Glucose-6-phosphate dehydrogenase in small intestine of rabbit: biochemical properties and subcellular localization.

Biochemical properties and cellular and subcellular distribution patterns of glucose-6-phosphate dehydrogenase (G6PD) were investigated in small intestine of rabbits. The specific activity of G6PD in fresh homogenates of small intestine was 19 +/- 9 IU/g protein. This value did not change significantly after dialysis. The kinetic and electrophoretic properties of the partially purified enzyme were similar to those found in other rabbit tissues. Enzyme histochemical analysis of G6PD activity using the tetrazolium salt method showed high activity in epithelial cells of villi and crypts of Lieberkuhn. The activity in acinar cells of Brunner's glands was lower than that in epithelium, whereas cells of the muscularis externa showed a very low activity. Immunohistochemical analysis showed that the amounts of G6PD protein were lower in the epithelium than in Brunner's glands and muscularis externa. The differences between distribution patterns of activity and protein of G6PD may reflect the presence of inactive enzyme molecules in Brunner's glands and muscularis externa or posttranslational activation of G6PD in epithelium. Electron microscopic immunocytochemical analysis performed with gold-labelled antibodies showed the presence of G6PD protein throughout the cytoplasm and at smooth endoplasmic reticulum in enterocytes. In Paneth cells and cells of Brunner's glands, G6PD was found in the cytoplasm, at rough endoplasmic reticulum and Golgi complex. Immunolabelling was not found in mitochondria or nuclei. Our findings show that G6PD is heterogeneously distributed in cells of the small intestine and that the enzyme is associated with rough and smooth endoplasmic reticulum to support synthetic functions in these compartments by NADPH production.

Heterogeneous distribution of glucose-6-phosphate dehydrogenase in lingual epithelium.

Lingual epithelium undergoes oxidative stress and apoptosis with consequent renewal of superficial keratinized cells by proliferation and differentation of the stem cells of the basal germinative layer. In 3 distinct areas of lingual epithelium of rat and rabbit, the anterior third, central third and posterior third, we determined the activity of hexose monophosphate shunt enzymes and antioxidant enzymes, which are essential for support of cell proliferation and differentation. Enzymatic assays of the epithelium showed that glucose-6-phosphate dehydrogenase (G6PD) activity was highest in the anterior third, whereas activity of glutathione peroxidase, 6-phosphogluconate dehydrogenase, glutathione reductase, superoxide dismutase and catalase was similar over all areas. Histochemical localization of activity and immunohistochemical localization of protein of G6PD showed that all types of papillae had a similar G6PD content; moreover, the presence of different G6PD isoforms in the 3 areas was excluded by electrophoretic analysis. We conclude that the higher G6PD activity in the anterior part of the epithelium is due only to the anatomical organization of the epithelial surface of this area, in which many filiform and fungiform papillae are arranged in a compact manner, which corresponds with a higher number of proliferating and differentiating cells. These processes need products of G6PD activity. This study indicates that G6PD is a good marker for the number of differentiating cells in tongue epithelium.

Iron release and oxidant damage in human myoblasts by divicine.

Divicine is an aglycone derived from vicine, a glucosidic compound contained in fava beans (Vicia faba major or broad beans). In this study, we investigated the effect of divicine on cultured human myoblasts from normal subjects, in order to see if the drug may induce signs of oxidant stress in these cells. Myoblasts incubated 24 hours in the presence of 1 mM divicine, showed an increase of carbonyl groups and 4-hydroxynonenal (4-HNE) bound to cell proteins, as well as a significant release of iron and lactate dehydrogenase in the culture medium. Desferrioxamine (DFO), an iron chelator, significantly prevented protein oxidation and formation 4-HNE adducts. Our results can be interpreted as indicating that divicine autooxidizes both at extracellular level and into myoblasts thus inducing the release of free iron, which initiates oxidation of cellular proteins and lipids. DFO protects the cells by subtracting the free iron both at intracellular and extracellular level.

High glucose-6-phosphate dehydrogenase activity contributes to the structural plasticity of periglomerular cells in the olfactory bulb of adult rats.

