Transformation ofAspergillus flavus to study aflatoxin biosynthesis

Aflatoxin contamination of agricultural commodities continues to be a serious problem in the United States. Breeding for resistant genotypes has been unsuccessful and detoxification of food sources is not economically feasible. New strategies for control may become apparent once more is known about the biosynthesis and regulation of aflatoxin. Although the biosynthetic pathway of aflatoxin has been extensively studied, little is known about the regulation of the individual steps in the pathway. We have developed a genetic transformation system forAspergillus flavus that provides a new and expedient approach to studying the biosynthesis of aflatoxin and its regulation. Through the use of this genetic transformation system, genes for aflatoxin biosynthesis can be identified and isolated by the complementation of aflatoxin negative mutants. In this paper we discuss molecular strategies for studying the regulation and biosynthesis of aflatoxin.     

Mn2+ reduces Yz + in manganese-depleted Photosystem II preparations

Manganese in the oxygen-evolving complex is a physiological electron donor to Photosystem II. PS II depleted of manganese may oxidize exogenous reductants including benzidine and Mn²⁺. Using flash photolysis with electron spin resonance detection, we examined the room-temperature reaction kinetics of these reductants with Y_z ⁺, the tyrosine radical formed in PS II membranes under illumination. Kinetics were measured with membranes that did or did not contain the 33 kDa extrinsic polypeptide of PS II, whose presence had no effect on the reaction kinetics with either reductant. The rate of Y_z ⁺ reduction by benzidine was a linear function of benzidine concentration. The rate of Y_z ⁺ reduction by Mn²⁺ at pH 6 increased linearly at low Mn²⁺ concentrations and reached a maximum at the Mn²⁺ concentrations equal to several times the reaction center concentration. The rate was inhibited by K⁺, Ca²⁺ and Mg²⁺. These data are described by a model in which negative charge on the membrane causes a local increase in the cation concentration. The rate of Y_z ⁺ reduction at pH 7.5 was biphasic with a fast 400 s phase that suggests binding of Mn²⁺ near Y_z ⁺ at a site that may be one of the native manganese binding sites.Abbreviations PS II Photosystem II - Y_D tyrosine residue in Photosystem II that gives rise to the stable Signal II EPR spectrum - Y_z tyrosine residue in Photosystem II that mediates electron transfer between the reaction center chlorophyll and the site of water oxidation - ESR electron spin resonance - DPC diphenylcarbazide - DCIP dichlorophenolindophenol     

Higher order structure is present in the yeast nucleus: autoantibody probes demonstrate that the nucleolus lies opposite the spindle pole body

A panel of sera from 892 autoimmune patients was screened by indirect immunofluorescence on mammalian cells. Seventy-three sera were identified that recognize the nucleolus. Three of these sera appear to stain the nucleolus in yeast, suggesting that they recognize highly conserved antigens. These three sera also immunoprecipitate mammalian U3 snRNA-containing particles, which reside in the nucleolus and have been implicated in rRNA processing. Double immunofluorescence experiments with anti-nucleolus and anti-tubulin antibodies revealed a novel form of non-random nuclear organization in yeast. The spindle pole body and the nucleolus — both of which are associated with the nuclear envelope — preferentially localize at opposite ends of the nucleus. Organization of these and other components into specific regions of the nucleus may be important for optimizing their proper function.     

Effect of acetaminophen on hepatic content and biliary efflux of glutathione disulfide in mice

The increased expiration of ethane and pentane by mice treated with hepatotoxic doses of acetaminophen suggests the possibility of oxidant mechanisms associated with the necrosis. However, studies in rats are not consistent with oxidant stress mechanisms causing the damage, because acetaminophen given to rats does not increase GSSG efflux, a sensitive index of intrahepatic oxidant stress. To compare the extent of oxidant stress generated by acetaminophen in mice versus rats, hepatic content and biliary efflux of GSSG and GSH in mice have been examined. Bile was collected from anesthetized male ICR mice before and after intraperitoneal administration of acetaminophen (325 mg/kg, 2.15 mmol/kg), t-butyl hydroperoxide (TBHP) (1.5 mmol/kg), diethyl maleate (400 mg/kg, 2.33 mmol/kg, in corn oil) or saline (control) and GSH and GSSG were measured by the enzymatic recycling method of Tietze. An increase in biliary GSSG efflux was produced by t-butyl hydroperoxide, but not by the other agents. Biliary GSH/GSSG ratios decreased in acetaminophen-treated animals, presumably reflecting the marked depletion of hepatic GSH, since a similar decrease was observed with non-hepatotoxic doses of diethyl maleate. The failure of acetaminophen to increase the hepatic content or biliary efflux of GSSG in ICR mice is not consistent with the view that oxidant stress mechanisms cause the damage, despite the increases in alkanes expired after acetaminophen administration in this specific animal model.     

