Mycologia
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DOI: 10.3852/mycologia.99.4.562
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Mycologia, 99(4), 2007, pp. 562-568.
© 2007 by The Mycological Society of America

Evidence that a wortmannin-sensitive signal transduction pathway regulates aflatoxin biosynthesis


Joo-Won Lee

     Department of Pharmacology, Institute of Biomedical Science, Hanyang University, Seoul, South Korea

Ludmila V. Roze

     Department of Food Science and Human Nutrition, Michigan State University, East Lansing, Michigan

John E. Linz 1

     Department of Food Science and Human Nutrition, Department of Microbiology and Molecular Genetics, National Food Safety and Toxicology Center, 234B GM Trout Building, Michigan State University, East Lansing, Michigan 48823

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

A signal transduction pathway involving cAMP and protein kinase A (PKA) regulates aflatoxin accumulation and nor-1 and ver-1 (aflatoxin structural genes) promoter function in Aspergillus parasiticus by modulating expression of a key transcriptional activator, AflR. To understand the function of this pathway in greater detail we treated A. parasiticus in culture with wortmannin, a frequently used probe of phosphatidyl inositol (PI)-3 kinase activity. A. parasiticus D8D3 (nor-1::GUS reporter) and I4 (ver- ::GUS reporter) were grown on a defined solid growth medium (GMS agar) under aflatoxin-inducing conditions. GMS containing wortmannin (1 µM) reduced aflatoxin B1 accumulation up to 15-fold accompanied by a similarly large decrease in ver-1 and nor-1 promoter activity. Wortmannin inhibited growth (colony diameter) and asexual sporulation but to a much smaller extent. Wortmannin treatment increased intracellular cAMP levels up to 25-fold; total PKA activity also increased within 10 min of wortmannin exposure. These data support a regulatory model in which PI-3 kinase activity modulates intracellular cAMP accumulation and PKA activity. This in turn regulates AflR expression and activity, aflatoxin gene expression and aflatoxin accumulation.

Key words: aflatoxin, Aspergillus parasiticus, phosphatidyl inositol (PI) 3-Kinase, phosphodiesterase, signal transduction, wortmannin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Aflatoxins are synthesized predominantly by Aspergillus parasiticus and A. flavus as they grow on a variety of food and feed crops including corn, cotton seed, tree nuts and peanuts (Cotty 1994Go). Aflatoxins are among the most potent naturally occurring carcinogens known and they pose a significant threat to human and animal health (Cary et al 2000Go). One potential mechanism to control aflatoxin contamination is to block aflatoxin gene expression on susceptible plant tissues.

Expression of many aflatoxin genes is positively regulated by the Zn(II)2Cys6, binuclear cluster transcriptional regulator AflR (Brown et al 1999Go) under the influence of at least two signal transduction pathways (Hicks et al 1997Go, Tag et al 2000Go, Shimizu et al 2003Go). A G-protein (FadA)-mediated cAMP signaling pathway employs flbA, fluG, fadA and pkaA gene products and modulates AflR activity at the transcriptional level. A RasA-mediated pathway also influences AflR activity at the post-transcriptional level (Shimizu et al 2003Go); the mechanistic details and the environmental stimuli that trigger these pathways are not clearly understood.

Phosphatidylinositol 3-kinases (PI 3-K) catalyze the phosphorylation of inositol in phosphoinositides and regulate diverse cellular pathways including growth factor signaling cascades, apoptosis, membrane trafficking and other cellular functions in mammalian cells (Shepherd et al 1988Go, Rondinone et al 2000Go, Zhao et al 2000Go, Jackson et al 2004Go, Lindemans and Coffer 2004Go, Shimaya et al 2004Go, Vanhaesebroeck et al 2005Go). In yeast PI 3-K encoded by VPS34 of Saccharomyces cerevisiae and pik3+ of Schizosaccharomyces pombe, mediates sorting of proteins from the Golgi apparatus to the vacuole (Schu et al 1993Go, Stack et al 1995Go, Onishi et al 2004).