Glucose-6-phosphate dehydrogenase (G6PD) activity, assayed spectrophotometrically, was found to be higher in the olfactory bulb (OB) than in other brain areas of adult rats [P. Ninfali, G. Aluigi, W. Balduini, A. Pompella, Glucose-6-phosphate dehydrogenase is higher in the olfactory bulb than into other brain areas, Brain Res. 744 (1997) 138-142]. Histochemical demonstration of G6PD activity in cryostat sections of OB, analyzed with optical microscopy, revealed a marked and well defined line of formazan deposition in the internal part of the glomerular layer (Glm), indicating that G6PD was much higher in cells distributed along the glomeruli. Electron microscope analysis showed that G6PD activity was mainly concentrated in cytoplasm and dendrites of periglomerular cells, the interneurons which span glomeruli and connect olfactory nerves with mitral/tufted cells. Since G6PD regulates the flux through the hexose monophosphate shunt (HMS) pathway, which provides NADPH for reductive biosynthesis and pentose phosphates for nucleic acid formation, it can be concluded that high G6PD activity in periglomerular neurons is functional to their differentiating capability. This result is consistent with the occurrence of structural plasticity events in the OB of adult rats.

Postnatal expression of glucose-6-phosphate dehydrogenase in different brain areas.

The activity of glucose-6-phosphate dehydrogenase (G6PD) was studied in five brain areas of rats aged 5 to 90 days. The areas studied were: the olfactory bulb (OB), cortex, hippocampus, striatum and septum. The G6PD activity increased more than 2-fold from 5 to 90 days in the OB, while it was almost constant in the other areas. At every stage of development, the G6PD activity was significantly higher in the OB than in the other areas. The G6PD pattern was compared with 6-phosphogluconate dehydrogenase (6PGD), glutathione reductase (GR); glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD) in order to find synergistic interactions among activities of these enzymes during development. Over the considered period, the activity of 6PGD increased significantly in the OB, while no significant difference in activity was detected in the other areas. GR increased significantly and progressively at each developmental stage in all areas. GPX showed a progressive increase in the OB, while in other areas a significant increase was detected at 90 days only. CAT and SOD showed a different and independent pattern which differed from the G6PD pattern. CAT showed the highest level of activity at 5 days then progressively decreased or was constant until 90 days; SOD had the highest value at 5 days, than it decreased at 10 days and increased from 10 to 90 days. In all areas, G6PD activity showed three electrophoretic bands, whose relative activity changed with development. At histochemical level, we found a marked G6PD activity in the periglomerular zone of the OB, which increased with age, while other areas showed a homogeneous staining. The present results demonstrate that G6PD activity increases in the OB during the developmental stages and there is a coordinated simultaneous activation of 6PGD, GPX and GR. It is likely that this enzyme induction increases the antioxidant defense of periglomerular cells that are subject to a rapid renewal and thus much more exposed to oxidant stress.

Variability of oxygen radical absorbance capacity (ORAC) in different animal species.

The oxygen radical absorbance capacity (ORAC) was measured both in whole (ORAC-T) and deproteinized (ORAC-AS) plasma samples of human, pig, cow, rabbit, dog, cat, sheep, horse, dolphin, turkey, guinea-hen and chicken. In the 12 species, ORAC-T data, expressed as micromoles of peroxyl radicals trapped by 11 of sample, were found scattered between 8,600 and 23,000 micromol/l. The species with the highest ORAC-T values were cat among mammals and chicken among avies. ORAC-AS values ranged between 600 and 2000 micromol/l, with the highest values found in dolphin and sheep among mammals, while chicken was first among avies. In the 12 species, the relative contribution of ORAC-AS in relation to ORAC-T ranged from 5% to 20%. Protein SH-groups and uric acid were measured in plasma of all species, but no significant correlation was found between thiols and ORAC-T values or between uric acid and ORAC-AS values. Our results show that: (1) the ORAC method is reproducible and sensitive enough to be used in the comparison of the peroxyl-radical absorbance capacity of protein and non-protein plasma components in different animal species; (2) both in mammals and in avies, there is a deep intra-class heterogeneity of ORAC-T and ORAC-AS values; (3) by considering most species, plasma proteins and lipoproteins account for about 85-90% of the overall peroxyl-radical trapping capacity. In the dolphin only, the protein contribution decreases to 80%; (4) uric acid accounts for about one-half of the ORAC-AS value in human, guinea-hen and for about one-third in chicken, while it provides a very limited contribution in other species. We conclude that species with the highest ORAC-T, like cat and chicken, or with the highest ORAC-AS, like dolphin, are interesting models to study the reasons of such a marked antioxidant defense in the plasma.

