Ochratoxin A occurrence in slaughter-pigs in Sweden and its use as a tool for feed screening programs.

The occurrence of ochratoxin A in pig blood can be used as a measure of ochratoxin A contamination of the feed. A method for ochratoxin A analysis in pig blood, suitable for screening programs, is presented. The relationship between ochratoxin A contents in the blood of pigs and the feed given is investigated with feeding experiments. Blood samples from pigs originating from 279 herds slaughtered in nine slaughter-houses in Sweden were analyzed for ochratoxin A. In total, 14% of the investigated pigs contained greater than or equal to 2 ng ochratoxin A per ml blood. The highest level found was 215 ng/mL. Correlations between the occurrence of ochratoxin A in the blood and parameters concerning the feed are investigated.

Human exposure to ochratoxin A in areas of Yugoslavia with endemic nephropathy.

Ochratoxin A is a mycotoxin with pronounced nephrotoxic potency in all species of single-stomach animals studied; it is a major disease determinant of porcine nephropathy and a disease occurring endemically in several countries. This disease is comparable with Balkan (endemic) nephropathy, suggesting a common causal relationship. Ochratoxin A has been found in foodstuffs in many countries, but the highest frequency of ochratoxin A contamination in foods (10.3% of 1,553 samples of foodstuffs) was encountered in an area of Yugoslavia, where Balkan (endemic) nephropathy is prevalent. Detection of ochratoxin A in human blood samples confirmed the prevalent exposure to this food contaminant. Relative risk calculations indicated a tendency to an association between this mycotoxin and Balkan (endemic) nephropathy, supporting the hypothesis of a causal role of ochratoxin A in this disease.

Molecular cloning of Bacillus sphaericus penicillin V amidase gene and its expression in Escherichia coli and Bacillus subtilis.

The Bacillus sphaericus gene coding for penicillin V amidase, which catalyzes the hydrolysis of penicillin V to yield 6-aminopenicillanic acid and phenoxyacetic acid, has been isolated by molecular cloning in Escherichia coli. The gene is contained within a 2.2-kilobase HindIII-PstI fragment and is expressed when transferred into E. coli and Bacillus subtilis. The expression in B. subtilis carrying the recombinant plasmid is approximately two times higher than in the original B. sphaericus strain. A comparison of the purified enzyme from B. sphaericus and the expressed gene product in E. coli minicells suggests that the native enzyme consists of four identical subunits, each with a molecular weight of 35,000.

Efficient secretion and purification of human insulin-like growth factor I with a gene fusion vector in Staphylococci.

A novel approach for production of small polypeptides, using a staphylococcal protein A vector, is described. This system is used to express, secrete and purify human insulin-like growth factor I (IGF-I). A fusion protein consisting of protein A and IGF-I is recovered in high yield by passing the culture medium through an IgG affinity column. Using site-specific mutagenesis an acid labile asp-pro cleavage site was introduced at the fusion point between the two proteins. The protein A "tail" can thereby be removed from the affinity purified fusion protein by chemical cleavage releasing biologically active IGF-I molecules.

Ochratoxin A in blood of slaughter pigs.

The global ochratoxin A contamination of Swedish feed cereals was studied by analysis of pig blood samples from 122 different herds. The samples were collected at seven Swedish slaughterhouses. The ochratoxin A analysis showed 21% of the samples to contain greater than or equal to 2 ng ochratoxin A per ml. Samples from Visby showed a significantly higher frequency of contamination compared with the rest of the country.

Intermediary Metabolism in Clostridium acetobutylicum: Levels of Enzymes Involved in the Formation of Acetate and Butyrate.

The levels of seven intermediary enzymes involved in acetate and butyrate formation from acetyl coenzyme A in the saccharolytic anaerobe Clostridium acetobutylicum were investigated as a function of time in solvent-producing batch fermentations. Phosphate acetyltransferase and acetate kinase, which are known to form acetate from acetyl coenzyme A, both showed a decrease in specific activity when the organism reached the solvent formation stage. The three consecutive enzymes thiolase, beta-hydroxybutyrylcoenzyme A dehydrogenase, and crotonase exhibited a coordinate expression and a maximal activity after growth had ceased. Only low levels of butyryl coenzyme A dehydrogenase activity were found. Phosphate butyryltransferase activity rapidly decreased after 20 h from 5 to 11 U/mg of protein to below the detection limit (1 mU/mg). Butyrate no longer can be formed, and the metabolic flux may be diverted to butanol. Butyrate kinase showed a 2.5- to 10-fold increase in specific activity after phosphate butyryltransferase activity no longer could be detected. These results suggest that the uptake of acetate and butyrate during solvent formation can not proceed via a complete reversal of the phosphate transferase and kinase reactions. The activities of all enzymes investigated as a function of time in vitro are much higher than the metabolic fluxes through them in vivo. This indicates that none of the maximal activities of the enzymes assayed is rate limiting in C. acetobutylicum.