PI 3-K activates different downstream effectors depending on cell type; these include Gi in platelets ( Jackson et al 2004Go) and protein kinase B (PKB, akt) in granulocytes (Lindemans and Coffer 2004Go), adipocytes (Shimaya et al 2004Go) and hepatocytes (Zhao et al 2000Go). In hepatocytes, PI 3-K-dependent activation of phosphodiesterase (PDE3B) mediates downstream physiological effects by stimulating the catalytic conversion of cAMP to 5'AMP; this decreases intracellular cAMP and protein kinase A (PKA) activity (Zhao et al 1997Go, Zhao et al 1998Go, Rondinone et al 2000Go, Zhao et al 2000Go). Because we observed previously a connection between intracellular cAMP levels, PKA activity and regulation of aflatoxin biosynthesis in A. parasiticus (Roze et al 2004Go), we determined whether PI 3-K might play a role in this regulatory scheme.

Wortmannin, a secondary metabolite produced by isolates of Fusarium and Penicillium, has been used extensively to probe the functional role of PI 3-K in signal transduction pathways in a variety of mammalian cell types as well as in whole mice (Shepherd et al 1988Go, Woscholski et al 1994Go, Rondinone et al 2000Go, Zhao et al 2000Go, Jackson et al 2004Go, Lindemans and Coffer 2004Go, Shimaya et al 2004Go, Vanhaesebroeck et al 2005Go). In the current study we analyzed the effects of wortmannin on aflatoxin accumulation, aflatoxin gene expression, cAMP accumulation and PKA activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Fungal strains and growth conditions.— – Aspergillus parasiticus D8D3 (Chiou et al 2002) carrying a nor-1 promoter:: GUS reporter fusion (uidA; encodes ß-D-glucuronidase) was used in all experiments. A. parasiticus I4 (Miller 2003Go) carrying a ver-1 promoter::GUS reporter fusion was used to analyze effects of wortmannin on ver-1 promoter activity. A chemically defined glucose minimal salts (GMS) agar medium supplemented with 5 µM Zn2+ was used in all experiments (Roze et al 2004Go, Miller et al 2005Go); small Petri dishes (60 x 15 mm) were used in these experiments. To measure treatment effects on aflatoxin accumulation, sporulation and colony growth, two culture protocols were used: (i) Standard growth. To measure effects of extended treatment, conidiospores (104) of D8D3 and I4 were center-inoculated on GMS medium, GMS containing vehicle only (5 µL DMSO) or GMS containing wortmannin (1 µM) and incubated at 30 C in the dark 5 d. (ii) Colony transfer. To allow measurement of effects immediately after treatment or after extended treatment, conidiospores were center-inoculated onto sterile cellophane disks placed on the solid GMS agar medium and grown 48 h at 30 C in the dark. The cellophane then was peeled from the agar surface and transferred to fresh GMS containing cAMP (5 mM), wortmannin (1 µM), both compounds or vehicle (5 µL DMSO) and incubated at 30 C in the dark an additional 72 h (5 d total). cAMP, wortmannin or vehicle were added to agar media just before it was allowed to solidify.

Determination of aflatoxin accumulation.— – The fungal mycelium and agar medium from colonies grown with standard growth or colony transfer protocols were harvested and extracted three times with 5 mL of chloroform. These three extracts were pooled, evaporated with nitrogen gas and the dried residue was resuspended in 70% methanol for analysis by thin layer chromatography (TLC; Roze et al 2004Go) and by enzyme-linked immunosorbent assay (ELISA; Pestka 1988Go). Three colonies from three separate plates (nine colonies total) were analyzed. These methods have been used extensively in our laboratory (Lee et al 2004Go, Chiou et al 2004Go, Roze et al 2004aGo, bGo, Miller et al 2005Go).

Determination of conidiopspore number and colony growth.— – Sterile, distilled water containing 0.005% Triton x 100 (1 mL) was added to the surface of GMS agar plates. Conidiospores were harvested from colonies grown with standard or colony transfer protocols by scraping the agar surface with a bent glass rod and counted with a hemacytometer. To determine radial fungal growth, colony diameter from colonies grown with standard or colony transfer protocols was measured in two perpendicular directions at regular time intervals and the average of the two measurements calculated as described by Roze et al (2004a)Go.

