Biomedical subjects

T E Cleveland

Publications and source records attributed to T E Cleveland.

67 records · Page 4Linked to original sources

PubMed1991

Aflatoxin production via cross-feeding of pathway intermediates during cofermentation of aflatoxin pathway-blocked Aspergillus parasiticus mutants.

Cofermentation of Aspergillus parasiticus strains (SRRC 163 and SRRC 2043) blocked at different steps in the aflatoxin B1 (AFB1) biosynthetic pathway in a synthetic liquid medium or on seeds (cottonseed, corn kernels, and peanuts) resulted in production of AFB1. Strain SRRC 2043 accumulated O-methylsterigmatocystin (OMST), a late precursor in AFB1 biosynthesis, whereas SRRC 163 accumulated averantin, an early precursor in the pathway. Strain SRRC 2043 secreted large amounts of OMST in culture relative to the amounts of several other pathway intermediates secreted into media (by other AFB1 pathway-blocked strains). AFB1 production occurred even when colonies of SRRC 163 and SRRC 2043 strains (producing no detectable AFB1) were grown together on an agar medium while physically separated from each other by a filter membrane (0.22-micron pore size). In addition, when mycelia of strain SRRC 163 were added to culture filtrates (containing no mycelia but containing secreted OMST) of strain SRRC 2043, AFB1 production occurred. The results suggested a chemical (rather than genetic) mechanism of complementation for AFB1 production between AFB1 pathway-blocked strains, since no mycelial contact was required between these strains for AFB1 production. The mechanism for chemical complementation involves secretion of OMST by SRRC 2043 and subsequent absorption and conversion of OMST to AFB1 by mycelia of strain SRRC 163.

Aflatoxin B1↗

PubMed1990

Variation in polygalacturonase production among Aspergillus flavus isolates.

Pectinase production by Aspergillus flavus was determined by measuring clear zones formed around colonies stained with ruthenium red. Several isolates produced red zones instead of clear zones. Red zones were reproduced with pectinesterase and correlated with absence of specific polygalacturonases. Of 87 isolates tested, 15 produced red zones.

Aflatoxins↗

PubMed1990

Evidence for de novo synthesis of an aflatoxin pathway methyltransferase near the cessation of active growth and the onset of aflatoxin biosynthesis in Aspergillus parasiticus mycelia.

The accumulation of both activity and protein of a methyltransferase (MTase) from Aspergillus parasiticus, which catalyzes conversion of sterigmatocystin to O-methylsterigmatocystin in the aflatoxin pathway, was detected in fungal mycelia slightly before the onset of aflatoxin biosynthesis in the same cultures. MTase protein was identified in mycelial postmicrosomal (soluble protein) fractions by electrophoresis and subsequent immunoblotting using antiserum raised against purified MTase protein; MTase activity was determined by measuring the rate of conversion of sterigmatocystin to O-methylsterigmatocystin in the presence of soluble protein fractions. Using the above technique, it was determined that MTase protein as well as MTase activity increased sharply in mycelia 30 to 45 h after inoculation, shortly after which, mycelial growth rate began to decline. During the subsequent time interval (45 to 70 h after inoculation), a sharp increase in aflatoxin levels was detected in the culture medium. Results obtained from an experiment in which cycloheximide was added to cultures at various times to inhibit protein synthesis and from an experiment in which mycelial proteins were radiolabelled to identify newly synthesized proteins indicated that accumulation of MTase activity and protein in late growth phase mycelia is due to de novo protein synthesis.

Aflatoxins↗

PubMed1989

Enzymes in aflatoxin B1 biosynthesis: strategies for identifying pertinent genes.