Complete sequence of the staphylococcal gene encoding protein A. A gene evolved through multiple duplications.

The gene coding for protein A from Staphylococcus aureus has been isolated by molecular cloning, and a subclone containing an 1.8-kilobase insert was found to give a functional protein A in Escherichia coli. The complete nucleotide sequence of the insert, including the structural gene and the 5' and 3' flanking sequences, has been determined. Starting from a TTG initiator codon, an open reading frame comprising 1527 nucleotides gives a preprotein of 509 amino acids and a predicted Mr = 58,703. The structural gene is flanked on both sides by palindromic structures followed by a stretch of T residues, suggesting transcriptional termination signals. Thus, it appears that protein A is translated from a monocistronic mRNA. The sequence reveals extensive internal homologies involving a 58-amino acid unit, responsible for IgG binding, repeated 5 times and an 8-amino acid unit, possibly responsible for binding to the cell wall of S. aureus, repeated 12 times. Comparisons between the repeated regions show a marked preference for silent mutations, indicating an evolutionary pressure to keep the amino acid sequence preserved. The structure of the gene also suggests how the gene has evolved.

An improved positive selection plasmid vector constructed by oligonucleotide mediated mutagenesis.

An Escherichia coli plasmid vector, pUN121, has been constructed which allows for positive selection of transformants harboring DNA inserts. The positive selection of transformants harboring DNA inserts. The vector is based on plasmid pTR262 (Roberts et al. Gene, 12, (1980), 123-127) in which the tetracycline resistance gene is under transcriptional control of the repressor protein coded by the phage lambda cI gene. This plasmid has been rearranged, using in vitro recombinant techniques including oligonucleotide mediated mutagenesis to yield a smaller plasmid (4.4 kb) with unique cloning sites for EcoRI, XmaI and SmaI in addition to the unique HindIII and BclI sites. The plasmid has a functional ampicillin resistance gene and the new restriction sites (EcoRI, XmaI and SmaI) when used for cloning, give rise to tetracycline resistant transformants.

Gene fusion vectors based on the gene for staphylococcal protein A.

Two plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusion, have been constructed. These vectors will allow fusion of any gene to the protein A gene, thus giving hybrid proteins which can be purified, in a one-step procedure, by IgG affinity chromatography. As an example of the practical use of such vectors, the protein A gene has been fused to the lacZ gene of Escherichia coli. E. coli strains containing such plasmids produce hybrid proteins with both IgG binding and beta-galactosidase activities. The hybrid protein(s) can be immobilized on IgG-Sepharose by its protein A moiety with high efficiency without losing its enzymatic activity and they can be eluted from the column by competitive elution with pure protein A. The fused protein(s) also binds to IgG-coated microtiter wells which means that the in vivo product can be used as an enzyme conjugate in ELISA tests.

Enzymatic synthesis of (+)- and (-)-bisdechlorogeodin with sulochrin oxidase from Penicillium frequentans and Oospora sulphurea-ochracea.

Sulochrin oxidase is a blue copper-containing glycoenzyme that catalyzes a stereospecific formation of bisdechlorogeodin from sulochrin. The enzyme has been isolated from Penicillium frequentans and Oospora sulphurea-ochracea which catalyzes the formation of (+)-form and (-)-form of bisdechlorogeodin respectively. The Penicillium enzyme has a molecular weight of 157,000 and contains 19.5% of carbohydrates. Amino acid and carbohydrate compositions are given. The enzyme has probably a dimeric structure containing 6 Cu-atoms. Apparent Km-values of various substrates are presented. The Oospora enzyme has a molecular weight of 128,000 and except for its stereospecificity its properties are very similar to those of the Penicillium enzyme.

The distribution of the NADPH regenerating mannitol cycle among fungal species.

The mannitol cycle is an important NADPH regenerating system in Alternaria alternata. The cycle is built up to the following enzymes: mannitol 1-phosphate dehydrogenase, mannitol 1-phosphatase, mannitol dehydrogenase and hexokinase. The net reaction of one cycle turn is: NADH + NADP+ + ATP leads to NAD+ + NADPH + ADP + Pi. The enzymes needed for an operating cycle were found in Aspergillus, Botrytis, Penicillium, Pyricularia, Trichothecium, Cladosporium and Thermomyces all genera belonging to Fungi Imperfecti. The only genus of this class lacking the cycle was Candida. No genera from the classes Basidiomycetes and Phycomycetes showed any mannitol 1-phosphate dehydrogenase or mannitol 1-phosphatase activities. The genera investigated, belonging to Ascomycetes, Gibberella, Ceratocystis and Neurospora all lacked mannitol 1-phosphate dehydrogenase. It was concluded that the mannitol cycle is an important and widespread pathway for NADH oxidation and NADP+ reduction in the organisms belonging to the class Fungi Imperfecti.

