Glutathione reductase fromSaccharomyces cerevisiae undergoes redox interconversionin situ andin vivo

Redox interconversion of glutathione reductase was studiedin situ withS. cerevisiae. The enzyme was more sensitive to redox inactivation in 24 hour-starved cells than in freshly-grown ones. While 5 μM NADPH or 100 μM NADH caused 50% inactivation in normal cells in 30 min, 0.75 μM NADPH or 50 μM NADH promoted a similar effect in starved cells. GSSG reactivated the enzyme previously inactivated by NADPH, ascertaining that the enzyme was subjected to redox interconversion. Low EDTA concentrations fully protected the enzyme from NADPH inactivation, thus confirming the participation of metals in such a process. Extensive inactivation was obtained in permeabilized cells incubated with glucose-6-phosphate or 6-phosphogluconate, in agreement with the very high specific activities of the corresponding dehydrogenases. Some inactivation was also observed with malate, L-lactate, gluconate or isocitrate in the presence of low NADP⁺ concentrations. The inactivation of yeast glutathione reductase has also been studiedin vivo. The activity decreased to 75% after 2 hours of growth with glucono-δ-lactone as carbon source, while NADPH rose to 144% and NADP⁺ fell to 86% of their initial values. Greater changes were observed in the presence of 1.5 μM rotenone: enzymatic activity descended to 23% of the control value, while the NADH/NAD⁺ and NADPH/NADP⁺ ratios rose to 171% and 262% of their initial values, respectively. Such results indicate that the lowered redox potential of the pyridine nucleotide pool existing when glucono-δ-lactone is oxidized promotesin vivo inactivation of glutathione reductase.     

Oscillations in glycolysis: multifactorial quantitative analysis in muscle extract

A multifactorial quantitative analysis of oscillations in glycolysis was conducted in the postmicrosomal supernatant of rat muscle homogenates incubated in the presence of yeast hexokinase. Oscillations in adenine nucleotides, D-fructose 1,6-bisphosphate, triose phosphates, L-glycerol 3-phosphate, ³HOH generation from D-[5-³H]glucose, NADH and L-lactate production were documented. The occurrence of such oscillations were found to depend mainly on the balance between the consumption of ATP associated with the phosphorylation of D-glucose, as catalyzed by both yeast and muscle hexokinase, and the net production of ATP resulting from the further catabolism of D-fructose 6-phosphate, as initiated by activation of phosphofructokinase. The oscillatory pattern was suppressed in the presence of D-fructose 2,6-bisphosphate. It is proposed that the quantitative information gathered in this study may set the scene for further studies in extracts of cells other than myocytes, e. g. hepatocytes and pancreatic islet cells, in which no oscillation of glycolysis was so far observed.     

Enzyme-linked immunosorbent assays for the measurement of blood group A and B glycosyltransferase activities

ELISA assays have been developed for (1–3)N-acetylgalactosaminyltransferase (blood group A transferase) and (1–3)galactosyltransferase (blood group B transferase) activities. In these assays, microtitre plates coated with the bovine serum albumin conjugate of a synthetic Fuc1–2Gal-R acceptor substrate are incubated with the appropriate nucleotide donor (UDP-GalNAc or UDP-Gal) and human serum as the enzyme source. The resulting trisaccharide products Fuc1–2(GalNAc1–3)Gal-R-BSA or Fuc1–2(Gal1–3)Gal-R-BSA are detected and quantified with monoclonal antibodies selected not to cross-react with the substrate structure. With less than a microliter of human serum, product formation is proportional to enzyme concentration and to time of incubation of up to 90 min.     

The effects of aflatoxin B1 on immature germinating maize (Zea mays) embryos

Immature maize (Zea mays L.) embryos were treated with aflatoxin B₁ concentrations, ranging from 0.1 g ml^–1 to 25 g ml^–1. Below 5 g ml^–1 aflatoxin B₁, root and shoot elongation was not significantly inhibited. Ultrastructurally, root tip cells showed little deterioration, except a possible diffused clearing in mitochondria and plastids. As the toxin concentration was increased above 5 gml^–1, shoot, and particularly root elongation, was progressively inhibited. Associated with this, there was an apparent decrease in the ribosome population. Furthermore, membranes, particularly the vacuolar membrane, became abnormal and vacuolar distension occurred. At 20 and 25 g ml^–1, these effects were exacerbated, and mitochondria and plastid structure was disrupted. At these concentrations, there was evidence of a disruption in lipid metabolism. The results are discussed in the context of known aflatoxin effects on cellular control mechanisms and ultrastructure in animal systems.     

Role of cell-mediated immunity in the resistance to experimental sporotrichosis in mice

The in vitro activity of several new imidazoles, cloconazole, sulconazole, butoconazole, isoconazole and fenticonazole, were compared with those of amphothericin B, flucytosine, and three azoles: econazole, miconazole and ketoconazole against isolates of pathogenic Candida. A total of 186 clinical isolates of 10 species of the genus Candida and two culture collection strains were tested by an agar-dilution technique. Isoconazole was the most active azole, followed by butoconazole and sulconazole. Differences between some of the species in their susceptibility to the antifungal agents were noted. Sulconazole and cloconazole had the highest activity in vitro against 106 isolates of C. albicans. Butoconazole and isoconazole were also very active against isolates of C. albicans, and were the most active azole compounds against 80 isolates of Candida spp.     