Recent work on the aflatoxin biosynthetic pathway is reviewed, with special emphasis on the enzymes of the late stages of the pathway involving conversion of sterigmatocystin (ST) to aflatoxin B1 (AFB1) through an O-methylsterigmatocystin intermediate. Two enzyme activities were discovered in subcellular fractions of cell-free extracts of a mutant strain of Aspergillus parasiticus (SRRC 163): 1) A post-microsomal methyltransferase (MT) catalyzed conversion of ST to OMST, and 2) a microsomal-associated activity (oxido-reductase) converted OMST to AFB1. The 168 KDa, anionic MT was purified to homogeneity and characterized (two subunits, 110 KDa and 58 KDa). Preliminary evidence indicated the presence of a cationic isozyme of the MT in mycelial extracts. The oxido-reductase has been partially purified and characterized. Polyclonal antibodies were prepared to the anionic MT and the enzyme's amino acid composition determined. A cDNA library has been constructed from mRNA isolated from Aspergillus parasiticus mycelia during the onset of AFB1 biosynthesis for the purpose of identifying the genes responsible for aflatoxin biosynthesis.

Aflatoxin B1↗

PubMed1988

Fate of the methyl group during the conversion of sterigmatocystin into O-methylsterigmatocystin and aflatoxin B1 by cell-free preparations of Aspergillus parasiticus.

Cell-free extracts of fungal mycelia of two aflatoxin non-producing isolates of Aspergillus parasiticus (SRRC 163 and SRRC 2043) were utilized for the study of enzyme activities involved in the latter stages of aflatoxin biosynthesis. The post-microsomal fractions (105,000 x g supernatant) of both SRRC 163 and SRRC 2043 were able to convert sterigmatocystin (ST) into O-methylsterigmatocystin (OMST); whereas the microsomal (105,000 x g pellet) preparation of only SRRC 163 was able to convert OMST into aflatoxin B1 (AFB1). S-Adenosylmethionine (SAM) was the primary substrate for the ST to OMST (methyltransferase) enzymatic conversion; [3H]OMST of specific activity 0.93 Ci/mmol was obtained in a reaction containing the [3H]SAM substrate (specific activity 1 Ci/mmol). After the terminal enzymatic conversion of OMST into AFB1, none of the radiolabel of the methyl group from OMST was found in AFB1. It is postulated that the methylation of ST may be required for subsequent enzymatic oxidation of OMST to aflatoxin B1.

Aflatoxin B1↗

PubMed1988

Purification and characterization of a methyltransferase from Aspergillus parasiticus SRRC 163 involved in aflatoxin biosynthetic pathway.

A five step scheme has been developed for the purification of a methyltransferase (MT) from mycelia of 3-day old Aspergillus parasiticus (SRRC 163), which catalyzes one step in the aflatoxin biosynthetic pathway. The S-adenosylmethionine (SAM) requiring MT activity is essential for the conversion of sterigmatocystin (ST) to O-methylsterigmatocystin (OMST) prior to being converted to aflatoxin B1. The purification of the MT was carried out from cell-free extracts by CDR (Cell Debris Remover, a cellulosic weak anion exchanger, Whatman) treatment, QMA ACELL, Hydroxylapatite-Ultrogel, PBE 94 chromatofocusing and FractoGel TSK HW-50F filtration chromatography. The purified enzyme was only about 0.1% of the total extractable proteins. The pI of the protein was about 5.0 as judged by chromatofocusing. Results of gel filtration chromatography indicated the approximate molecular mass of the native protein to be 160-KDa. SDS-polyacrylamide gel electrophoresis revealed two protein subunit bands of molecular masses approximately 110-KDa and 58-KDa. The molar extinction coefficient of the enzyme at 280 nm was estimated to be 7.87 X 10(4) M-1 cm-1 in 50 mM potassium phosphate buffer (pH 7.5). The reaction catalyzed by the MT was optimum at pH 7.5 and between 25-35 degrees C. The Km of the enzyme for ST and SAM was determined to be 1.8 microM and 42 microM, respectively with an estimated turnover number of the enzyme for ST of 2.2 X 10(-2) per sec.

Aflatoxins↗

PubMed1987

Wound-inducible expression of a potato inhibitor II-chloramphenicol acetyltransferase gene fusion in transgenic tobacco plants.

