Postnatal differentiation of sex-specific distribution patterns of G6Pase, G6PDH and ME in the rat liver

The activity of the liver enzymes G6Pase, G6PDH and ME was studied in rats of 2-9 weeks old by histochemical means. In addition, G6PDH and ME activity was quantitatively determined in homogenates. In the 2nd and 3rd week G6Pase is similarly distributed in both sexes: while in the periportal zone high activity is demonstrable, the perivenous zone shows only low activity. After this period a nearly homogeneous distribution pattern becomes evident in all animals. Sex difference occurs after the 6th week: in the livers of male rats the periportal "maximum" is sometimes combined with a second peak in the perivenous area, in females a steep gradient emerges with high activity in the periportal zone and a low one in the perivenous zone. In the first postnatal weeks G6PDH activity is very low in parenchymal cells, but very prominent in Kupffer cells. Around the 5th week there is an increase, predominantly in the perivenous zone of both sexes. While there is again a further decrease demonstrable in male rats, the G6PDH activity of female rats rises to high adult values. This increase seems to be restricted to the perivenous zone. ME can be demonstrated at first in leucocytes. In the course of the 3rd week there is an increase of activity in both sexes: ME is demonstrable in parenchymal cells of the perivenous area and in scattered hepatocytes of the periportal area. In male rats, the perivenous activity is diminished towards the end of the investigation period, in females, however, a high activity remains in the perivenous zone. The data show that in females the activity of NADP dependent enzymes is high in the perivenous zone, so it may be assumed that a lipogenic area is situated around the terminal efferent vessels. Because of the sex difference this area may be hormone-dependent. The lipogenic area is situated opposite to the gluco(neo)genic area which corresponds to the periportal zone.     

Postnatal differentiation of sex-specific distribution patterns of G6Pase,G6PDH and ME in the rat liver

Summary The activity of the liver enzymes G6Pase, G6PDH and ME was studied in rats of 2–9 weeks old by histochemical means. In addition, G6PDH and ME activity was quantitatively determined in homogenates. In the 2nd and 3rd week G6Pase is similarly distributed in both sexes: while in the periportal zone high activity is demonstrable, the perivenous zone shows only low activity. After this period a nearly homogeneous distribution pattern becomes evident in all animals. Sex difference occurs after the 6th week: in the livers of male rats the periportal maximum is sometimes combined with a second peak in the perivenous area, in females a steep gradient emerges with high activity in the periportal zone and a low one in the perivenous zone. In the first postnatal weeks G6PDH activity is very low in parenchymal cells, but very prominent in Kupffer cells. Around the 5th week there is an increase, predominantly in the perivenous zone of both sexes. While there is again a further decrease demonstrable in male rats, the G6PDH activity of female rats rises to high adult values. This increase seems to be restricted to the perivenous zone. ME can be demonstrated at first in leucocytes. In the course of the 3rd week there is an increase of activity in both sexes: ME is demonstrable in parenchymal cells of the perivenous area and in scattered hepatocytes of the periportal area. In male rats, the perivenous activity is diminished towards the end of the investigation period, in females, however, a high activity remains in the perivenous zone. The data show that in females the activity of NADP dependent enzymes is high in the perivenous zone, so it may be assumed that a lipogenic area is situated around the terminal efferent vessels. Because of the sex difference this area may be hormone-dependent. The lipogenic area is situated opposite to the gluco(neo)genic area which corresponds to the periportal zone.Parts of this study were presented as an Inaugural Dissertation to the Medical Faculty of the University of Freiburg by H.H.Supported by grants from the Deutsche Forschungsgemeinschaft (Sa 127/7 and the SFB 46 (Molgrudent)     

Wound healing in the liver with particular reference to stem cells.