Measurement of intracellular cAMP and PKA activity in D8D3.— – Colonies of D8D3 were grown 48 h with the colony transfer protocol. However, after transfer to fresh GMS containing cAMP (5 mM), wortmannin (1 µM), both compounds, or vehicle only (5 µL DMSO), colonies were harvested after 30 min, 1 h or 2 h to measure intracellular cAMP or after 10 min, 30 min or 1 h to measure PKA activity.

For intracellular cAMP the mycelium was collected at appropriate times (see above), immediately frozen in liquid N2 and stored at 280 C until analysis. The frozen mycelium was ground in liquid N2 in a mortar and the ground mycelium was suspended in 0.1 N HCl. cAMP was quantified with a competitive immunoassay (Direct cAMP Enzyme Immunoassay Kit, Assay Designs Inc., Ann Arbor, Michigan) according to the manufacturer’s instructions. Intracellular cAMP was determined in two independent experiments; data are reported as the mean ± standard error.

For PKA activity the mycelium was harvested at appropriate times (see above) and crude cell extracts were prepared as described by Roze et al (2004)Go. PKA activity was measured with the Pep Tag Assay Kit for nonradioactive detection of PKA (Promega, Madison, Wisconsin) according to the manufacturer’s instructions. A volume of crude extract containing 60 µg protein was used to measure PKA activity with PepTag A1 peptide (kemptide peptide tagged with fluorescent dye) as substrate for PKA, and the phosphorylation reaction was incubated at room temperature for 10 min. cAMP-dependent protein kinase catalytic subunit provided by the kit was used as positive control. The phosphorylation reaction without cAMP-dependent protein kinase catalytic subunit and without protein extract served as negative control. Total PKA activity in the presence of exogenously added 5 µM cAMP was determined. This experiment was conducted three times with similar results. Data from one representative PKA activity measurement are presented (FIG. 2Go); fluorescence intensity of bands on the 0.8% agarose gel was measured by densitometry with Kodak 1D 3.6 software (Kodak, New Haven, Connecticut). Protein concentrations were determined by the Bradford method (Bradford 1976Go).


Figure 2
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  		FIG. 2. Effect of wortmannin treatment on total PKA activity. Two µg of Pep Tag peptide substrate were incubated with fungal lysates (generated after 10 min, 30 min or 1 h of wortmannin treatment) for 10 min at room temperature. The reaction was stopped by heating to 95 C for 10 min and the samples were resolved on a 0.8% agarose gel. We measured PKA activity in three independent experiments with similar results; representative data from one experiment are shown. Phosphorylated peptide moved toward the anode (see Lane 1; positive control). Nonphosphorylated peptide moved toward the cathode (see Lane 8, negative control). Lane 1: positive control (protein kinase catalytic subunit); Lanes 2–4: crude lysate from colonies grown on GMS medium and incubated for indicated time; Lanes 5–7: crude lysate from colonies grown on GMS plus wortmannin (1 µM) and incubated for indicated time; Lane 8: negative control, no extract.

 
Analysis of GUS reporter activity.— – A. parasiticus D8D3 (nor- 1::GUS) and I4 (ver-1::GUS) were cultured at 30 C for 72 h on cellophane overlaid onto GMS agar medium in the presence or absence of 1 µM wortmaninn. Fungal protein extracts were obtained and GUS activity measured with 50 µg fresh total fungal protein as described by Miller et al (2005)Go. ß-glucuronidase (GUS) activity was expressed as pmole 4-methyl umbeliferone/min/Cg protein. Three colonies from three different plates (nine colonies total) were analyzed by this procedure.