A potato inhibitor II gene (IIK) was isolated from a library of potato genes in lambda bacteriophage. An 8-kilobase-pair (kbp) insert was identified using a tomato inhibitor II cDNA as a hybridization probe, and a 2.6-kbp fragment containing the gene was subcloned into the plasmid pUC13 and characterized. The nucleotide sequence of the isolated gene exhibited 87% identity with the wound-inducible tomato inhibitor II cDNA sequence. The amino acid sequence of inhibitor IIK, deduced from the potato gene, exhibited 84% identity with the tomato inhibitor II protein. A 1000-bp restriction fragment from the 5' flanking region of the gene was fused to the open reading frame of the chloramphenicol acetyltransferase (CAT) gene. This fusion was terminated in two ways: (i) with a terminator sequence from the potato inhibitor II gene and (ii) with a terminator from the 6b gene of Ti plasmid pTiA6. These chimeric genes were transferred into tobacco cells via a binary Ti vector system, and transgenic plants were regenerated. The CAT gene was expressed in leaves of transformed plants in response to wounding when fused with the inhibitor IIK promoter and terminator regions. The chimeric gene containing the 6b terminator did not express CAT in response to wounding. The wound-inducible expression of CAT activity was systemic and was induced in tissues distal to the wounded tissues. The time course of wound induction of CAT activity in transgenic tobacco leaves is similar to that found for wound-inducible inhibitor I and II mRNAs in tomato leaves. These results demonstrate that sequences necessary and sufficient for wound inducibility are present within approximately 1000 bp of the control regions of the inhibitor IIK genes and that wound-inducible components of tobacco leaf cells can regulate these sequences.

Journal Article↗

PubMed1987

Conversion of a new metabolite to aflatoxin B2 by Aspergillus parasiticus.

A new metabolite which could be converted to aflatoxin (AF) B2 was detected during cofermentation analysis of two nonaflatoxigenic strains (SRRC 2043 and SRRC 163) of Aspergillus parasiticus. SRRC 2043, which accumulates the xanthone O-methylsterigmatocystin (OMST), a late precursor in the AFB1 pathway, was observed to accumulate another chemically related compound (HOMST; molecular weight, 356); SRRC 163 is blocked early in the pathway and accumulates averantin. During cofermentation of the two strains, levels of OMST and HOMST were observed to be greatly reduced in the culture, with simultaneous production of AFB1, AFB2, and AFG1. Intact cells of SRRC 163 were able to convert pure OMST or its precursor, sterigmatocystin, to AFB1 and AFG1 without AFB2 accumulation; the same cells converted isolated HOMST to AFB2 with no AFB1 or AFG1 production. The results indicate that AFB2 is produced from a separate branch in the AF biosynthetic pathway than are AFB1 and AFG1; AFB2 arises from HOMST, and AFB1 and AFG1 arise from sterigmatocystin and OMST.

Aflatoxins↗

PubMed1987

Appearance of enzyme activities catalyzing conversion of sterigmatocystin to aflatoxin B1 in late-growth-phase Aspergillus parasiticus cultures.

Two activities involved in terminal pathway conversion of sterigmatocystin to aflatoxin B1 were isolated from an aflatoxin-nonproducing mutant of Aspergillus parasiticus (avn-1), and the time course of appearance of the activities in culture was determined. Subcellular fractionation of fungal mycelia resolved the two activities into a postmicrosomal activity which catalyzed conversion of sterigmatocystin to O-methylsterigmatocystin and a microsomal activity which converted O-methylsterigmatocystin to aflatoxin B1. The two activities were absent in 24-h-old cells, increased to optimum levels during the stationary phase, and then declined.

Aflatoxin B1↗

PubMed1987

Individual reaction requirements of two enzyme activities, isolated from Aspergillus parasiticus, which together catalyze conversion of sterigmatocystin to aflatoxin B1.

Individual reaction requirements were determined for each of two enzyme activities present in Aspergillus parasiticus mycelia which together catalyze conversion of sterigmatocystin (ST) to aflatoxin B1 (AFB1). A postmicrosomal activity (PMA) catalyzed conversion of ST to O-methylsterigmatocystin (OMST) and a microsomal activity (MA) catalyzed conversion of OMST to AFB1. PMA was stimulated two- to three-fold in the presence of S-adenosylmethionine. Addition of NADPH promoted the maximum MA; this activity was not detected when FAD, FMN, NAD, or NADH were utilized individually as cofactors in reaction mixtures. A substantial amount (62%) of MA was lost during isolation of the microsomal fraction, but the activity was completely restored by reconstitution with a heat-treated (100 degrees C) postmicrosomal fraction. The reaction catalyzed by MA was optimum at pH 7.0 and at 17-23 degrees C, whereas the PMA reaction was optimum at pH 8.0-8.5 and at 35-40 degrees C. Apparent Km values of approximately 2.6 X 10(-6) M (for ST) and 6.6 X 10(-7) M (for OMST) were determined for PMA and MA, respectively.