The efficiency of liver regeneration in response to the loss of hepatocytes is widely acknowledged, and this is usually accomplished by the triggering of normally proliferatively quiescent hepatocytes into the cell cycle. However, when regeneration is defective, tortuous ductular structures, initially continuous with the biliary tree, proliferate and migrate into the surrounding hepatocyte parenchyma. In humans, these biliary cells have variously been referred to as ductular structures, neoductules and neocholangioles, and have been observed in many forms of chronic liver disease, including cancer. In experimental animals, similar ductal cells are usually called oval cells, and their association with impaired regeneration has led to the conclusion that they are the progeny of facultative stem cells. Oval cells are of considerable biological interest as they may represent a target population for hepatic carcinogens, and they may also be useful vehicles for ex vivo gene therapy for the correction of inborn errors of metabolism. This review proposes that the liver harbours stem cells that are located in the biliary epithelium, that oval cells are the progeny of these stem cells, and that these cells can undergo massive expansion in their numbers before differentiating into hepatocytes. This is a conditional process that only occurs when the regenerative capacity of hepatocytes is overwhelmed, and thus, unlike the intestinal epithelium, the liver is not behaving as a classical, continually renewing, stem cell-fed lineage. We focus on the biliary network, not merely as a conduit for bile, but also as a cell compartment with the ability to proliferate under appropriate conditions and give rise to fully differentiated hepatocytes and other cell types.     

Different distribution patterns of the two mannose 6-phosphate receptors in rat liver.

Two mannose 6-phosphate receptors, cation-dependent and -independent receptors (CDMPR and CIMPR), play an important role in the intracellular transport of lysosomal enzymes. To investigate functional differences between the two in vivo, their distribution was examined in the rat liver using immunohistochemical techniques. Positive signals corresponding to CIMPR were detected intensely in hepatocytes and weakly in sinusoidal Kupffer cells and interstitial cells in Glisson's capsule. In the liver acinus, hepatocytes in the perivenous region showed a more intense immunoreactivity than those in the periportal region. On the other hand, positive staining of CDMPR was detected at a high level in Kupffer cells, epithelial cells of interlobular bile ducts, and fibroblast-like cells, but the corresponding signal was rather weak in hepatocytes. In situ hybridization analysis also revealed a high level of expression of CIMPR mRNAs in hepatocytes and of CDMPR mRNA in Kupffer cells. By double immunostaining, OX6-positive antigen-presenting cells in Glisson's capsule were co-labeled with the CDMPR signal but were only faintly stained with anti-CIMPR. These different distribution patterns of the two MPRs suggest distinct functional properties of each receptor in liver tissue.     

The subcellular distribution and properties of aldehyde dehydrogenases in rat liver

1. Kinetic experiments suggested the possible existence of at least two different NAD(+)-dependent aldehyde dehydrogenases in rat liver. Distribution studies showed that one enzyme, designated enzyme I, was exclusively localized in the mitochondria and that another enzyme, designated enzyme II, was localized in both the mitochondria and the microsomal fraction. 2. A NADP(+)-dependent enzyme was also found in the mitochondria and the microsomal fraction and it is suggested that this enzyme is identical with enzyme II. 3. The K(m) for acetaldehyde was apparently less than 10mum for enzyme I and 0.9-1.7mm for enzyme II. The K(m) for NAD(+) was similar for both enzymes (20-30mum). The K(m) for NADP(+) was 2-3mm and for acetaldehyde 0.5-0.7mm for the NADP(+)-dependent activity. 4. The NAD(+)-dependent enzymes show pH optima between 9 and 10. The highest activity was found in pyrophosphate buffer for both enzymes. In phosphate buffer there was a striking difference in activity between the two enzymes. Compared with the activity in pyrophosphate buffer, the activity of enzyme II was uninfluenced, whereas the activity of enzyme I was very low. 5. The results are compared with those of earlier investigations on the distribution of aldehyde dehydrogenase and with the results from purified enzymes from different sources.     

