P. A. J. De Boer

Arginine-metabolizing enzymes in the developing rat small intestine.

Before weaning, arginine biosynthesis from citrulline most likely takes place in the small intestine rather than in the kidney. We studied the expression of ornithine cycle enzymes in the rat small intestine during perinatal development. The spatiotemporal patterns of expression of ornithine aminotransferase, carbamoylphosphate synthetase, ornithine transcarbamoylase, argininosuccinate synthetase, argininosuccinate lyase, and arginase mRNAs were studied by Northern blot analysis and in situ hybridization. In addition, the expression of carbamoylphosphate synthetase and argininosuccinate synthetase protein was studied by immunohistochemistry. Before birth, the developmentally more mature proximal loops of the intestine expressed the mRNAs at higher concentrations than the more distal loops. After birth, this difference was no longer obvious. The mRNAs of argininosuccinate synthetase and argininosuccinate lyase, the enzymes that metabolize citrulline to arginine, were detectable only in the upper part of the villi, whereas the other mRNAs were concentrated in the crypts. The distribution of argininosuccinate synthetase protein corresponded with that of the mRNA, whereas carbamoylphosphate synthetase protein was present in all enterocytes of the crypts and villi. Hepatic arginase mRNA could not be detected in the enterocytes. The spatial distribution of the respective mRNAs and proteins along the villus axis of the suckling small intestine indicates that the basal enterocytes synthesize citrulline, whereas the enterocytes in the upper half of the villus synthesize arginine.

Expression patterns of mRNAs for ammonia-metabolizing enzymes in the developing rat: the ontogenesis of hepatocyte heterogeneity

SummaryThe expression patterns of the mRNAs for the ammonia-metabolizing enzymes carbamoylphosphate synthetase (CPS), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were studied in developing pre- and neonatal rat liver byin situ hybridization.In the period of 11 to 14 embryonic days (ED) the concentrations of GS and GDH mRNA increases rapidly in the liver, whereas a substantial rise of CPS mRNA in the liver does not occur until ED 18. Hepatocyte heterogeneity related to the vascular architecture can first be observed at ED 18 for GS mRNA, at ED 20 for GDH mRNA and three days after birth for CPS mRNA. The adult phenotype is gradually established during the second neonatal week, i.e. GS mRNA becomes confined to a pericentral compartment of one to two hepatocytes thickness, CPS mRNA to a large periportal compartment being no longer expressed in the pericentral compartment and GDH mRNA is expressed over the entire porto-central distance, decreasing in concentration going from central to portal. Comparison of the observed mRNA distribution patterns in the perinatal liver, with published data on the distribution of the respective proteins, points to the occurrence of posttranslational, in addition to pretranslational control mechanisms in the period of ontogenesis of hepatocyte heterogeneity.Interestingly, during development all three mRNAS are expressed outside the liver to a considerable extent and in a highly specific way, indicating that several organs are involved in the developmentally regulated expression of the mRNAs for the ammonia-metabolizing enzymes, that were hitherto not recognized as such.

Expression patterns of mRNAs for α-fetoprotein and albumin in the developing rat: the ontogenesis of hepatocyte heterogeneity

SummaryIn developing and normal adult rat liver the expression patterns of the mRNAs for α-fetoprotein (AFP) and albumin (ALB) were analysed byin situ hybridization using specific35S-labelled complementary DNA probes. In the developing liver AFP and ALB mRNA are found from embryonic day (ED) 11 and 12, respectively, onward. At ED 20 the first signs of a zonal distribution of these mRNAs across the liver lobule can be observed, AFP mRNA concentration being higher in the pericentral area and ALB mRNA concentration higher in the periportal area. This distribution pattern of reciprocal, overlapping gradients of mRNA can be clearly recognized in the neonatal period. In the adult liver AFP mRNA can no longer be detected and similar to the neonatal situation, ALB mRNA is expressed across the entire porto-central distance decreasing in concentration going from the portal to the central area.Transient extra-hepatic expression of AFP mRNA is found in the embryonic heart and in the epithelial lining of intestine and lung furthermore, AFP and ALB mRNA are found to be transiently expressed in the developing renal tubules. Similar expression patterns have been observed for other liver-characteristic mRNAs (Moormanet al., 1990), suggesting that common regulatory factors are operative during development.

The histone H5 variant in Xenopus laevis

The presumptive histone H5 of Xenopus laevis has been characterized by SDS and acid-urea-Triton polyacrylamide gel electrophoresis and compared with chicken histone H5. Chicken H5 has a lower electrophoretic mobility compared to that of Xenopus H5 in both gel systems. It is shown, using a polyclonal antiserum against chicken H5, that the Xenopus histone H5 is immunologically related to chicken histone H5. Monoclonal antibodies have been prepared to the Xenopus histone types H5 and H1A, that do not cross-react, as determined by their reactivity in an enzyme linked immunosorbent assay and by their ability to react with either H1A or H5 in an immunochemical test on total erythrocyte histones that are transferred to nitrocellulose after fractionation by SDS- or acid-urea polyacrylamide gel electrophoresis. As all nuclei of erythrocytes from adult Xenopus laevis can be shown to contain histone H1A and H5, these monoclonal antibodies can be used to further delineate the role of H5 in tissue differentiation.

