Robert R. Cardell

Candida albicans translocation across the gut mucosa following burn injury.

Normal guinea pigs were challenged intragastrically with Candida albicans 1 hr prior to a 30 or 50% flame burn to determine if burn injury increased translocation of the yeasts across gut mucosa. Tissues were harvested between 3 and 24 hr postburn and cultured on Sabouraud dextrose agar. Control animals (no yeast challenge) showed no yeast in intestinal homogenates or in the mesenteric lymph nodes (MLN). At a dose of 1 X 10(9) yeasts, they did not escape from the gut lumen, with either a 30 or 50% burn. At a dose of 2 to 4 X 10(10) organisms, they translocated to the MLN in 92% of the 50%-burned animals (P less than 0.001), 75% of the 30%-burned animals (P less than 0.05), and 12.5% of unburned animals. The ileal mucosa appeared to be the most susceptible site for yeast invasion. To observe the penetration through the gut mucosa and/or translocation to other tissues, yeasts were labeled with biotin before administration, and tissues were stained with avidin-peroxidase diaminobenzidine sequence. With biotinylated yeasts, phagocytized organisms were observed in large numbers in the lamina propria and mesenteric lymph nodes but they were not viable upon culture. Toluidine blue staining of semithin sections revealed that translocated yeasts were located selectively in the lymphoid follicles of the MLN, entrapped by macrophages.

Hepatic lobular patterns of phosphoenolpyruvate carboxykinase, glycogen synthase, and glycogen phosphorylase in fasted and fed rats.

The goal of this study was to localize phosphoenolpyruvate carboxykinase (PEPCK), glycogen synthase (GS), and glycogen phosphorylase (GP) in the liver lobule by immunocytochemical techniques and to describe the effects of feeding and fasting on the distribution and quantity of these enzymes. Livers from ad lib fed and overnight fasted normal adult male rats were frozen in liquid nitrogen after transcardial perfusion with 30% sucrose. Serial cryostat sections of tissue were collected on slides, fixed by immersion in 4% paraformaldehyde, and incubated with antibodies against PEPCK, GS, and GP. Antibodies to these enzymes were visualized with a gold-conjugated secondary antibody and a silver enhancement technique. Fed animals demonstrated a periportal to pericentral gradient of PEPCK. Fasting increased the periportal content of PEPCK, induced the midlobular and centrilobular cells to express the enzyme, and steepened the periportal to pericentral gradient. The increase of PEPCK was confirmed by Western blot analysis. GS and GP were distributed throughout the lobule in the fed animal but often showed a centrilobular pattern, and fasting did not alter the lobular distribution of either enzyme. Western blot analysis revealed no changes in the amount of these enzymes in the fed or fasted state. The cellular distribution of the three enzymes is similar to that of hepatic glycogen, in that the immunoreactive material has a clumped appearance in the periportal hepatocytes and is more dispersed in the pericentral cells. On fasting the periportal hepatocytes lose the dense compact localization of the enzymes and the protein becomes more homogeneously distributed throughout the cytosol. Further studies are needed to elucidate the functional significance of the regional heterogeneity of the glycogen-metabolizing enzymes and the molecular mechanisms regulating their gene expression.

Maternal malnutrition does not affect fetal hepatic glycogen synthase ontogeny

Maternal malnutrition late in pregnancy results in the reduced storage of fetal hepatic glycogen in the final days of gestation and an accentuation of normal birth-related hypoglycemia. It was of interest to determine whether or not low glycogen levels resulted when maternal malnutrition disrupted the normal ontogeny of fetal hepatic glycogen synthase, an important glycogenic enzyme. A defect in this enzyme would be expected to seriously affect prenatal and postnatal glycogen synthesis. For this study, livers were removed from fetuses from malnourished (50% of normal dietary intake) mice, as well as fromad libitum-fed mice, and used for the determination of hepatic glycogen, glycogen synthase activity, and glycogen synthase protein levels. In this paper we report that maternal dietary restriction late in pregnancy produces growth-retarded fetuses with severely reduced hepatic glycogen levels, but the normal ontogenic changes in the quantity and activity of hepatic glycogen synthase were not affected. It is especially significant that the accumulation of glycogen synthase occurred despite the minimal level of natural substrate available for the enzyme. These results suggest that the accumulation and activity of hepatic glycogen synthase during late gestation is related to developmental events rather than levels of substrate or glycogen.

