ureteric muscle were butyrylcholinesterase-positive and merged into a single longitudinal layer at the level of the UVJ and form the superficial trigone distally to the ureteric orifices, and (3) the muscularis mucosae is a discontinuous butyrylcholinesterase-positive layer in the bladder that is absent from the trigone. No evidence of any muscular connection was found between the ureter and bladder musculature.The anatomy of the UVJ as observed by us suggests the following model of the ureteric peristalsis. The urine bolus arrives in the ureteric lumen at the UVJ level. The ureter can only shorten its length, slides freely in its tunnel, and discharges the urine bolus in the bladder cavity. Ureteric constriction due to the peristalsis and thickening of the contracted portion of the ureter prevents the upstream leakage. Distal spreading of the ureteric "peristalsis" in the superficial trigone increases the submucosal ureteric length and prevents reflux.

A striking example of the relationship between regulation of transcription and phenotype is the central role of the Y-chromosomal gene Sry in mammalian sex determination. Sry is the founding member of a large family of so-called Sox genes. During murine embryogenesis, the transcriptional activator Sox-4 is expressed at several sites, but in adult mice expression is restricted to immature B and T lymphocytes. Using targeted gene distruption, we have found that SOX-4(-/-) embryos succumb to circulatory failure at day E14. This was a result of impaired development of the endocardial ridges (a specific site of Sox-4 expression) into the semilunar valves and the outlet portion of the muscular ventricular septum. The observed range of septation defects is known as 'common arterial trunk' in man. We studied haemopoiesis in lethally irradiated mice reconstituted with SOX-4(-/-) fetal liver cells and found that a specific block occurred in B-cell development at the pro-B cell stage. In line with this, the frequency and proliferative capacity of IL-7-responsive B cell progenitors in fetal liver were severely decreased in vitro.

Abstract The histogenesis of the separation between atrial and ventricular myocardium at the atrioventricular junction in the developing human heart has been investigated immunohistochemically by using monoclonal antibodies specific for atrioventricular cushion tissue, mesenchymal cells, atrial and ventricular myocardium, and myocardium of the primary ring. It was found that the insulation between the muscle masses of atrium and ventricle is established by the fusion of the tissues of the atrioventricular sulcus (located at the epicardial side of the junctional myocardium) with those of the atrioventricular cushions (located at the endocardial side of the junctional myocardium). This process takes place at the ventricular margin of the myocardium of the atrioventricular canal. The separation of atrial and ventricular myocardium starts at ≈7 weeks of development in the anteromedial portion of the right atrioventricular junction and is largely completed around the 12th week of development. The only remaining myocardial continuity between atrial and ventricular myocardium is the atrioventricular axis of conduction. Our findings show that the nonmuscular part of the developing leaflets of the atrioventricular valves derives from the atrioventricular cushions and that the tissues of the atrioventricular groove do not contribute to the development of these leaflets.

Carbamoylphosphate synthetase I (CbmPS) is first expressed in rat hepatocytes shortly before birth. After birth, expression of CbmPS gradually becomes confined to the hepatocytes surrounding the portal veins. To obtain insight into the spatiotemporal regulation of its expression, the rat CbmPS gene was isolated and characterized. The gene is 110 kb in length and contains 38 exons. The basal promoter comprises the first 161 nucleotides upstream of the transcription-initiation site. Determination of the state of methylation of the 5' portion of the gene identified a CCGG sequence at -6.3 kb that is selectively demethylated in adult tissues which express CbmPS. This site remains methylated before birth, however, despite recruitment of all hepatocytes for CbmPS synthesis, indicating that its demethylation is a consequence of rather than a condition for expression of CbmPS. Transient expression assays revealed that the region surrounding the CCGG site at 6.3 kb functions as an enhancer. In FTO-2B hepatoma cells and Rat-1 fibroblasts, this enhancer is constitutively active when tested in front of the basal viral thymidine kinase promoter. When tested in front of the basal CbmPS promoter in hepatoma cells, however, the activity of this enhancer is dependent on the presence of glucocorticoids. In Rat-1 fibroblasts, the presence of both glucocorticoids and cyclic AMP is required for full activity, suggesting that the hepatocyte-specific expression of CbmPS is related to tissue-specific differences in the sensitivity to cyclic AMP. Matrix-attachment regions (MAR) are present upstream and downstream of the CbmPS gene. The downstream MAR defines the 3' boundary of the gene. The upstream MAR is located midway between the basal promoter and the enhancer, and may function as a hinge point to facilitate the positioning of the enhancer in the vicinity of the basal promoter.

