Immunogold-silver staining method for light and electron microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies

We used the immunogold-silver staining method (IGSS) for detection of lymphocyte cell surface antigens with monoclonal antibodies in light and electron microscopy and compared this procedure with the immunogold staining method. Two different sizes of colloidal gold particles (5 nm and 15 nm) were used in this study. Immunolabeling on cell surfaces was visualized as fine granules only by IGSS in light microscopy. The labeling density (silver-gold complexes/cell) and diameters of silver-enhanced gold particles on cell surfaces were examined by electron microscopy. Labeling density was influenced not by the enhancement time of the physical developer but by the size of the gold particles. However, the development of shells of silver-enhanced gold particles correlated with the enhancement time of the physical developer rather than the size of the colloidal gold particles. Five-nm gold particles enhanced with the physical developer for 3 min were considered optimal for this IGSS method because of reduced background staining and high specific staining in the cell suspensions in sheep lymph. Moreover, this method may make it possible to show the ultrastructure of identical positive cells detected in 1-micron sections counterstained with toluidine blue by electron microscopy, in addition to the percentage of positive cells by light microscopy.     

Hyperglycemia associated with increased hepatic glycogen phosphorylase and phosphoenolpyruvate carboxykinase in rats following subchronic exposure to malathion

We examined the effects of subchronic exposure to malathion, an organophosphorous (OP) insecticide, on plasma glucose and hepatic enzymes of glycogenolysis and gluconeogenesis in rats in vivo. Malathion was administered orally at doses of 100, 200 and 400 ppm for 4 weeks. At the end of the specified treatment (18 h fasting after the last dose of malathion), the liver was removed. The activities of glycogen phosphorylase (GP) and phosphoenolpyruvate carboxykinase (PEPCK) were analyzed in the homogenate. Four weeks administration of malathion at doses of 100 ppm, 200 ppm, and 400 ppm increased plasma glucose concentrations by 25% (P < 0.01), 17% (P < 0.01), and 14% (P < 0.01) of control, respectively. Malathion also increased hepatic PEPCK activity by 25% (100 ppm, P < 0.01), 16% (200 ppm, P < 0.01), and 21% (400 ppm, P < 0.01) of control, respectively. In addition, malathion increased hepatic GP by 22% (100 ppm, P < 0.01), 41% (200 ppm, P < 0.01), and 32% (400 ppm, P < 0.01) of controls. We conclude that exposure of rats to malathion as a widely used OP in subchronic exposure, which resembles human exposure, may induce diabetes associated with stimulation of hepatic gluconeogenesis and glycogenolysis in favor of glucose release into the blood. The possible mechanisms including increased energy production to detoxification, depressed paraoxonase activity, and increased production of cyclic nucleotides are discussed.     

Postmaturity in the rat: phosphorylase, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase activities in the fetal liver.

Liver glucose-6-phosphatase and phosphoenolpyruvate carboxykinase activities were increased in the postmature rat fetus (23.5 day old) when compared to term rat fetus (21.5 day old). Postmaturity was without effect on liver phosphorylase activity. The three liver enzyme activities were also determined in normal 2 day old neonates. These results are correlated with the mobilisation of fetal liver glycogen occuring during postmaturity in the rat and are discussed in relation to the secretory pattern of the pancreatic hormones.     

Electron microscopic demonstration of glycogen and phosphorylase activity in rat hepatocytes

Synopsis The effect of fixation with a bicarbonate-buffered solution of paraformaldehyde and polyvinyl pyrrolidone (PVP) on the ultrastructural demonstration of glycogen and phosphorylase activity in rat hepatocytes has been studied. Phosphorylase was demonstrated by the precipitation of liberated phosphate ions with ferrous ions. 7.5% PVP was included in all steps in the procedure before post-fixation in osmium tetroxide.Glycogen particles were well preserved. Structures connecting membranes and glycogen particles were also evident. Phosphorylase activity was rapidly inhibited by the fixative; the fixation time was, therefore, kept very short. The final reaction product was localized on glycogen particles and on endoplasmic membranes in association with glycogen particles. The results support the view that endoplasmic membranes are involved in the metabolism of glycogen in hepatocytes.Paper presented at a symposium The changing directions of carbohydrate histochemistry at the Fifth International Congress of Cytochemistry and Histochemistry in Bucharest, Romania on 1 September 1976.     

Electron microscopic demonstration of rat liver glycogen phosphorylase activity with iron (Fe++)

Summary In this study a new electron microscopic method for the demonstration of liver glycogen phosphorylase activity has been presented.Prior to incubation the liver samples were shortly fixed in cold paraformaldehyde. Inorganic phosphate, liberated in the reaction catalyzed by the enzyme, were precipitated with iron (Fe⁺⁺) present in the incubating medium. Postfixation was performed in glutaraldehyde and osmium tetroxide.The ferrous phosphate precipitate was detected electron microscopically in unstained sections.The precipitate was mainly localized to endoplasmic membranes but also in glycogen particles. The method is imperfect in demonstrating phosphorylase activity bound to glycogen particles because of poor preservation of glycogen during treatment.     

