Study of the Ochratoxin A effect on Aspergillus parasiticus growth and aflatoxin B1 production.

Aflatoxin B1 (AFB1) is a carcinogenic metabolite produced by certain Aspergillus species. Ochratoxin A (OTA) is a metabolite of Aspergillus ochraceus and Penicillium verrucosum. AFB1 and OTA are amongst the most frequent combinations of mycotoxins found in plant products. Thus, synergistic effects or interactions between the two mycotoxins could be taking place. The aim of the present study was to investigate the effect of OTA on Aspergillus parasiticus growth and AFB1 production in yeast extract sucrose (YES) medium at concentrations of 0.16, 1.6 and 16 ng OTA flask(-1). The AFB1 extracted from cultures and purified with immunoaffinity columns was then quantitated by HPLC. The recovery and detection limit of the method were 95.3% and 0.02 ng AFB1 mL(-1), respectively. Maximum AFB1 productions in cultures with OTA were observed from 9 to 12 days (76.09-82.52 ng AFB1 flask(-1)) while in control cultures (without OTA) maximum production (197.2 ng AFB1 flask(-1)) was observed on 14th day. Maximum AFB1 levels in cultures with OTA were reduced by a percentage of 58-61% compared to control cultures. Furthermore AFB1 levels in cultures with OTA were practically (92%) degraded after 18 days of incubation. Conclusively when OTA is present the production of AFB1 by A. parasiticus in YES medium is inhibited.

The alpha- and beta-subunits are required for expression of catalytic activity in the hetero-dimeric glucosidase II complex from human liver.

The alpha- and beta-subunits of the hetero-dimeric glucosidase II complex from human liver were cloned and expressed in COS-1 cells. The 4106 bp full-length cDNA for the alpha-subunit contained a 2835 bp ORF encoding a 107 kDa polypeptide. The 2095 bp cDNA for the beta-subunit encodes a approximately 60 kDa protein in a continuous 1605 bp ORF. The alpha- and beta-subunits each contain two potential Asn-Xaa-Thr/Ser acceptor sites, with only one site in the alpha-subunit (Asn97) being glycosylated. Additional lambda-clones were isolated for each subunit containing in-frame insertions/deletions within the coding region, indicating alternative splicing. Analysis of different human tissues revealed approximately 4.4 kb and approximately 2.4 kb transcripts for alpha- and beta-subunit, respectively, consistent with their full-length cDNA. Coexpression of the alpha- and beta-subunits in COS-1 cells resulted in >4-fold increase of glucosidase II activity. An inactive protein was obtained, however, after transfection with the alpha-subunit alone, showing that both subunits are essential for expression of active glucosidase II. The observation that the enzyme, previously purified from pig liver and lacking the beta-subunit, was catalytically active indicates that the beta-subunit is involved in alpha-subunit maturation rather than being required for enzymatic activity once the alpha-subunit has acquired its mature form. The alpha-subunit is expressed in COS-1 cells as an ER-located protein, whether inactive or part of a catalytically active complex. This suggests that ER-localization of the alpha-subunit, when associated with the dimeric enzyme complex, is mediated by the C-terminal HDEL-signal in the beta-subunit, whereas the apparently incompletely folded form of the inactive alpha-subunit could be retained in the ER by the putative "glycoprotein-specific quality control machinery. "