Statistical analyses.— – These were conducted with Sigma Stat software, version 2.0 ( Jandel Corp.). The Student t-test was used to analyze statistical differences between control and experimental treatments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Effects of wortmannin, a PI 3-K antagonist, on AFB1 accumulation, sporulation, and growth.— – A. parasiticus D8D3 was grown 5 d with the standard growth protocol on solid GMS medium containing 1 µM wortmannin, 5 µL DMSO (vehicle control) or no addition (negative control). Wortmannin treatment reduced AFB1 accumulation (as measured by ELISA) up to 15-fold (2.1 µg/colony) compared to vehicle control (30.1 µg/colony) and up to 20-fold compared to negative control (40.8 µg/colony). TLC analysis (not shown) supported these observations. Wortmannin treatment also reduced colony diameter (growth) and asexual sporulation approximately 10–15% (not shown).

A similar experiment was conducted with the colony transfer protocol. Transfer of fungal colonies to GMS plus wortmannin reduced aflatoxin accumulation approximately eightfold compared to vehicle control (TABLE IGo). Treatment with 5 mM cAMP alone stimulated AFB1 accumulation approximately 35-fold compared to vehicle control while addition of wortmannin plus cAMP stimulated AFB1 accumulation only approximately eightfold. Treatment with cAMP, wortmannin, or both compounds had a much smaller effect on sporulation and colony growth.


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  		TABLE I. Effect of wortmannin on A. parasiticus growth, sporulation and toxin accumulation using the colony transfer protocol

 
Effect of wortmannin on nor-1 and ver-1 promoter activity.— – To determine whether wortmannin treatment affected aflatoxin accumulation at the level of gene expression, A. parasiticus D8D3 (nor-1::GUS reporter) and I4 (ver-1::GUS reporter) were grown 72 h with the standard growth protocol on GMS agar medium containing wortmannin (1 µM) or 5 µL DMSO (vehicle control). Wortmannin treatment reduced nor-1 and ver-1 promoter activity as measured by GUS activity up to 10-fold compared to vehicle control (FIG. 1Go), a decrease similar in magnitude to the observed 15-fold down-regulation of AFB1 accumulation.


Figure 1
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  		FIG. 1. Effect of wortmannin on nor-1 and ver-1 promoter activity. A. parasiticus strains D8D3 or I4 carrying nor- 1::GUS or ver-l ::GUS reporter constructs, respectively, were grown on GMS agar medium with wortmannin (1 µM) or vehicle (5 µL DMSO) for 72 h. GUS activity was analyzed by a fluorescence-based assay. Bars represent the mean of GUS activity in three independent colonies ± standard error. * = statistically significant difference between control and experimental treatment (P < 0.05) as analyzed by Student t-test.

 
Effect of wortmannin on intracellular cAMP levels in A. parasiticus.— – Because we observed that modulation of intracellular cAMP levels strongly influences nor-1 and ver-1 promoter activity (Roze et al 2004Go) we analyzed the effect of wortmannin treatment on the accumulation of this cyclic nucleotide. We measured intracellular cAMP levels in D8D3 grown on GMS agar media containing wortmannin (1 µM), cAMP (5 mM), cAMP and wortmannin, or 5 µL DMSO (vehicle control) with the colony transfer protocol (TABLE IIGo). Wortmannin treatment increased intracellular cAMP concentrations approximately 25-fold 2 h after transfer compared to vehicle control. Similar to previous studies, increases in intracellular cAMP to nonphysiologically high levels (approximately 25 000-fold) were observed with exogenous cAMP (5 mM) either with or without addition of wortmannin; however wortmannin treatment maintained these high intracellular cAMP levels up to 2 h while cells treated with exogenous cAMP only returned to the 30 min levels by 2 h, supporting the idea that wortmannin prevents cAMP turnover (metabolism).


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  		TABLE II. Intracellular cAMP levels in A. parasiticus using the colony transfer protocol

 
Effect of wortmannin treatment on PKA activity.— – To determine whether increased intracellular cAMP accumulation (in response to wortmannin treatment) resulted in activation of PKA, A. parasiticus D8D3 was grown on GMS agar medium and transferred to medium containing wortmannin (1 µM) or 5 µL DMSO (vehicle control). PKA activity was measured 10 min, 30 min and 1 h after the shift with a fluorescence-based assay (FIG. 2Go). We observed a 30% increase in total PKA activity within 10 min (compared to vehicle control); PKA activity returned to control levels by 30 min after the shift.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Our data clearly demonstrate that wortmannin inhibits aflatoxin accumulation and aflatoxin gene expression at the level of promoter function. Wortmannin treatment also stimulates cAMP accumulation and PKA activity. Based on these observations we propose that a wortmannin-sensitive signal transduction pathway modulates intracellular cAMP levels and PKA activity and regulates aflatoxin biosynthesis in A. parasiticus.