Bacteriophage Transport in Sandy Soil and Fractured Tuff

Bacteriophage transport was investigated in laboratory column experiments using sandy soil, a controlled field study in a sandy wash, and laboratory experiments using fractured rock. In the soil columns, the phage MS-2 exhibited significant dispersion and was excluded from 35 to 40% of the void volume but did not adsorb. Dispersion in the field was similiar to that observed in the laboratory. The phage f2 was largely excluded from the porous matrix of the two fractured-rock cores studied, coming through 1.2 and 2.0 times later than predicted on the basis of fracture flow alone. Because of matrix diffusion, nonsorbing solutes were retarded by over a factor of three relative to fracture flow. The time for a solute tracer to equilibrate with the porous matrix of 6.5-cm-diameter by 25-cm-long cores was measured in days. Results of both granular-medium and fractured-rock experiments illustrate the inability of a solute tracer to provide estimates for dispersion and effective porosity that are applicable to a colloid. Bacteriophage can be used to better estimate the maximum subsurface transport rate of colloidal contaminants through a porous formation.     

Interferon effects upon fluorouracil metabolism by HL-60 cells

In order to better understand the synergistic antiproliferative effects of interferon in combination with fluorouracil (FUra), we studied effects of alpha 2-interferon upon FUra induced inhibition of thymidylate synthase of HL-60 cells. The 50% inhibitory dose for FUra decreased from approximately 75 microM to 10 microM following interferon treatment, as measured by whole cell activity assays. Enhanced FUra inhibition of cytosolic [3H] - FdUMP binding of interferon treated cells was also noted. FdUMP accumulation following FUra treatment increased over 10 fold in interferon treated cells, but dUMP did not increase. These results suggest that interferon can sensitize cells to FUra inhibition of thymidylate synthase by enhancing accumulation of FdUMP.     

Molecular genetics of rotifers: preliminary restriction napping of the mitochondrial genome of Brachionus plicatilis

Methods are presented to extract and purify mitochondrial DNA from the rotifer Brachionus plicatilis. The mtDNA obtained is of sufficient purity for digestion with restriction endonucleases. EcoR I restriction patterns are presented for 4 geographically separated clones. A restriction map based on digestion with 5 different restriction enzymes is included for one of these clones. Finally, use of mtDNA analysis for studies on the population structure and biogeography of rotifers is discussed.     

A re-appraisal of two members of the genus Notholca from the Andes,with a note on the fine structure of the lorica of N. foliacea (Ehrenberg)

Rotifers described from the Andes by Murray (1913) and De Beauchamp (1939) as Notholca foliacea (Ehrenberg) are reviewed and re-assessed as Notholca walterkostei De Paggi, 1982. They are compared with N. foliacea and details of the lorica of this species seen with the scanning electron microscope are presented.     

Pregnancy-associated changes in oligomannose oligosaccharides of human and bovine uromodulin (Tamm-Horsfall glycoprotein)

The urinary glycoprotein uromodulin (Tamm-Horsfall glycoprotein) exhibits a pregnancy-associated ability to inhibit antigen-specific T cell proliferation, and the activity is associated with a carbohydrate moiety [Muchmore and Decker (1985) Science 229:479–81; Hessionet al., (1987) Science 237:1479–84; Muchmore, Shifrin and Decker (1987) J Immunol 138:2547–53]. We report here that the Man₆₍₇₎GlcNAc₂-R glycopeptides derived from uromodulin inhibit antigen-specific T cell proliferation by 50% at 0.2–2 M, and further studies, reported elsewhere, confirm that oligomannose glycopeptides from other sources are also inhibitory, with Man₉GlcNAc₂-R the most inhibitory of those tested [Muchmoreet al., J Leukocyte Biol (in press)]. In this work, we have extended the observation of pregnancy-associated inhibitory activity to a second species, and have compared the oligomannose profile of Tamm-Horsfall glycoprotein (nonpregnant) with that of uromodulin (pregnant) derived from both human and bovine sources. Surprisingly, there was a pregnancy-associated decrease in the total content of oligomannose chains due predominantly to a reduction in Man₅GlcNAc₂-R and Man₆GlcNAc₂-R. Man₇GlcNAc₂-R, which did not decrease with pregnancy, comprised a significantly greater proportion of the total oligomannose chains in pregnant vs. nonpregnant samples from both species (human; 34.6% vs. 25.9%: bovine; 14.4% vs. 7.2%).     

Influence of environmental factors on 2,4-dichlorophenoxyacetic acid degradation by Pseudomonas cepacia isolated from peat

A Pseudomonas cepacia, designated strain BRI6001, was isolated from peat by enrichment culture using 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon source. BRI6001 grew at up to 13 mM 2,4-D, and degraded 1 mM 2,4-D at an average starting population density as low as 1.5 cells/ml. Degradation was optimal at acidic pH, but could also be inhibited at low pH, associated with chloride release from the substrate, and the limited buffering capacity of the growth medium. The only metabolite detected during growth on 2,4-D was 2,4-dichlorophenol (2,4-DCP), and degradation of the aromatic nucleus was by intradiol cleavage. Growth lag times prior to the on-set of degradation, and the total time required for degradation, were linearly related to the starting population density and the initial 2,4-D concentration. BRI6001, grown on 2,4-D, oxidized a variety of structurally similar chlorinated aromatic compounds accompanied by stoichiometric chloride release.