Interaction of 5-azacytidine and dexamethasone in the control of hepatic tyrosine aminotransferase.

The nucleoside analog 5-azacytidine is able to induce tyrosine aminotransferase several-fold in the liver of suckling rats. Bilateral adrenalectomy abolishes this inducing effect. The drug also decreases significantly the concentration of cytosolic glucocorticoid receptor accompanied by an increase of the glucocorticoid receptor concentration in the nuclei. The antiglucocorticoid RU 486 which abolishes the induction of tyrosine aminotransferase and serine dehydratase by dexamethasone very effectively is not able to inhibit either the induction of tyrosine aminotransferase or the translocation of the glucocorticoid receptor into the nuclei by 5-azacytidine.

Interaction of adrenal and pancreatic hormones in the control of hepatic enzymes during development.

In the liver of suckling rats, the synthesis of hepatic tyrosine aminotransferase, serine dehydratase, and phosphofructokinase 2 as well as of renal beta-glucosidase is controlled by the circulating concentrations of adrenal and pancreatic hormones. Glucagon is capable of stimulating enzyme synthesis only in the presence of a steroid hormone. Dexamethasone and estradiol have been found to exert a permissive function on the inducibility of the studied enzymes by glucagon. Between the hormones of the adrenal medulla and glucagon antagonistic effects in enzyme induction were observed. Obviously, this antagonism is mediated by the alpha 1-adrenergic signal transferring system. A characteristic age dependence of enzyme induction by dexamethasone has been established. This might be correlated to alterations in the degree of methylation of the respective promoters. The methylation inhibitor 5-azacytidine influences significantly the enzyme induction by glucocorticoid hormones.

Origin of the enhanced activity of lysosomal beta-galactosidase in serum and skin in progressive systemic sclerosis.

In 14 patients with progressive systemic sclerosis (PSS) the activities of acid lysosomal glycosidases (alpha-, beta-galactosidase, beta-glucosidase, beta-glucuronidase, and beta-N-acetyl-glucosaminidase) were determined fluorometrically in serum, leukocytes, and skin tissue. The beta-galactosidase was the only enzyme which exhibited a significantly elevated activity in PSS serum and skin but not leukocytes, as compared to the control. The activity patterns of the studied glycosidases in serum were similar to those found in skin, but differ from the distribution of glycosidase activities in leukocytes. In cultured dermal fibroblasts derived from PSS patients, an elevated intracellular activity of beta-galactosidase was detected. These results suggest that the increased beta-galactosidase activity in the serum originates from the skin fibroblasts.

Stability of ketone bodies in serum in dependence on storage time and storage temperature.

The serum concentrations of beta-hydroxybutyrate and acetoacetate as well as the beta-hydroxybutyrate/acetoacetate ratio are important parameters for the differential diagnosis of certain inborn errors of metabolism. Acetoacetate, however, is an unstable compound which becomes rapidly decarboxylated. At a storage temperature of -20 degrees C about 40% of the acetoacetate is lost within 7 days and after 40 days storage at this temperature virtually all of the acetoacetate has become degraded. At -80 degrees C the decomposition of acetoacetate occurs with a much slower rate and only 15% of the initial acetoacetate is lost after 40 days storage. The rate constants for the decarboxylation reaction were found to be (6.4 +/- 2.9) * 10(-5) [min(-1)] at -20 degrees C and (0.4 +/- 0.3) * 10(-5) [min(-1)] at -80 degrees C. In contrast, beta-hydroxybutyrate is very stable during storage and hence should be used as main parameter for the evaluation of ketonemia. If determination of acetoacetate and/or of the beta-hydroxybutyrate/acetoacetate ratio is necessary, an assay immediately after collecting the serum samples is recommended. Otherwise, the serum samples should be frozen as soon as possible and stored at -80 degrees C during transport and storage.

Changes of glycogen phosphorylase isozyme pattern, in rat tissues during pre- and postnatal development.

Quantitative estimates of M- and L-type phosphorylase subunits in adult and developing rat tissues have been obtained by immunotitration of tissue extracts with subunit-specific antibodies. It has been shown that M-type subunits occur in all studied tissues with exception of adult liver and placenta. L-type subunits account for most of the total phosphorylase activity in liver and bone marrow but are present also in a variety of other adult and in most of the fetal tissues examined. During maturation, selective increases in the expression of phosphorylase isozymes occur in certain tissues such as liver, brain, heart and skeletal muscle. The hitherto unknown hybrid LM has been produced by in vitro hybridization of M- and L-subunits.

Effect of prenatal insulin and glucagon injection on the glycogen content of rat placenta and fetal liver.

The placental glycogen content per g wet weight decreases after the 16th day of gestation but remains nearly constant till the 21st day of gestation when expressed per placenta as whole organ. Injection of insulin to rat fetuses at the 21st day of gestation gives rise to hypoglycemia and an increase of the glycogen content in liver and in placenta. Intrauterine glucagon application makes fetuses hyperglycemic in consequence of a mobilization of the placental glycogen stores. Hepatic glucose production appears in later stages because of the low activity of glucose 6-phosphatase as demonstrated by the effect of glucagon on the hepatic glucose 6-phosphate concentration.