In mammalian hepatocytes, binding of insulin to the insulin receptor triggers phosphorylation of PI 3-K, which appears to be in direct contact with this protein; leptin also stimulates PI 3-K through a similar pathway (Rondinone et al 2000Go, Zhao et al 2000Go). Activated PI 3-K leaves the receptor and phosphorylates downstream targets, including phosphodiesterase PDE3B, resulting in modulation of intracellular cAMP levels and PKA activity; function of this pathway is associated with the antilipolytic effects of PI 3-K activation.

We propose that wortmannin treatment blocks aflatoxin synthesis in A. parasiticus by interfering with a PI 3-K-mediated signaling pathway analogous to that described for mammalian hepatocytes (Rondione et al 2000, Zhao et al 2000Go). In our proposed model wortmannin blocks PI 3-K activity and PI 3-K fails to activate phospodiesterase. As a result intracellular cAMP accumulates to high physiological levels, which stimulates PKA activity. In response AflR expression and activity decrease, which down-regulates nor-1 and ver-1 promoter activity and aflatoxin accumulation. Consistent with this model we observed increases in intracellular cAMP and PKA activity accompanied by decreases in nor-1 and ver-1 promoter activities. In future work we will test additional predictions of this model by measuring PI 3-K, phosphodiesterase and adenylate cyclase activities with the standard growth and colony transfer protocols. Because wortmannin has been demonstrated to modulate Polo-like kinases (Liu et al 2004Go) and phospholipase A2 activity (Cross et al 1995Go) in vitro, we cannot rule out the possibility that wortmannin affects other kinases in A. parasiticus in addition to PI 3-K.

Lafon et al (2005)Go recently reported that a heterotrimeric G protein composed of GanB ({alpha}subunit), SfaD (ß-subunit) and GpgA ({gamma}-subunit) enables A. nidulans to sense the presence of a fermentable carbon source (like glucose) in the growth medium. This triggers a transient cAMP signal that activates PKA that in turn activates conidiospore germination. Keller’s group previously proposed a model in which the G{alpha} subunit fadA stimulates PKA activity resulting in cell growth. The fluG and flbA gene products, in response to unknown stimuli, act upstream of fadA to block PKA activity and the growth pathway; this in turn activates AflR expression and activity, aflatoxin gene expression and aflatoxin accumulation (Hicks et al 1997Go). Based on our studies (Roze et al 2004aGo, bGo) and studies and observations of Lafon (Lafon et al 2005Go) we expand the Keller model.

In A. parasiticus aflatoxin synthesis is stimulated by the presence of glucose (or other preferred carbon sources) in the growth medium (reviewed in Miller and Linz 2006Go); this is known as the "glucose effect". Nonpreferred carbon sources such as peptone and lactose do not stimulate aflatoxin synthesis. It seems reasonable to suggest that a signaling pathway analogous to that reported in A. nidulans might enable A. parasiticus to sense glucose levels in the growth medium. We propose that during active growth, "sufficient" glucose in the medium stimulates the growth pathway via FadA. As glucose in the medium is depleted, intracellular cAMP levels and PKA activity decline, AflR expression and activity increase, resulting in aflatoxin gene expression and aflatoxin accumulation. Furthermore we propose that the decline in G protein activity activates a parallel pathway that dampens the growth pathway and enhances aflatoxin synthesis. We propose that the decline in FadA activity activates PI-3K, which stimulates phosphodiesterase (PDE). This causes an even larger decline in intracellular cAMP, further activating expression and activity of AflR. Future work will be focused on providing details of this proposed regulatory scheme and on using this information to reduce or eliminate aflatoxin accumulation on food or feed crops.


   FOOTNOTES
 
Accepted for publication April 3, 2007.