Methods for studying the glucose-6-phosphate dehydrogenase activity in brain areas.

This paper reports on the protocols for the spectrophotometric determination of the enzymatic activity, the electrophoretic pattern and the cytochemical assay of glucose-6-phosphate dehydrogenase (G6PD) in different areas of rat brain. For the spectrophotometric assay we used the method of Glock and McLean. Non-denaturing polyacrylamide gel electrophoresis was performed using 1.5 mm thick slab-gel, with a 7.5% acrylamide separating gel and a 4% acrylamide stacking gel. NADP (0.01 mM) was included at the cathode buffer. The cytochemical assay was performed on unfixed cryostat sections, by incubating the slides for 5 min at 37 degrees C in the staining medium. A combination of these techniques offers a means for the study of the effect of age, drugs, oxidizing or reducing agents on the G6PD activity in different brain areas.

Glucose-6-phosphate dehydrogenase activity is higher in the olfactory bulb than in other brain areas.

The activity of antioxidant enzymes was measured in the olfactory bulb (OB) of rat and compared with cortex, hippocampus, striatum and septum. Glutathione reductase, glutathione peroxidase, catalase and superoxide dismutase were not significantly different in the five brain areas, while glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase activities were four times higher in the OB than in the other areas. This picture prompted us to explore the reasons of the marked increase of G6PD, since it is the enzyme that regulates the operation of the hexose monophosphate shunt. A first approach was to analyze the G6PD electrophoretic pattern. The analysis revealed that the high G6PD activity of the bulb was neither due to new isoenzymes nor to a modification of the equilibrium between the G6PD dimers. We secondly hypothesized an induction of G6PD activity in the OB by oxidant stress. The assay of markers of the oxidant stress, such as thiobarbituric acid reactive substances, oxidized and reduced glutathione, did not confirm this hypothesis. A third approach was the cytochemical analysis of cryostat sections of OB. By this method we identified a particular cell type which was very rich in G6PD and located at the border of the glomerular layer. Thus, we attributed the high G6PD activity of the OB to the consistent presence of periglomerular cells, that probably need a high G6PD activity for their regulatory function in the neurochemical transmission.

Inhibition of murine AIDS by combination of AZT and dideoxycytidine 5'-triphosphate.

SUMMARY: A combination of antiretroviral drugs acting on different cell types (lymphocytes and macrophages) was evaluated in a murine retrovirus-induced immunodeficiency model of AIDS (MAIDS). In a first experiment, C57BL/6 mice were infected with a single i.p. administration of LP-BM5 and treated with 0.125 or 0.25 mg/ml AZT in drinking water for 3 months. AZT treatment was found to reduce lymphadenopathy (60 and 65 percent, respectively), splenomegaly (37 and 50 percent, respectively), and hypergammaglobulinemia (6 and 50 percent, respectively). Furthermore, at the highest AZT concentration, BM5d proviral DNA content in lymph nodes and in the spleen showed a reduction of 78 and 70 percent, respectively, compared to untreated animals. In a second experiment, infected mice were treated with AZT (0.25 mg/ml in drinking water) and with 2',3'-dideoxycytidine 5'-triphosphate (ddCTP) encapsulated into autologous erythrocytes for macrophage protection. Combined treatments resulted in a further reduction of lymphadenopathy (a further 33 percent with respect to the single treatment of AZT) and splenomegaly (a further 28 percent respect to the single treatment of AZT) but not of gammaglobulinemia. Proviral DNA in lymph nodes and spleen showed a reduction of 82 and 77 percent, respectively, compared to infected mice. Stimulation index of T cells was also significantly increased in animals receiving both treatments versus AZT only. In conclusion, the selective administration of antiviral drugs that preferentially protect different cell types seems to provide additional advantages compared to single-agent therapy.