Primary structure of the histone H2A and H2B genes and their flanking sequences in a minor histone gene cluster of Xenopus laevis

The study of histone gene expression has proved to be particularly useful in obtaining insight into eukaryotic gene regulation [ 11. The differential expression of the histone multigene family in oogenesis and early development is especially well documented [2,3]. We have focussed our work on histone gene expression in Xenopus laevis, since the study of oogenesis and development of this vertebrate species has given a wealth of information both at the molecular level, especially the regulation of specific genes, and the cellular level [4]. Sl mapping experiments indicate that at least the H3 gene present in the clone Xl-hi-l is not expressed during oogenesis and early embryogenesis.

Developmental changes in the expression of the liver-enriched transcription factors LF-B1, C/EBP, DBP and LAP/LIP in relation to the expression of albumin, α-fetoprotein, carbamoylphosphate synthase and lactase mRNA

SummaryExpression of α-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 α-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.

Immunohistochemical distribution of the histone H10/H5 variant in various tissues of adult Xenopus laevis

The cellular distribution of the histone H1(0)/H5 variant has been examined immunohistochemically in various tissues of adult Xenopus laevis, using monoclonal antibodies against this variant that was isolated from erythrocyte nuclei. The H1(0)/H5 variant appears not to be erythrocyte-specific and appears to be present in all cell types of liver, stomach, and skin. In contrast, in oocyte nuclei the H1(0)/H5 variant cannot be detected, whereas they do contain H1; the nuclei of spermatogenic cells contain the H1(0)/H5 variant, but probably less than the somatic cells. In Xenopus no H1(0) variant distinct from H5 seems to occur and the H1(0)/H5 variant apparently may perform a functional role related to mammalian H1(0).

Zonal distribution of peroxisomal 3-oxoacyl-CoA thiolase mRNA in liver from rats treated with di-(2-ethylhexyl) phthalate

Treatment of rats with di-(2-ethylhexyl)phthalate leads to a dramatic increase in peroxisomal 3-oxoacyl-CoA thiolase RNA, the concentration being higher in the pericentral than in periportal hepatocytes. These findings indicate that the production of peroxisomal thiolase and the zonal distribution of the enzyme are regulated at a pretranslational level.

Regulation of Hepatocyte-Specific Gene Expression in Cultures of Human Embryonic Hepatocytes

The aim of this study was to see whether the rat embryo can serve as a model system for hepatocyte-specific gene expression in the human embryo. Carbamoylphosphate synthetase was used as a hepatocyte-specific marker molecule. Despite the earlier developmental appearance of this enzyme in human than in murine liver, the hormonal regulation of gene expression in cultures of embryonic hepatocytes was found to be the same. Therefore, a relatively early developmental appearance of regulatory hormones rather than differences in regulatory mechanisms of gene expression appears to be responsible for the early accumulation of the enzyme in human liver, when compared to murine liver.

Dextranmarkierung auf agargel

Abstract This study concerns the use of dextran as an indicator for the zone of zero mobility. Wieme, and Bennett and Boursnell had described techniques for marking by dextran. A modification and combination of those techniques have been proposed: the protein sample and the reference mixture are run on the same agar plate, then the gel is fixed and dried, the protein bands are stained with amidoblack. Finally the dextran part of the agar gel is treated with an alkaline permanganate solution; with potassium bisulfite, the brown background of the periphery is decolorized. After this the agar plate can be scanned and the migration distances can be measured; besides, it is possible to calculate the percentage composition of the sample on the same agar plate. The migration distances of the reference—transferrin and of dextran have been measured; the difference between them (called “TD-distance”) have been calculated. The variabilities of the TD-distances have been estimated for two techniques of marking with dextran. The proposed technique has been tested and found to reduce the variability of the calculated TD-distances.This study concerns the use of dextran as an indicator for the zone of zero mobility. Wieme, and Bennett and Boursnell had described techniques for marking by dextran. A modification and combination of those techniques have been proposed: the protein sample and the reference mixture are run on the same agar plate, then the gel is fixed and dried, the protein bands are stained with amidoblack. Finally the dextran part of the agar gel is treated with an alkaline permanganate solution; with potassium bisulfite, the brown background of the periphery is decolorized. After this the agar plate can be scanned and the migration distances can be measured; besides, it is possible to calculate the percentage composition of the sample on the same agar plate. The migration distances of the reference—transferrin and of dextran have been measured; the difference between them (called “TD-distance”) have been calculated. The variabilities of the TD-distances have been estimated for two techniques of marking with dextran. The proposed technique has been tested and found to reduce the variability of the calculated TD-distances.