Two Dutch patients with liver phosphorylase kinase (PhK) deficiency were studied for abnormalities in the PhK liver alpha (alpha L) subunit mRNA by reversed-transcribed-PCR (RT-PCR) and RNase protection assays. One patient, belonging to a large Dutch family that expresses X-linked liver PhK deficiency, had a C3614T mutation in the PhK alpha L coding sequence. The C3614T mutation leads to replacement of proline 1205 with leucine, which changes the composition of an amino acid region, containing amino acids 1195-1214 of the PhK alpha L subunit, that is highly conserved in different species. The patient showed normal levels of PhK alpha L mRNA. The second patient, from an unrelated family, was found to have a TCT (bp 419-421) deletion in the PhK alpha L coding sequence, resulting in a phenylalanine 141 deletion. The same deletion was found in the PhK alpha L coding sequence from lymphocytes of the patient's mother, together with a normal PhK alpha L coding sequence. The phenylalanine that is absent in the PhK alpha L coding sequence of the second patient is a highly conserved amino acid between species. Both the C3614T mutation and the TCT (bp 419-421) deletion were not found in a panel of 80 control X chromosomes. On the basis of these results, it is postulated that the mutations found are responsible for liver PhK deficiency in the two patients investigated.

Background Some of the problems concerning the origin of the inlet component of the definitive right ventricle were resolved in a previous study in which we showed it to be derived exclusively from the embryonic right ventricle. Questions remain, however, concerning the relative contributions of endocardial cushion tissue and myocardium to the definitive valvar apparatus guarding the right atrioventricular orifice and the origin of the valvar leaflets. Methods and Results The formation of the tricuspid valve was studied by scanning electron microscopic and immunohistochemical techniques. Concurrent with the development of the right atrioventricular connection, a myocardial ridge forms at the boundary between the atrioventricular canal and the embryonic right ventricle. It grows to become a myocardial gully that funnels atrial blood beneath the lesser curvature of the initial heart tube toward the middle of the right ventricle. Fenestrations in the floor of the gully create an additional inferior opening in the funnel, transforming its initial anterior rim into the septomarginal trabeculation. The septum formed by the fusion of the endocardial ridges of the outflow tract becomes myocardialized in its inferior portion to form, in part, the outlet septum and, in part, the supraventricular crest. The smooth atrial surface of the tricuspid valvar leaflets develops from endocardial cushion tissue. The leaflets become freely movable, however, only after delamination of the tension apparatus within the myocardium. The inferior and septal leaflets derive from the gully and the ventricular septum, their delamination being a single, continuous process. The anterosuperior leaflet forms by delamination from the developing supraventricular crest. Conclusions The leaflets of the tricuspid valve develop equally from the endocardial cushion tissues and the myocardium. The myocardium contributing to the valve comes from two sources, the tricuspid gully complex and the developing supraventricular crest. These findings facilitate the understanding of several congenital malformations.

Changes in protein and mRNAs for enzymes of glutamine metabolism were determined in rat kidney cortex at different times after induction of NH4Cl acidosis. After NH4Cl, phosphoenolpyruvate carboxykinase (PEPCK) mRNA increased 16-fold by 10 h (P < 0.05) and then returned to control levels by 30 h. In situ hybridization (ISH) showed that PEPCK mRNA was confined to medullary rays; after NH4Cl, expression of PEPCK expanded throughout the cortex, reaching a maximal intensity at 10 h. Phosphate-dependent glutaminase (PDG) and glutamate dehydrogenase (GDH) mRNAs increased 8- and 2.6-fold, respectively (both P < 0.05), by 10 h before decreasing; the increased expression was confirmed by ISH. Immunohistochemistry showed that increased PEPCK, PDG, and GDH protein occurred at variable times after the rise in mRNAs. The increase was confined to proximal tubules and was sustained, a finding noted also by Western blot analysis. In contrast, glutamine synthase protein and mRNA, confined to deep cortex and outer medullar, did not change after NH4Cl. These studies reveal striking changes in PEPCK and PDG mRNAs in rat renal cortex during acidosis. The ISH pattern suggested that increased amounts of PEPCK were synthesized in recruited cells which contained little enzyme under physiological conditions. mRNA levels for PEPCK, PDG, and GDH peaked at 10 h before returning to control levels. Despite the decrease in mRNAs, a sustained increase in proteins was noted.