Fluorescent labeling of the nucleotide site in cytosolic rat liver phosphoenolpyruvate carboxykinase

Reaction of rat liver phosphoenolpyruvate carboxykinase (GTP: oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32) with the alkylating fluorescent probe N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (1,5-I-AEDANS), results in complete loss of enzymatic activity. One mole of the fluorescent reagent is incorporated per mole of the inactivated enzyme. When the modification is carried out in the presence of GDPMn, the enzyme retains 97% of its activity with almost no incorporation of label. The specificity of the reaction is further supported by the detection of a unique fluorescent peptide from the trypsin-treated modified enzyme. Fluorescence emission of enzyme-bound AEDANS shows a broad band centered at 470 nm and presents a monoexponential decay with a lifetime of 19 ns. These data indicate that the probe-binding site is considerably less polar than water and similar in polarity to ethanol. Anisotropy determinations give evidence for restricted rotational freedom for AEDANS bound to the rat carboxykinase, while acrylamide quenching studies reveal limited accessibility to the probe site. The results are consistent with specific labeling of rat liver phosphoenolpyruvate carboxykinase at or near the GDP site. The characteristics of the nucleotide-binding sites of rat liver and yeast (ATP) phosphoenolpyruvate carboxykinase are compared.     

Daily rhythms of glycogen synthetase and phosphorylase activities in rat liver: Influences of food and light

In rats fed during the night time (from 8 p.m. to 8 a.m.) the activities of liver glycogen synthetase $\text{I}$ and phosphorylase $\text{a}$ varied rhythmically during a 24 hour period. There was an inverse relationship between their levels; the level of synthetase $\text{I}$ rose to a maximum at around 6 a.m. and that of phosphorylase $\text{a}$ attained the peak value at around 6 p.m. Eye enucleation of rats did not affect significantly the daily rhythms of the enzymes. However, when food was offered only during the day time, the phases of both enzyme rhythms were shifted by about 12 hours. On starvation for 24 hours, the glycogen level was reduced almost to nil, but the daily rhythms of the enzymes were retained. It is thus very likely that the daily variations of the enzyme activities are not merely a passive effect of food intake, and that food can be a synchronizer or zeitgeber which sets up the characteristic rhythms of glycogen metabolism in the liver.     

A reactive cysteine in avian liver phosphoenolpyruvate carboxykinase

The modification of avian phosphoenolpyruvate carboxykinase by a variety of sulfhydryl reagents leads to inhibition. The inhibition is related to the loss of 1 highly reactive cysteine residue of the 13 cysteines present in the enzyme. Inhibition by reagents which yield a mixed disulfide was rapidly reversed by thiols. Reagents specific for vicinal sulfhydryl configurations were not potent inhibitors. The cysteine-modified enzyme continues to bind Mn2+ with the same stoichiometry and dissociation constant as the native enzyme. All of the substrates also bind to thiol-modified inactive enzyme. The modification of the reactive cysteine with the spin-labeled iodoacetate derivative leads to inactive enzyme with spin label stoichiometrically incorporated. The EPR spectrum showed an immobilized spin label on the enzyme. EPR studies of the perturbation of the phosphoenolpyruvate carboxykinase-bound spin label by bound Mn2+ showed a dipolar interaction between the two spins, estimated to be 10 A apart. The perturbation of the 1/T1 and 1/T2 values of the 31P resonances of ITP by spin-labeled enzyme indicates that this portion of the nucleotide binds 8-10 A from the spin label. These results indicate that the reactive cysteine is close to but not at the active site of the enzyme. The thiol group must be free and in its reduced form for the enzyme to be active. Perhaps modification of this group prevents conformational change(s) upon ligand binding necessary for the catalytic process.     

Reduction in phosphoenolpyruvate carboxykinase in rat liver parenchymal cells following experimentally induced cholestasis

The effect of experimentally induced cholestasis on the amount of phosphoenolpyruvate carboxykinase (PEPCK) was studied immunohistochemically in rat liver parenchyma. In control liver, the enzyme was mainly localized periportally and, although the enzyme content was much reduced, this distribution pattern was maintained up to 2 weeks after ligation of the common bile duct. At 4 and 8 weeks after ligation the enzyme content in parenchymal cells remained low, but became distributed homogeneously throughout the liver parenchyma. This suggests that after bile duct ligation, gluconeogenesis from lactate is impaired. This may well be the cause of the adaptive changes to enhance the glycogenolytic capacity of parenchymal cells to maintain as far as possible a constant blood glucose level.