Ochratoxin A in blood from slaughter pigs in Sweden: use in evaluation of toxin content of consumed feed.

Samples of pig blood, intended for ochratoxin A analysis, were collected from pigs of 279 randomly selected herds. The samples were obtained at nine different slaughterhouses from different areas of Sweden. Pigs from 47 herds (16.8% of the total) exhibited ochratoxin A in amounts of greater than or equal to 2 ng of ochratoxin A per ml of blood. One sample each from a single pig per herd identified herds contaminated with ochratoxin A in amounts exceeding three times the detection limit of the method (3 x 2 ng of ochratoxin A per ml of blood = 6 ng of ochratoxin A per ml of blood). There was a good agreement between ochratoxin A concentrations in the blood from different pigs within the same herd (correlation coefficient = 0.80). The ochratoxin A concentration in pig blood was used as an estimate of the ochratoxin A content of the consumed feed. This method showed that feed from grain produced on-farm contained higher concentrations of ochratoxin A than commercial feed preparations. No geographical variation of ochratoxin A occurrence within Sweden was detected.

Ochratoxin A in pig blood: method of analysis and use as a tool for feed studies.

A procedure is presented for screening the quality of feed in respect to ochratoxin A contamination based upon the analysis of ochratoxin A in pig blood. Representative samples from large feed lots may be obtained by using pigs as in vivo sample collectors which enrich the toxin and forms homogeneous samples in the blood. The spectrofluorometric procedure for ochratoxin A analysis (K. Hult and S. Gatenbeck, J. Assoc. Off. Anal. Chem. 59:128-129, 1976) has been adapted to pig blood and has been simplified to involve only three extraction steps. A volume of 2.5 ml of blood or plasma is needed, and the detection limit is 2 ng of ochratoxin A per ml. The disappearance of ochratoxin A from pig blood as a function of time has been studied. A feeding experiment with ochratoxin A has been performed, and the time course of the concentration of ochratoxin A in blood has been followed during the experiment.

Production of NADPH in the mannitol cycle and its relation to polyketide formation in Alternaria alternata.

The enzymes mannitol-1-phosphate dehydrogenase, mannitol-1-phosphatase, mannitol dehydrogenase and hexokinase participate in an enzymatic cycle in the fungus Alternaria alternata. One turn of the cycle gives the net result: NADH + NADP+ + ATP leads to NAD+ + NADPH + ADP + Pi. The cycle alone can meet the total need of NADPH formation for fat synthesis in the organism. A polyketide producing strain of A. alternata shows a lower mannitol oxidation as well as a lower fat synthesis than a nonproducing mutant, supporting the hypothesis that polyketide formation is favoured at limiting NADPH production. It is further suggested that the mannitol cycle is regulating the glycolytic flux by substrate withdrawal from phosphofructokinase.

Analysis of ochratoxin B alone and in the presence of ochratoxin A, using carboxypeptidase A.

A method is described for ochratoxin B analysis, which is adapted to the earlier described method of ochratoxin A analysis, using carboxypeptidase A (K. Hult and S. Gatenbeck, J. Assoc. Off. Anal. Chem. 59:128-129, 1976). The fluorescence spectra of ochratoxins A and B coincide too much to allow direct discrimination of the two compounds. A method using the differences in kinetic parameters of the enzymatic hydrolysis of the two compounds is suggested for the analysis of mixtures of the ochratoxins.

Intermediates in the barnol biosynthesis in Penicillium baarnense.

By using appropriate 14C-labelled phenolic substances as precursors in feeding experiments the following steps in barnol biosynthesis have been established: acetate + malonate leads to 2,4-dihydroxy-6-ethyl-5-methylbenzaldehyde leads to 1,3-dihydroxy-4,6-dimethyl-2-ethylbenzene leads to barnol. P. baarnense can also reduce orcylaldehyde and 2,4-dihydroxy-5,6-dimethylbenzaldehyde to dimethylresorcinol and trimethylresorcinol, respecitively.

Degradation of ochratoxin A by a ruminant.

The fate of ochratoxin A during incubation with contents from the four stomachs of the cow was studied. It was concluded that ochratoxin A was cleaved into the nontoxic ochratoxin alpha and phenylalanine by the contents from all but the abomasum.

A spectrophotometric procedure, using carboxypeptidase A, for the quantitative measurement of ochratoxin A.

In the method described, ochratoxin A is eleaved into ochratoxin alpha (free isocoumarin chromophore) and phenylaline, using carboxypeptidase. Detection is based on the difference in fluorescence excitation spectra of ochratoxin A (380 NM, maximum) and ochratoxin alpha (340 nm, maximum). The quantitation of ochratoxin A is based on the loss of fluorescence intensity at 380 nm. The method has been used for the quantitative determination of as little as 4 mug ochratoxin A/kg barley and barley meal but it could be extended to other products.