Influence of the placenta on fetal plasma insulin and neonatal hepatic enzyme induction in the rat.

In order to study the placental role in controlling glucose metabolism and neonatal induction of tyrosine aminotransferase in rat liver, a comparison was made between the artificially delivered-rat feto-placental unit and the newborn littermates. The immunoreactive serum insulin in the feto-placental unit remains as high as in utero for at least 1 h whereas in the newborn rat it decreases rapidly after delivery. From Caesarian section to 6 h later, in contrast to the newborn littermates, the feto-placental unit remains hyperglycaemic. Upon glucagon injection the feto-placental unit shows a delay in the increase of the hepatic tyrosine aminotransferase activity in comparison with the newborn rat.

Fructose breath hydrogen test--is it really a harmless diagnostic procedure?

Usage of hydrogen breath tests has become one of the standard procedures in diagnosing chronic unspecific abdominal pain. These tests are said to be of sufficient specificity and sensitivity, are easily done, non-invasive and are more often practiced in outpatients. A 13-year-old boy is reported with chronic unspecific abdominal pain and growth retardation and so far misdiagnosed hereditary fructose intolerance (HFI), who developed life-threatening adverse effects during the fructose breath hydrogen test. It is concluded that the possibility of HFI should be excluded first by a carefully explored dietary history before the fructose breath test is performed under medical supervision. If there is any suspicion of HFI, a molecular genetic analysis should be preferred.

Cloning and characterization of another lignin peroxidase gene from the white-rot fungus Phanerochaete chrysosporium.

Lignin peroxidase (LiP) isozymes of Phanerochaete chrysosporium are encoded by a large family of closely related genes, whose total number is still unknown. Among genomic clones, obtained using the polymerase chain reaction to clone the LiP gene LPOA from Phanerochaete chrysosporium strain BKM-F 1767, another LiP gene was found. This gene, HG3, showed more than 95% nucleotide homology to those LiP gene variants which encode LiP isozyme H8. The gene encodes a protein of 372 amino acids, including the typical leader sequence for secretion, that is identical to the LiP isozyme H8 except for 6 amino acid substitutions.

Developmental changes of glycogen phosphorylase b isozymes in rat tissues.

The isozyme distribution of glycogen phosphorylase b was studied in various fetal, neonatal, and adult rat tissues by means of discontinuous polyacrylamide gel electrophoresis in the presence of glycogen in the separating gel. The brain-type isozyme BB being the predominant form in fetal rat tissues is replaced during maturation either partially (brain and kidney) or completely (skeletal muscle and liver) by the isozymes MM or LL, respectively. In the various organs, the developmental formation of the adult phosphorylase isozyme pattern does not proceed simultaneously. The definitive isozyme pattern is expressed in heart muscle at the 19th day of gestation. The isozyme transitions in liver and skeletal muscle are finished about two days after birth, while at this time in the brain just the muscle-type subunit M appears and forms the hybrid MB. The isozyme distribution of the maturated rat brain (BB, MB, MM) is not expressed before the 9th day of postnatal life.

Pig kidney phosphofructokinase. Purification to homogeneity and qualitative basic characterization.

Phosphofructokinase has been purified from pig kidney by extraction with phosphate buffer at pH 8, followed by alcohol treatment, affinity chromatography on matrix-bound Cibacron blue F3G-A, and gel chromatography on Sepharose 6B. Using sodium dodecyl sulphate electrophoresis the enzyme was found to be homogeneous and to have a specific activity of about 80 units/mg protein. Like other phosphofructokinases, at pH 7.0 the enzyme exhibits a sigmoidal dependence in its activity on the fructose 6-phosphate concentration and is strongly inhibited by ATP. The degree of citrate inhibition is influenced by the concentration of the two substrates. ATP strengthens and fructose 6-phosphate relieves the inhibition by citrate. AMP and cAMP are able to overcome the ATP inhibition. The ADP activation curve is biphasic. The molecular weight of the subunit of pig kidney phosphofructokinase was determined to be 88 000 by means of sodium dodecyl sulphate electrophoresis.

[Microcalorimetric determination of thermochemical parameters of the phosphofructokinase reaction]. / Mikrokalorimetrische Bestimmung der thermochemischen Parameter der Phosphofruktokinase-Reaktion

A calorimetric procedure for determining deltaH, deltaG, deltaS and Keq of a bimolecular reaction with two or more products is described. By using this method the thermodynamic parameters of the phosphofructokinase reaction are determined. At pH 7.0 and 25 degrees C a reaction enthalpy of-6.96kcal/mole was found after correction for the neutralization enthalpy of the buffer and of the enthalpy difference of the magnesium complexes of ATP and ADP, respectively. The free energy of the phosphofructokinase reaction has been found under these conditions to be -3.96kcal/mole.

