Guanine deaminase in rat liver and mouse liver and brain

1. The guanine deaminase in rat liver supernatant preparations was resolved into two fractions, A and B, on DEAE-cellulose columns. The two differed in electrophoretic mobility and in various properties. The most noteworthy distinction between A and B components was that the enzyme A activity showed a sigmoid dependence on substrate concentration whereas the enzyme B showed classical Michaelis-Menten kinetics. The K(m) value of enzyme A for guanine was 5.3mum and that of enzyme B 20mum. 2. The entire guanine deaminase activity of mouse liver was contained in the 15000g supernatant of iso-osmotic homogenates. 3. A reinvestigation of the behaviour of rat brain 15000g supernatant guanine deaminase isoenzymes revealed that one enzyme had sigmoidal kinetics and the other enzyme showed a hyperbolic response. 4. Of the guanine deaminase in mouse brain iso-osmotic sucrose homogenate 80% was recovered in the 15000g supernatant and the rest from the particles. The supernatant guanine deaminase was resolvable into two fractions on DEAE-cellulose columns. One enzyme showed sigmoidal kinetics whereas the other showed a hyperbolic response to increasing substrate concentration; the K(m) values for the reaction with guanine were respectively 5 and 66mum. 5. The particulate fractions of mouse liver and brain were devoid of any overt inhibitory activity.     

Phosphorylation and inactivation of liver glycogen synthase by liver protein kinases

A rapid method for purifying glycogen synthase a from rat liver was developed and the enzyme was tested as a substrate for nine different protein kinases, six of which were isolated from rat liver. The enzyme was phosphorylated on a 17-kDa CNBr fragment to approximately 1 phosphate/87-kDa subunit by phosphorylase b kinase from muscle or liver with a decrease in the activity ratio (-Glc-6-P/+Glc-6-P) from 0.95 to 0.6. Calmodulin-dependent glycogen synthase kinase from rabbit liver produced a similar phosphorylation pattern, but a smaller activity change. The catalytic subunit of beef heart cAMP-dependent protein kinase incorporated greater than 1 phosphate/subunit initially into a 17-kDa CNBr peptide and then into a 27-30-kDa CNBr peptide, with an activity ratio decrease to 0.5. Glycogen synthase kinases 3, 4, and 5 and casein kinase 1 were purified from rat liver. Glycogen synthase kinase 3 rapidly phosphorylated liver glycogen synthase to 1.5 phosphate/subunit with incorporation of phosphate into 3 CNBr peptides and a decrease in the activity ratio to 0.3. Glycogen synthase kinase 4 produced a pattern of phosphorylation and inactivation of liver synthase which was very similar to that caused by phosphorylase b kinase. Glycogen synthase kinase 5 incorporated 1 phosphate/subunit into a 24-kDa CNBr peptide, but did not alter the activity of the synthase. Casein kinase 1 phosphorylated and inactivated liver synthase with incorporation of phosphate into a 24-kDa CNBr peptide. This kinase and glycogen synthase kinase 4 were more active against muscle glycogen synthase. Calcium-phospholipid-dependent protein kinase from brain phosphorylated liver and muscle glycogen synthase on 17- and 27-kDa CNBr peptides, respectively. However, there was no change in the activity ratio of either enzyme. The following conclusions are drawn. 1) Liver glycogen synthase a is subject to multiple site phosphorylation. 2) Phosphorylation of some sites does not per se control activity of the enzyme under the assay conditions used. 3) Liver contains most, if not all, of the protein kinases active on glycogen synthase previously identified in skeletal muscle.     

Expression of liver and placental alkaline phosphatases in Chang liver cells

This paper presents evidence that a protein characteristic of differentiated liver cells, liver alkaline phosphatase, is synthesized by the Chang liver cell line. Liver alkaline phosphatase was demonstrated by immumochemical assay, ³²P-labeling and polyacrylamide gel electrophoresis, immunofluorescence microscopy, and the fluorescence-activated cell sorter. The synthesis of the liver enzyme by the Chang liver cells is interpreted to indicate fidelity of the Chang cells to their origin from human liver tissue. Chang liver cells also synthesize a phosphatase which is similar if not indentical to the placental alkaline phosphatase. Since a placental-type alkaline phosphatase has been observed in a number of non-trophoblastic cell lines and also in some neoplasms, it does not seem reliable as an index of the origins of the cell line. Because of the claims that Chang liver cells are actually HeLa cells, HeLa cells were studied in tandem with the Chang cells. The results showed that the HeLa cells do not make the liver type phosphatase. The data are discussed in relation to the question of HeLa cell contamination of the Chang cell line and the validity of criteria normally used to identify cell lines.     