Expression of alpha-fetoprotein, carbamoylphosphate synthase and albumin, that are generally accepted markers for the hepatic phenotype, require a distinct set of transcription factors. We investigated by in situ hybridization whether this set of transcription factors, LF-B1, C/EBP, DBP and LAP/LIP, is expressed coordinately in the liver during embryonic development and to what extent they are also expressed elsewhere. Our results demonstrate that mRNA levels of all transcription factors tested are significantly above background in the whole embryo and are either reduced or enhanced in expression during subsequent development. Interestingly, cardiac mesoderm, which induces prehepatic endoderm to liver formation, is temporarily permissive to its own signals, showing enhanced expression of these transcription factors and, as a result, the hepatocyte-specific genes alpha-fetoprotein and carbamoylphosphate synthase. In addition, these transcription factors and many liver-specific structural genes rise concomitantly in intestine and kidney just before birth, suggesting the expression of hepatogenic factors in these tissues as well. Despite the extrahepatic expression of these transcription factors, expression of albumin remains confined to the liver at all developmental stages.

Many enzymes are distributed heterogeneously within the liver lobule. The factors that play a determining role in the establishment and maintenance of these heterogeneous expression patterns have not yet been identified. To investigate whether the composition of the afferent hepatic blood plays a crucial role in the maintenance of the heterogeneity of gene expression of the parenchymal cells within the liver lobule, we changed the source of the afferent hepatic blood by microsurgical techniques. Three different groups of experimental animals were studied: rats with livers that are perfused with portal blood only (ligation of the hepatic artery), with caval blood only (portocaval transposition and ligation of the hepatic artery) and arterial blood only (portocaval shunt, arterialization of the distal end of the portal vein and ligation of the hepatic artery). To study differences in gene expression patterns, we chose enzymes that have a heterogeneous expression pattern within the liver lobule: the periportally located enzymes carbamoylphosphate synthase, succinate dehydrogenase, phosphoenolpyruvate carboxykinase and the pericentrally located enzymes glutamine synthase, glutamate dehydrogenase and NADPH-cytochrome P-450 reductase. To eliminate the potential interference of the long half-lives of some of these proteins on the interpretation of the results, we also studied the distribution of the mRNAs of carbamoylphosphate synthase, glutamine synthase, glutamate dehydrogenase and phosphoenolpyruvate carboxykinase. The animals were studied 2 wk after the operations. On the basis of their changes in body weight the animals were in steady state for at least a week. The patterns of gene expression of the enzymes studied did not change, regardless of the source of the altered afferent hepatic blood. The changes in gene expression that were observed in animals that did not regain their preoperative weight were shown to be caused by a limited intake of food. This study demonstrates that the physiological position of the liver within the circulation (i.e., between the gastrointestinal tract and the systemic circulation) is not as critical as is often stated and is certainly not essential for the maintenance of liver cell heterogeneity. The data suggest that the direction of the bloodstream (i.e., the existence of an upstream and a downstream compartment) is a major determinant of zonation of gene expression.

We have studied factors regulating the rate of protein degradation in cultured hepatocytes obtained from 17-day-old fetal, 7-day-old suckling, and 20-day-old weanling rats. At all three stages of development 60-70% of protein degradation was sensitive to inhibition by amino acids and 3-methyladenine, an inhibitor of macroautophagy, indicating a major role of the lysosomes in proteolysis under these conditions. A combination of dibutyryl cyclic AMP and dexamethasone strongly stimulated proteolysis in hepatocytes from weanling, but not from fetal and suckling rats. The stimulatory effect of these compounds was eliminated at high amino acid concentrations in the culture medium. Cultured perinatal hepatocytes responded to exposure to dibutyryl cyclic AMP and dexamethasone by de novo synthesis of mRNA for carbamoyl-phosphate synthase and for phosphoenolpyruvate carboxykinase, demonstrating that the developmental change in the effect of dibutyryl cyclic AMP and dexamethasone on proteolysis was due to a developmental change in the regulation of proteolysis. An analysis of the changes in intracellular amino acid concentrations in response to variations in the extracellular amino acid concentrations at all three stages of development showed that of all amino acids that could be identified, only Ile, Leu, Lys, Phe, and Tyr are implicated as possible regulators of hepatic proteolysis. Dibutyryl cyclic AMP and dexamethasone did not affect the intracellular concentrations of these amino acids, showing that hormonal regulation of proteolysis is not mediated by changes in intracellular concentrations of these amino acids. It is concluded that the lack of sensitivity of the proteolytic system to catabolic hormones in the period around birth, combined with higher circulating plasma amino acid concentrations, are mechanisms contributing to the low rate of intrahepatic proteolysis in vivo in the perinatal period and thus to the rapid growth of the liver in this period.