Biochemistry of liver development in the perinatal period.

Just before birth, changes occur in the metabolic capacities of rat liver so that the animal can adapt to changes in the substrate supply. In utero, glucose is the main energy-generating fuel and the liver metabolism is directed towards glucose degradation. The activities of the rate-limiting enzymes of glycolysis, hexokinase and phosphofructokinase, are high. In preparation for post-natal life, when the continuous glucose supply from the mother is interrupted, very large amounts of glycogen are stored in the late fetal liver. With the intake of the fat-rich and carbohydrate-poor milk diet, the animal develops the ability to synthesize glucose de novo from non-carbohydrate precursors. During suckling, metabolic energy is derived mainly from the beta-oxidation of fatty acids, which in turn is an essential prerequisite for the high rate of gluconeogenesis, by yielding acetyl-CoA for the activation of pyruvate carboxylase and by generating a high NADH/NAD ratio for the shift of the glyceraldehyde 3-phosphate dehydrogenase reaction in the direction of glucose formation.--The developmental adaptation of metabolism and the process of enzymatic differentiation are closely connected with the maturation of the endocrine system and the changes in the concentration of circulating hormones. The neonatal regulation of phosphoenolpyruvate carboxykinase and of tyrosine aminotransferase by variations in the hormonal milieu around birth, and also the interaction of hormonal and nutritional factors in the induction of serine dehydratase and glucokinase at the end of the suckling period, will be discussed in detail.

Biochemical and clinical observations in four patients with fructose-1,6-diphosphatase deficiency.

Three boys and one girl suffering from inherited fructose-1,6-diphosphatase (FDPase) deficiency are reported. All four patients had less than 25% residual hepatic FDPase activity. While in two out of three patients the enzyme deficiency was also expressed in leucocytes, one patient had a normal enzyme activity. Remarkably, three patients had pronounced neonatal hyperbilirubinaemia requiring exchange transfusion.

[Hemoglobin A1 in hypoglycemias in childhood]. / Hämoglobin A1 bei Hypoglykämien im Kindesalter.

The average HbA1 concentrations of 20 patients with hypoglycaemic diseases were not significantly different from metabolically healthy controls. There was also no correlation between the HbA1 and the blood glucose levels of these patients. Remarkably, two patients which responded to therapy with an increase of blood glucose showed also an increase of HbA1.

Purification and properties of pig kidney glutamate dehydrogenase.

Glutamate dehydrogenase from pig kidney has been purified to homogeneity by means of affinity chromatography on matrix bound Cibacron Blue F3G-A and gel chromatography on Sepharose 6B. The enzyme exhibits allosteric properties with the substrates alpha-ketoglutarate, ammonium, and NADH, respectively. GTP is a strong inhibitor which strengthened the cooperative interactions between the ammonium binding sites. ADP as an activator relieves the inhibition by GTP. Like glutamate dehydrogenase from bovine liver, glutamate dehydrogenase from pig kidney shows the ability of self-association, too. The sedimentation coefficient increases from 13.5 S at 0.07 mg protein/ml to 19.4 S at 1.32 mg protein/ml. In the sodium dodecylsulphate gel electrophoresis the enzyme migrates as a single band with a molecular-weight at 51000.

Developmental changes of glycosidase activities in rat renal cortex.

The activities of beta-glucosidase as well as of alpha- and beta-galactosidase increase in the renal cortex of the rat during the early postnatal development. However, while the two galactosidases reach adult values around the end of the second week of life, beta-glucosidase activity exhibits a second steep increase after the third post-uterine week. This rise in activity correlates with the functional maturation of kidney and is confined nearly exclusively to the cytosolic isozyme. The effects of taurocholate, Triton X-100, and dexamethasone on beta-glucosidase isozymes were studied. The results suggest that taurocholate should be included in the assay medium for the measurement of the beta-glucocerebrosidase activity in the diagnosis of Gaucher's disease. For the determination of the total beta-glucosidase activity in samples containing mainly the lysosomal isozyme (perinatal kidney, isolated lysosomes) the use of Triton X-100 is recommended.

An improved method for adrenalectomy of suckling rats. The influence of thrombin treatment and deoxycorticosterone substitution on survival and on hepatic and renal enzyme activities.

Adrenalectomy of suckling rats is complicated by a high mortality rate, caused by the loss of blood (early mortality) and by the disturbed sodium-potassium balance (late mortality). Treatment of the abdominal cavity with a thrombin solution and a daily administration of deoxycorticosterone glucoside (DOC) decrease the total mortality remarkably. DOC treatment has no influence on renal beta-glucosidase and beta-galactosidase as well as on hepatic tyrosine aminotransferase activity, whereas hepatic serine dehydratase activity exhibits a time- and dosage-dependent response to this hormone. The DOC effect is very likely a consequence of the glucocorticoid-like action of the synthetic hormone, which competes with the endogenous glucocorticoids for the hepatic receptor molecules.