Nobu Akamatsu

Concurrent changes in sinusoidal expression of laminin and affinity of hepatocytes to laminin during rat liver regeneration

Distribution of fibronectin, laminin, and collagens type I, III, IV, and V in the lobular regions of regenerating rat liver was studied by indirect immunofluorescence. Little or no laminin was detected in sham-operated controls throughout the experimental period, while it was detected in sinusoids of regenerating liver as early as 6 h after partial hepatectomy (PH). After reaching a maximum at 24 h, it decreased and was barely detectable 6 days after PH. Changes in the other extracellular matrix (ECM) proteins were evident 3 days after PH, but not earlier than 24 h. Hepatocytes isolated from regenerating rat livers were tested in a short term assay for attachment to the substrates coated with the ECM proteins. The attachment of hepatocytes to laminin substrates increased 12 h after PH, reached a maximum at 24 h, and decreased to the control level 6 days after PH, while that of the control remained constant. The attachment to fibronectin substrates was not different between regenerating livers and controls at any time point. The attachment to collagen did not change earlier than 24 h after PH, but increased slightly 3 days after PH. Primary rat hepatocytes cultured on the substrates coated with the ECM proteins were determined for replicative DNA synthesis in response to epidermal growth factor. Both in normal liver and in regenerating liver 24 h after PH, laminin was one of the most effective substrates in supporting the responsiveness of hepatocytes to the growth stimulus. Taken together, these results suggest the importance of hepatocyte-laminin interaction during the early stage of liver regeneration possibly in growth stimulation of hepatocytes and/or maintenance of hepatocyte-specific functions.

UDP-N-acetylglucosamine-glycoproteinN-acetylglucosaminyltransferase in regenerating rat liver

The assay condition for N-acetylglucosaminyltransferase activities in rat liver microsomal fraction was developed. The enzyme activities towards endogenous acceptors within 48 h after partial hepatectomy were lower than in controls, exceeding the control level by 96 h, and then higher than in controls up to 240 h after the operation. The changes in N-acetylglucosaminyltransferase activities towards exogenous acceptor (UDP-2-acetamido-2-deoxy-D-glucose: glycoprotein 2-acetamido-2-deoxy-D-glucosyltransferase, EC 2.4.1.51) were consistent with those in the enzyme activities towards endogenous acceptors at 144 h, but not at 48 h, after the operation. The contents of protein and the levels of protein-bound hexosamine in the liver microsomes were decreased at early period of liver regeneration. These results suggest that the acceptor capacity of liver microsomal proteins is diminished during first 48 h of the regeneration. This may be responsible for the decreased transfer of the amino sugar to nascent glycoproteins. However, the enzyme activity was enhanced at 144 h and the level of endogenous acceptors may increase.

Synthesis in vitro of glycoprotein in regenerating rat liver.

The metabolism of glucosamine in regenerating rat liver was studied in liver slices. [1-14C] Glucosamine was incorporated into acid-soluble fraction, rapidly converted to UDP-N-acetylhexosamine and transferred to acid-insoluble fraction. Electrophoretic analysis revealed that most of the radioactive macromolecules released from the slices to the incubation medium were plasma glycoproteins. The incorporation of [1-14C] glucosamine into UDP-N-acetylhexosamine significantly increased from 6 h to 48 h after partial hepatectomy. On the contrary, the incorporation into acid-insoluble fractions of slice and medium decreased to about 50% of the control values. The rate of transfer of N-acetylhexosamine from UDP-N-acetylhexosamine to acid-soluble fractions also decreased at 12 h and 48 h respectively. This indicates that the transfer of N-acetylhexosamine to glycoproteins decreases during 48 h of liver regeneration. The enhancement of [1-14C] glucosamine incorporation into UDP-N-acetylhexosamine is due to an accumulation of the label in the larger pool of this compound. Evidently, some control mechanism may operate on the transfer of N-acetylhexosamine to glycoproteins in regenerating rat liver.