Appearance of a nonfunctional isozyme of hepatic glycogen synthase in late gestation

Glycogen levels, glycogen synthase activities, and glycogen synthase protein levels were determined in liver tissues obtained from 14- to 19-day-old fetal mice, newborn mice, and adult mice. The results of these experiments demonstrate a significant increase in the quantity of hepatic glycogen synthase beginning at Day 17 of gestation and reaching adult levels at birth. However, during the same time period, there is a dramatic decrease in total glycogen synthase activity suggesting that the accumulating glycogen synthase molecules are unable to transfer UDP-glucose to glycogen. These inversely coordinated changes in the quantity and activity of glycogen synthase are consistent with the suggestion that glycogen synthesis in the near-term fetal mouse is being maintained by preexisting enzyme, while accumulating enzyme molecules may represent a quiescent isozyme.

Endoplasmic Reticulum: Rough and Smooth

Publisher Summary This chapter discuses the morphology, biochemistry, and function of the endoplasmic reticulum (ER) in a variety of cell types. The ER maintains and generates its own membrane complex in addition to providing the components of membranes for other cellular compartments including plasma membrane, Golgi apparatus, lysosomes, and nuclear envelope. The location of the ER corresponds to the basophilic regions of the cell seen in the light microscope. The earliest observations of this organelle in thin sections described it as a continuous three-dimensional reticulum made up of a network of membrane-enclosed spaces in the form of tubules that were in continuity with the layers of flattened cisternae. The tubular elements free of ribosomes are referred to as smooth endoplasmic reticulum and the ribosome-studded component is called rough endoplasmic reticulum. Molecular genetics, monoclonal antibodies, in situ immunocytochemistry, complementary cDNA probes, as well as refinements in and development of new ultrastructural techniques, biochemical analyses, and laboratory equipment promise to greatly enhance current knowledge about the ER.

Optimal visualization of immunogold-silver staining of hepatic PEPCK with epipolarized light microscopy.

We used immunogold-silver staining to localize phosphoenolpyruvate carboxykinase in 10 microns cryosections of 4% paraformaldehyde perfusion-fixed normal male rat liver. The resolution and sensitivity of detection were improved by epipolarized light microscopy of 0.5 microns semi-thin plastic sections prepared from these pre-embedding immunogold-silver-enhanced 10-microns thick cryosections. Epipolarized light combined with transmitted light simultaneously demonstrated antigenic sites (visualized with epipolarized light illumination) and tissue morphology (revealed by transmitted light). To optimize the conditions for high resolution, an oil immersion objective lens (x 100) with adjustable iris diaphragm was used with different intensity settings for both light sources. Our observations indicate that if the intensity of the transmitted light is too high, the visibility of the gold-cored silver grains by epipolarized illumination is decreased; if the intensity of epipolarized light is too strong, haloes appear around the gold-cored silver particles. By adjusting the aperture in the objective lens and the neutral density filter in the transmitted light pathway to balance the intensities of transmitted and epipolarized light, an optimal image is obtained that shows the maximal number of antigenic sites and excellent morphology.

Morphometric analysis of hepatocytes from rats subjected to compound 48/80-induced anaphylactic shock.

Morphometric and biochemical techniques were used to analyze hepatic glycogen, endoplasmic reticulum, and mitochondrial matrix granules in rats treated with compound 48/80 to induce an anaphylactic-like state of shock. Thirty minutes after insult there was a significant decrease in glycogen and mitochondrial matrix granules, an increase in rough endoplasmic reticulum (RER), and no change in smooth endoplasmic reticulum (SER). Less glycogen in experimental rats substantiated a previously described glycogenolytic response to compound 48/80. The decrease in matrix granules implies a loss and/or shift in intramitochondrial calcium as occurs in epinephrine-induced glycogenolysis in the rat. Since other glycogenolytic agents, e.g. glucagon, and starvation stimulate an increase in SER presumably from RER, the present morphological data suggest the increase in RER may precede proliferation of SER from RER.