Aflatoxin B1↗

PubMed1982

Partial purification of proteinase inhibitors from wounded tomato plants.

Proteinase inhibitors were extracted from the upper leaves of tomato plants, Lycopersicon esculentum Mill., 48 hours after wounding single lower leaves. Inhibitors were partially purified by affinity chromatography and isoelectric focusing. Significantly higher levels of trypsin and chymotrypsin inhibitory activity were recovered from wounded plants than from unwounded controls. Several inhibitor peaks were partially resolved by isoelectric focusing of affinity column eluates from both wounded and control plants. Inhibitor activity associated with each peak was greater in wounded plants than in corresponding peaks of controls. Agar double diffusion immunological assays showed that inhibitors with basic isoelectric points (pI) of 9.5, 8.9, 8.3, 8.2, and 8.0 are serologically related to inhibitor I. Certain of these inhibitors (pI = 9.5, 8.2, and 8.0) reacted strongly with both inhibitors I and II antiserum. Three acidic proteinase inhibitors (pI = 6.5, 5.9, and 4.7), which accumulated due to wounding, also were isolated. These inhibitors are novel, since they were shown to be serologically unrelated to inhibitors I and II.

Journal Article↗

PubMedDate not supplied

Conversion of dihydro-O-methylsterigmatocystin to aflatoxin B2 by Aspergillus parasiticus.

Dihydro-O-methylsterigmatocystin (DHOMST) was identified in cultures of Aspergillus parasiticus SRRC 2043, an aflatoxin (AF) non-producer, by comparison of its mass spectrum to that of authentic DHOMST. In addition to DHOMST, SRRC 2043 is known to produce two other chemically related compounds in culture: O-methylsterigmatocystin (OMST) which is an AFB1 precursor and a hydrated-vinyl ether analogue of OMST (HOMST) which is an AFB2 precursor. In the present study, DHOMST also was shown to be an AFB2 precursor by demonstrating that DHOMST is converted to AFB2 by mycelia of an A. parasiticus mutant (SRRC 163) blocked early in the AF biosynthetic pathway; this conversion occurred at an even higher rate than observed for HOMST to AFB2 conversion by the same fungal strain. SRRC 2043 mycelia, although incapable of producing AF's when supplied with OMST, HOMST or DHOMST, were capable of converting HOMST to DHOMST. The results indicated that DHOMST can be placed after HOMST in the biosynthetic sequence which produces AFB2.

Aflatoxins↗

PubMedDate not supplied

Comparison of the enzymatic composition of cell-free extracts of non-aflatoxigenic Aspergillus parasiticus with respect to late stages of aflatoxin biosynthesis.

Cell-free extracts of fungal mycelia of two non-aflatoxigenic isolates of Aspergillus parasiticus (SRRC 163 and SRRC 2043) were examined for enzyme activities involved in the latter stages of aflatoxin biosynthesis. Post-microsomal fractions (105 Kxg supernatant) of both SRRC 163 (ATTC 56774) and SRRC 2043 were able to convert sterigmatocystin (ST) to O-methylsterigmatocystin (OMST); whereas the microsomal (105 Kxg pellet) preparation of only SRRC 163 was able to convert OMST to aflatoxin B1 (AFB1). A mixture of the microsomal fraction of SRRC 163 and post-microsomal fraction of SRRC 2043 converted ST to AFB1. Electrophoretic protein separations of the microsomal fractions of the two A. parasiticus isolates on non-denaturing or denaturing polyacrylamide gel electrophoresis (PAGE) provided equivalent protein profiles.

Aflatoxins↗