1 Corresponsing author. E-mail: jlinz{at}msu.edu


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254.[CrossRef][Medline]

Brown MP, Brown-Jenco CS, Payne GA. 1999. Genetic and molecular analysis of aflatoxin biosynthesis. Fungal Genet Biol 26:81–98.[CrossRef][Medline]

Cary JW, Linz JE, Bhatnagar D. 2000. Aflatoxin: biological significance and regulation of toxin synthesis. In: Cary J, Bhatnagar D, Linz JE, eds. Microbial Foodborne Diseases; Mechanisms of Pathogenesis and Toxin Synthesis. Lancaster, Pennsylvania: Technomic Publishing. p 317–362.

Chiou CH, Lee LW, Owens SA, Whallon JH, Klomparens KL, Townsend CA, Linz JE. 2004. Distribution and sub-cellular localization of the aflatoxin enzyme versicolorin B synthase in time-fractionated colonies of Aspergillus parasiticus. Arch Microbiol 182:67–79.[CrossRef][Medline]

Cotty PJ. 1994. Agriculture, aflatoxin and Aspergillus. In: Powell KA, Fenwick A, Peberdy JF, eds. The genus Aspergillus. New York: Plenum Press. p 1–27.

Cross MJ, Stewart A, Hodgkin MN, Kerr DJ, Wakelam MJ. 1995. Wortmannin and its structural analogue demethoxyviridin inhibit stimulated phospholipase A2 activity in Swiss 3T3 cells. Wortmannin is not a specific inhibitor of phosphatidylinositol 3-kinase. J Biol Chem 270:25352–25355.[Abstract/Free Full Text]

Hicks J, Yu JH, Keller NP, Adams TH. 1997. Aspergillus sporulation and mycotoxin production both require inactivation of the FadA G{alpha} protein-dependent signaling pathway. EMBO J 16:4916–4923.[CrossRef][Medline]

Jackson SP, Yap CL, Anderson KE. 2004. Phosphoinoside 3-kinases and the regulation of platelet function. Biochem Soc Trans 32:387–392.[CrossRef][Medline]

Lafon A, Seo JA, Han KH, Yu JH, d’Enfant C. 2005. The heterotrimeric G protein GanB({alpha} )-SfaD(ß)-GpgA({gamma}) is a carbon source sensor involved in early cAMP-dependent germination in Aspergillus nidulans. Genetics 171:71–81.[Abstract/Free Full Text]

Lee LW, Chiou CH, Klomparens KL, Cary JW, Linz JE. 2004. Sub-cellular localization of aflatoxin biosynthetic enzymes Nor-1, Ver-1, and OmtA in time fractionated colonies of Aspergillus parasiticus. Arch Microbiol 181: 204–214.[CrossRef][Medline]

Lindemans CA, Coffer PJ. 2004. Regulation of granulocyte apoptosis by phosphatidyl inositol-3-kinase. Bichem Soc Trans 32:480–484.[CrossRef]

Liu Y, Shreder KR, Gai W, Corral S, Rosenblum JS. 2004. Wortmannin, a widely used phosphoinositide 3-kinase inhibitor, also potently inhibits mammalian polo-like kinase. Chem Biol 12:99–107.

Miller M. 2003. Transcriptional regulation of the Aspergillus parasiticus aflatoxin biosynthetic pathway gene nor-1 [Doctoral dissertation]. East Lansing: Michigan State University.

Miller MJ, Linz JE. 2006. Regulation of mycotoxin biosynthesis. In: Shetty K, ed. Food Biotecnhology. 2nd ed. Boca Raton, Florida: Taylor & Francis Group, Chpt 3.9.