Alteration of rat liver proteoglycans during regeneration

Sepharose CL-6B column chromatography of crude extracts from the slices of regenerating rat livers after partial hepatectomy and sham-operated controls labeled with [35S]sulfuric acid revealed an enhancement of [35S]sulfate incorporation into proteoglycan fractions during regeneration. The 35S-labeled proteoglycans contained heparan sulfate (more than 80% of the total) and chondroitin/dermatan sulfate. The 35S-incorporation into both glycosaminoglycans increased to maxima 3-5 days after partial hepatectomy and decreased thereafter toward the respective control levels. When [35S]sulfuric acid was replaced by [3H]glucosamine, similar results were obtained. These results suggest that the maximal stimulation of proteoglycan synthesis in regenerating rat liver follows the maximal mitosis of hepatic cells 1-2 days after partial hepatectomy. The 35S-labeled proteoglycans from regenerating liver 3 days after partial hepatectomy and control were analyzed further. They were similar in chromatographic behavior on a gel filtration or an anion-exchange column and in glycosaminoglycan composition. Their glycosaminoglycans were indistinguishable in electrophoretic mobility. However, these proteoglycans were slightly but significantly different in their affinity to octyl-Sepharose and in the molecular-weight distribution of their glycosaminoglycans.

Three forms of rat liver glucosamine 6-phosphate acetylase and the changes in their levels during development

The three forms (Form I, II and III) of glucosamine 6-phosphate acetylase (glucosamine-phosphate acetyltransferase, EC 2.3.1.4) were present in rat liver. The enzyme activities changed separately during development, in which the successive epigenetic changes were suggested.

Glucosamine 6-phosphate acetylase in rat ascites hepatomas

The fetal type enzyme pattern of glucosamine 6-phosphate acetylase (glucosamine-phosphate acetyltransferase, EC 2.3.1.4) was present in rat ascites hepatomas. The levels of acid-soluble and protein-bound amino sugars in the hepatomas were determined.

Lipid intermediates in glycoprotein synthesis during rat liver regeneratio

Abstract 1. 1. In regenerating rat liver slices 24 hr after partial hepatectomy, the incorporations of d -[l-14C]glucosamine into oligosaccharide-lipid and protein fractions were decreased to 60–70% value of controls. 2. 2. The concentration of GDP-mannose in the liver 24 hr after partial hepatectomy was decreased to 60%. 3. 3. The incorporation of [14C]glucosamine into protein fraction in regenerating rat liver may be regulated at the step of lipid-intermediate synthesis.

Change in transferrin receptor distribution in regenerating rat liver

Partial hepatectomy results in an increase in the ability of the liver cells to bind 125I-labeled transferrin to surface receptors. Scatchard analysis of the binding of transferrin in regenerating rat liver indicates that this increase was due to an increase in the number of transferrin receptors on the cell surface. When we measured total cellular transferrin receptor number, we found that control and regenerating livers had identical amounts of receptor number. The increase in the surface receptor number is apparently due to the translocation of intracellular transferrin receptor to the cell surface.

Hexokinase of Angiostrongylus cantonensis: Presence of a glucokinase

1. Angiostrongylus cantonensis contains a glucokinase which was isolated by DEAE-cellulose column chromatography. 2. This enzyme has a much higher affinity toward glucose (apparent Km, 0.2 mM) than fructose (apparent Km, 85 mM). Glucose-6-phosphate (10 mM) does not inhibit glucose phosphorylation. 3. Molecular weight obtained by a molecular sieve chromatography (60,000) is also close to the value of mammalian glucokinase. 4. While Vmax value for mannose is one-third smaller than that for glucose, Km for mannose is rather lower than that for glucose. 5. In addition to the cytosol enzyme, a particle bound hexokinase is found in the worm.

Biosynthesis of N-methyl-L-glucosamine from D-glucose by Streptomyces griseus.

Biosynthesis of N-methyl-L-glucosamine moiety of streptomycin from D-glucose by Streptomyces griseus was studied. A mixture of D-[1-(14) C] glucose and D-[6(-3)H]glucose was given to the culture of S. griseus. The 3H/14C ratio found in N-methyl-L-glucosamine further supports a mechanism that the conversion of D-glucose to L-hexose is carried out without scission of carbon skeleton. When D-[1-14C]glucose and D-[3-3H]glucose were used, the fall of 3H/14C ratio in N-metyl-L-glucosamine showed that the hydrogen atom at C-3 plays a rôle in such a transformation.