Kinetic properties of hexose-monophosphate dehydrogenases. II. Isolation and partial purification of 6-phosphogluconate dehydrogenase from rat liver and kidney cortex

6-Phosphogluconate dehydrogenase (6PGDH) from rat-liver and kidney-cortex cytosol has been partially purified and almost completely isolated (more than 95%) from glucose-6-phosphate dehydrogenase activity. The purification and isolation procedures included high-speed centrifugation, 60–75% ammonium-sulphate fractionation, by which both hexose-monophosphate dehydrogenases activities were separated, and finally the protein fraction was applied to a chromatographic column of Sephadex G-25 equilibrated with 10 mM Tris-EDTA-NADP buffer, pH 7.6, to eliminate any contaminating metabolites. The kinetic properties of the isolated partially purified liver and renal 6PGDH were examined. The saturation curves of this enzyme in both rat tissues showed a typical Michaelis-Menten kinetic, with no evidence of co-operativity. The optimum pH for both liver and kidney-cortex 6PGDH was 8.0. The Km values of liver 6PGDH for 6-phosphogluconate (6PG) and for NADP were 157 M and 258 M respectively, while the specific activity measured at optimum conditions (pH 8.0 and 37°C) was 424.2 mU/mg of protein. NADPH caused a competitive inhibition against NADP with an inhibition constant (K_i) of 21 M. The Km values for 6PG and NADP from kidney-cortex 6PGDH were 49 M and 56 M respectively. The specific activity at pH 8.0 and 37°C was 120.7 mU/mg of protein. NADPH also competitively inhibited 6PGDH activity, with a K_i of 41 M. This paper describes a quick, easy and reliable method for the separation of the two dehydrogenases present in the oxidative segment of the pentose-phosphate pathway in animal tissues, eliminating interference in the measurements of their activities.Publication No 170 from Drugs, Environmental Toxics and Cell Metabolism research group. Department of Biochemistry and Molecular Biology, University of Granada, Granada, Spain     

Characterization of rat bone marrow cells. II. Analysis of surface antigens in small lymphocytes with particular reference to thymus antigen-carrying cells.

Rat bone marrow (BM) small cells were enriched by velocity sedimentation, further separated by means of free-flow electrophoresis, and characterized using T- and B-cell-specific surface markers. More than 80% of these cells were small lymphocytes by morphological criteria and reacted with lymphocyte-specific antisera. A minority of cells had high electrophoretic mobility (EPM), carried surface antigens characteristic of mature T cells, and lacked B-cell markers. These cells may represent recirculating T cells. A small number of cells were found with rat B lymphocyte-specific antigen (RBLA) and surface immunoglobulin (sIg) and had medium EPM. These cell fractions also contained “null” cells which were devoid of T- and B-cell-specific antigens. More than 80% of the BM small cells had low EPM and carried the three subspecificities of the Thy-1 antigen complex and the Thy-A antigens. These antigens were found at several-fold higher concentration on the surface of all thymocytes, but are lacking in most other lymphocytes. The thymus antigen-carrying BM cells of low EPM do not carry other T- and B-cell-specific markers found in thymocytes and peripheral T and B lymphocytes. These markers comprise the T-cell antigens RTLA (rat T-lymphocyte-specific antigen) and RHLA (antigens specific for rat T cells of high EPM) and the B-cell markers RBLA and sIg. Thus the majority of rat BM lymphocytes differ from all other lymphocytes of the T- and B-cell series which makes any classification on this basis purely speculative.     

Development of behavior and sex differences in infants; with reference to the formation of sex role

In order to observe the formation of sex role, an investigation was made on the development of behavior and sex differences in infants aged 1 to 6 at Fukuoka-city. The present study consisted of questionnaire on behavior of daily living and breeding, block construction test and coloring a drawing test. The parents instructed their infants to become masculine for males and feminine for females on the basis of traditional view. No sex differences were observed in construction ability of infants aged 4. Favorite colors were different according to sex.