Phospholipid-transfer proteins in human liver and primary liver carcinoma

Investigations have been carried out on phospholipid-transfer activity of the cytosol and the phospholipid composition of subcellular membranes from human liver and primary liver carcinoma. In both human liver and primary liver carcinoma cytosolic fractions, the transfer activity for phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin has been observed for the first time. The transfer rate of PC and PE in normal human liver was almost equal, whereas sphingomyelin-transfer activity was much slower. In carcinoma cells, the transfer activity for PE and PC was significantly enhanced, while sphingomyelin transfer remained unchanged. Comparative investigations with HepG2 cultured cells have revealed a high PE-transfer activity in this cell line. Parallel with the phospholipid-transfer activity modifications in neoplasic cells, changes in the phospholipid composition of microsomes and mitochondria have been observed. The content of PC and PE in hepatocarcinoma cells was decreased in microsomes, while in the mitochondria it was increased. The possible role of the phospholipid-transfer proteins in the maintenance of membrane composition and structure is discussed.     

清肝泄浊法治疗脂肪肝30例临床研究

目的:观察清肝泄浊法治疗脂肪肝的临床疗效.方法:治疗组30例口服自拟清肝泄浊汤,对照组24例口服复方益肝灵,观察肝功能及血脂变化.结果:治疗组30例,有效率90.0%,对照组30例,有效率36.6%.结论:清肝泄浊法治疗脂肪肝的疗效明显,适宜临床推广运用.     

Low-molecular-weight chromium-binding substance from chicken liver and American alligator liver

Low-molecular-weight chromium-binding substance (LMWCr), also known as chromodulin, is a chromium-binding oligopeptide proposed to have a function in chromium transport and insulin signaling in mammals. In this work, LMWCr has been isolated and purified for the first time from non-mammalian sources: chicken and American alligator. Milligram quantities of the oligopeptide can be obtained from kilogram quantities of liver. The LMWCr's from both sources are asparatate- and glutamate-rich oligopeptides which possess multinuclear chromium assemblies. The composition and physical and spectroscopic properties of the avian and reptilian LMWCr's are extremely similar to those of their mammalian analogues, suggesting the multinuclear sites of the biomolecule from all three classes of animal possess very similar structures. The chicken and alligator oligopeptides may possess intrinsic phosphotyrosine phosphatase activity.     

Lipogenic enzyme induction and incorporation of exogenous fatty acid into liver and liver nuclei

The incorporation of exogenous fatty acid into lipids of liver and liver nuclei of rats fed diets with or without fat was compared. When [3H]palmitic acid was injected into rats, more radioactivity was incorporated into triacylglycerols and phospholipids of liver and liver nuclei from rats fed the fat-free diet than from those fed the fat diet. The results were supported further by an autoradiographic study. On the other hand, the enzyme induction and quantity of malic enzyme mRNA were decreased by fat feeding. Other lipogenic enzymes were also coordinately decreased. Thus, it may be possible that exogenous fatty acid is involved in nuclear regulation in addition to cytosolic regulation of lipogenic enzyme induction.     

Potential neural progenitor cells in fetal liver and regenerating liver

From unfractionated embryonic mice liver cells, appreciable amount of spherical bodies containing nestin-positive cells were generated in the presence of neuronal growth factors. Following cultivation on poly-d-lysine/laminin-coated slips, approximately 70% of the cells expressed neuronal markers, and 16% had long processes. Functional analysis of these long-process-bearing cells with the whole-cell patch clamp method showed an inward current in response to glutamate, GABA, and serotonin as the neuronal characteristics. Furthermore, regenerating liver in adult mice also contained nestin-positive cells to the same extent as fetal liver. Regenerating liver could have potential as a source of neural cells for autologous transplantation.     

Fetal and adult liver stem cells for liver regeneration and tissue engineering

For the development of innovative cell-based liver directed therapies, e.g. liver tissue engineering, the use of stem cells might be very attractive to overcome the limitation of donor liver tissue. Liver specific differentiation of embryonic, fetal or adult stem cells is currently under investigation. Different types of fetal liver (stem) cells during development were identified, and their advantageous growth potential and bipotential differentiation capacity were shown. However, ethical and legal issues have to be addressed before using fetal cells. Use of adult stem cells is clinically established, e.g. transplantation of hematopoietic stem cells. Other bone marrow derived liver stem cells might be mesenchymal stem cells (MSC). However, the transdifferentiation potential is still in question due to the observation of cellular fusion in several in vivo experiments. In vitro experiments revealed a crucial role of the environment (e.g. growth factors and extracellular matrix) for specific differentiation of stem cells. Co-cultured liver cells also seemed to be important for hepatic gene expression of MSC. For successful liver cell transplantation, a novel approach of tissue engineering by orthotopic transplantation of gel-immobilized cells could be promising, providing optimal environment for the injected cells. Moreover, an orthotopic tissue engineering approach using bipotential stem cells could lead to a repopulation of the recipients liver with healthy liver and biliary cells, thus providing both hepatic functions and biliary excretion. Future studies have to investigate, which stem cell and environmental conditions would be most suitable for the use of stem cells for liver regeneration or tissue engineering approaches.