Tremorgenic mycotoxins produced by strains of Penicillium spp. isolated from toxic Poa huecu parodi

Seventeen strains of Penicillium spp. have been isolated from Poa huecu Parodi from the Zapala zone, exhibiting toxicity to sheet. The following strains have been identified: P. crustosum, cyclopium, notatum, palitans, puberulum, verrucosum, viridicatum and Penicillium spp. The toxigenic capacity of the strains was studied after growing them under suitable conditions. Toxins produced were analysed by thin layer chromatography (TLC). Penitrem A (PA) and Penitrem B (PB) neurotoxins were identified and quantitated in twelve strains; verruculogen (VERR) and fumitremorgen B (FTB) being present in one of them. The effect of these mycotoxins was studied in mice. Neurological symptoms characteristic of the intoxication by tremorgenic toxins and similar to those observed in sheep suffering from huecu's disease were observed. The possible role of these toxins as causative agents of huecu's disease is discussed.     

Distribution of histamine in the rat kidney during pregnancy and development

Summary An antiserum against conjugated histamine and two oligonucleotide probes that detect the mRNA encoding L-histidine decarboxylase (HDC) involved in histamine synthesis were used to study the appearance of histamine and its location in the kidneys of fetal, newborn and young postnatal rats and in the kidneys of pregnant rats. On embryonic days 16 and 18 (E16 and E18), some HA-immunoreactive (HA-ir) cells were found within the largest S-shaped bodies. Histamine was found to appear rapidly between the 18th and 20th embryonic days in the convoluted tubules of the kidneys. On postnatal day 0 (P0), the distal convoluted tubules and collecting ducts exhibited bright fluorescence, the intensity of which decreased quickly so that it was faint on day P4 and absent at later stages. In kidneys of pregnant rats HA-ir was found in the epithelium of both the Bowman's capsule, collecting ducts and in a few cells within the tubules. Nonuniform HA-ir was also detected within glomeruli. No evidence for the presence of L-histidine decarboxylase mRNA in kidneys of fetuses or pregnant rats was seen. It is concluded that distinct structures in the developing rat kidney contain histamine during a period around birth from day E20 to day P4. In the pregnant rat, the epithelium that is in direct contact with the urine flow is immunoreactive for histamine from day 16 to 20 of pregnancy. The results suggest that histamine is not synthesized locally in the kidneys but rather originates from other tissues.     

Modification of blood group A antigen expression in a pancreatic cancer cell line (PC-1) by inhibitors of N-glycan processing.

Pancreatic adenocarcinomas induced in Syrian hamsters by treatment with N-nitrosobis(2-oxopropyl) amine express blood group A antigen, which is absent in normal pancreatic cells. On membrane glycoproteins purified from tumors, blood group A antigen has been found to be expressed on multiantennary Asn-linked complex glycans. In this study, we investigated the effect of inhibitors of Asn-glycan processing on blood group A antigen bearing glycan structures in a cell line (PC-1) established from a primary induced pancreatic cancer. Expression of blood group A antigen on cells and in membrane preparations was blocked by treatment with 1-deoxymannojirimycin, an inhibitor of mannosidase I, but was retained after treatment with swainsonine, an inhibitor of mannosidase II. However, swainsonine treatment altered the glycan structure associated with blood group A antigen from an endoglycosidase H resistant type to a sensitive type, indicating that the blood group A structure might shift from a complex type to a hybrid type glycan by this treatment. These results demonstrate that Asn-linked glycans carry the major blood group A antigens in PC-1 cells.     

Indirect chiral separation and analyses in human biological fluids of the stereoisomers of a thienothiopyran-2-sulfonamide (TRUSOPT), a novel carbonic anhydrase inhibitor with two chiral centers in the molecule.

The indirect chiral separation of the four stereoisomers (1)-(4) of a novel carbonic anhydrase inhibitor with two chiral centers in the molecule is reported. The method is based on chemical derivatization of the secondary amino group of the inhibitor with chiral isocyanate, formation of diastereomeric urea derivatives, each with three chiral centers in the molecule, and their separation under nonchiral HPLC conditions. The attempts to separate racemic mixture (1) + (2) from its diastereomeric counterpart (3) + (4) under nonchiral conditions, and to separate enantiomers (1) and (2) directly on a chiral stationary phase (CSP) are also reported. The indirect method was utilized for the assessment of an in vivo inversion of configuration at either one or both chiral centers of the molecule of (1). Analyses of selected whole blood and urine samples from human subjects after multiple bilateral topical ocular dosing with (1) did not reveal the presence of any of the three possible stereoisomers (2)-(4) of (1) indicating that the inversion of configuration at neither one nor two chiral centers of (1) occurs in vivo.