———, Roze LV, Trail F, Linz JE. 2005. Role of cis-acting sites NorL, a TATA box, and AflR1 in nor-1 transcriptional activation in Aspergillus parasiticus. Appl Environ Microbiol 71:1539–1545.[Abstract/Free Full Text]

Onishi M, Koga T, Morita R, Nakamura Y, Nakamura T, Shimoda C, Takegawa K, Hirata A, Fukui Y. 2003. Role of phosphatidylinositol 3-phosphate in formation of forespore membrane in Schizosaccharomyces pombe. Yeast 20:193–206.[CrossRef][Medline]

Pestka JJ. 1988. Enhanced surveillance of food-borne mycotoxins by immunochemical assay. J Assoc Off Anal Chem 71:1075–1081.[Medline]

Rondinone CM, Carvalho E, Rahn T, Manganiello VC, Degerman E, Smith UP. 2000. Phosphorylation of PDE3B by phosphatidylinositol 3-kinase associated with the insulin receptor. J Biol Chem 275:10093–10098.[Abstract/Free Full Text]

Roze LV, Beaudry RM, Keller NP, Linz JE. 2004a. Regulation of aflatoxin synthesis by FadA/cAMP/protein kinase A signaling in Aspergillus parasiticus. Mycopathologia 158:219–232.[CrossRef][Medline]

———, Miller MJ, Rarick M, Mahianti N, Linz JE. 2004b. A novel cAMP response element, CRE1, modulates expression of nor-1 in Aspergillus parasiticus. J Biol Chem 279:27428–27439.[Abstract/Free Full Text]

Schu PV, Takegawa K, Fry MJ, Stack JH, Waterfield MD, Emr SD. 1993. Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. Science 260:88–91.[Abstract/Free Full Text]

Shepherd PR, Withers DJ, Siddle K. 1988. Phosphoinositide 3-kinase: the key switch mechanism in insulin signaling. Biochem J 333:471–490.

Shimaya A, Kovacina KS, Roth RA. 2004. On the mechanism for neomycin reversal of wortmannin inhibition of insulin stimulation of glucose uptake. J Biol Chem 279: 55277–55282.[Abstract/Free Full Text]

Shimizu K, Hicks JK, Huang TP, Keller NP. 2003. Pka, Ras, and RGS protein interactions regulate activity of AflR, a Zn(II)2Cys6 transcription factor in Aspergillus nidulans. Genetics 165:1095–1104.[Abstract/Free Full Text]

Stack JH, DeWald DB, Takegawa K, Emr SD. 1995. Vesicle-mediated protein transport: regulatory interactions between the Vps15 protein kinase and the Vps34 PtdIns 3-kinase essential for protein sorting to the vacuole in yeast. J Cell Biol 129:321–334.[Abstract/Free Full Text]

Tag A, Hicks J, Garifullina G, Ake C, Phillips TD, Beremand M, Keller NP. 2000. G-protein signaling mediates differential production of secondary metabolites. Mol Microbiol 38:658–665.[CrossRef][Medline]

Vanhaesebroeck B, Ali K, Bilancio A, Geering B, Foukas LC. 2005. Signaling by PI3K isoforms; insights from gene-targeted mice. Trend Biochem Sci 30:194–204.[CrossRef][Medline]

Woscholski R, Koddaki T, McKinoon M, Watefield MD, Parker PJ. 1994. A comparison of demthoxyviridin and wortmannin as inhibitors of phosphatidylinositol 3-kinase. FEBS Lett 342:109–114.[CrossRef][Medline]

Zhao AZ, Bornfeldt KE, Beavo JA. 1998. Leptin inhibits insulin secretion by activation of phosphodiesterase 3B. J Clin Invest 102:869–873.[Medline]

———, Shinohara MM, Huang D, Shimizu M, Eldar-Finkelman H, Krebs EG, Beavo JA, Bornfeldt BE. 2000. Leptin induces insulin-like signaling that antagonizes cAMP elevation by glucagon in hepatocytes. J Biol Chem 275:11348–11354.[Abstract/Free Full Text]

———, Zhao H, Teague J, Fujimoto W, Beavo JA. 1997. Attenuation of insulin secretion by insulin-like growth factor 1 is mediated through activation of phosphodiesterase 3B. Proc Nat Acad Sci USA 94:3223–3228.[Abstract/Free Full Text]

Zhou R, Linz JE. 1999. Enzymatic function of the nor-1 protein in aflatoxin biosynthesis in Aspergillus parasiticus. Appl Environ Microbiol 65:5639–5641.[Abstract/Free Full Text]





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