Biochemical, cytotoxic and pharmacokinetic properties of an immunotoxin composed of a mouse monoclonal antibody Fib75 and the ribosome-inactivating protein alpha-sarcin from Aspergillus giganteus.

An immunotoxin was synthesized by the attachment of alpha-sarcin, the ribosome-inactivating protein derived from the mould Aspergillus giganteus, to a monoclonal mouse IgG2 antibody Fib75. The alpha-sarcin immunotoxin exerted toxic effects in tissue culture against the EJ human bladder carcinoma cell line, expressing the antigen recognised by the Fib75 antibody, inhibiting the incorporation of [3H]leucine by 50% at a concentration of 0.46 nM. The cytotoxic effects of the alpha-sarcin immunotoxin were indistinguishable from those of a Fib75 immunotoxin made with ricin A chain. Fib75-alpha-sarcin was cleared from the circulation of the rat with biphasic kinetics following intravenous administration. The alpha- and beta-phase half-lives were 0.8 h and 6 h, respectively, similar to the serum half-lives of analogous Fib75 immunotoxins made with ribosome-inactivating proteins derived from plants. alpha-Sarcin was completely stable in physiological saline buffer at 37 degrees C, whereas the ribosome-inactivating activity of ricin A chain was gradually lost under identical conditions. alpha-Sarcin may be a valuable alternative to ricin A chain for the construction of therapeutic immunotoxins because of its smaller size and greater thermostability.

Acute toxicity of crude extracts of Aspergillus quadrilineatus isolated from acha (Digitaria exilis Stapf).

In a survey of fungi and mycotoxin conterminating acha (Digitaria exilis Stapf) in Plateau State of Nigeria, 96 fungal isolates were made. Screening of the fungi isolates for their mycotoxin-producing potentials showed that Aspergillus quadrilineatus (Thom and Raper) produced some of the most toxic mycotoxins. Two extracts of the Aspergillus quadrilineatus (the petroleum ether soluble extracts [PER] and the petroleum ether defatted crude extract [PEDCR]) were tested for acute toxicity in mice, chicks and cattle. The ip LD50 of PER in mice was 1148 mg/kg, and the oral LD50 of PEDCR was 566 mg/kg in mice and 556 mg/kg in chicks. The ip LD50 of PEDCR in mice was 21 mg/kg. The toxic signs of PER and PEDCR in mice and chicks included tachypnea, tachycardia, anorexia, somnolence, diarrhea, coma and death. The main postmortem findings were congestion of heart, liver, kidney and lungs and sloughing of the wall of stomach and hemorrhagic enteritis. The histopathologic findings in dead animals included edema and mild degeneration of the myocardium and necrosis of kidney tubular epithelial cells, hepatocytes and bronchioles. The only clinical observation in 1 calf orally dosed with a culture of Aspergillus quadrilineatus of maize was transient whole body tremors which occurred 1 h after dosing, tachycardia and profuse salivation. No significant histopathologic changes were found in the organs of the sacrificed calf.

Inhibition of HIV-reverse transcriptase activity by asterriquinone and its analogues.

Asterriquinone (ARQ; 2,5-bis-[1'-(1", 1"-dimethyl-2"-propenyl)- indol-3'-yl]-3,6-dihydroxy-1,4-benzoquinone) and its three analogues [i.e., 3,6-dihydroxy-2-[2'-(1", 1"-dimethyl-2"-propenyl)-indol-3'-yl]-5-[1', 7'- (1",1"-dimethylpropano)-indol-3'-yl]-1,4-benzoquinone (B1-4), 3,6-dihydroxy-2-[2'-(1", 1"-dimethyl-2"-propenyl)-indol-3'-yl]-5-indol-3'-yl-1,4-benzoquinone (C1-1) and 3,6-dihydroxy-2,5-diindol-3'-yl-1,4-benzoquinone (D-1)] were found to be strong inhibitors of the activity of reverse transcriptase from human immunodeficiency virus type-1. Under the reaction conditions employed, the enzyme activity was inhibited by more than 70% in the presence of 10 microM each of these compounds. The mode of inhibition by these compounds was competitive with respect to the template.primer, (rA)n.(dT)12-18, and noncompetitive with respect to the triphosphate substrate, dTTP. The Ki values of HIV-1 reverse transcriptase were determined to be 2.3, 1.5, 0.1 and 0.3 microM for ARQ, B1-4, C1-1 and D-1, respectively.

Fractionation of the ribosome inactivating protein preparations with triazine dyes.

Aspergillins are ribosome-inactivating proteins (RIPs), isolated from several strains of Aspergillus. The interaction between Cibacron Blue F3GA and two members of this family, alpha sarcin and mitogillin, and other RIPs of type I, was studied. Alpha sarcin retention depended on pH and ionic strength. By chromatography on Affi-Gel Blue in mild experimental conditions, mitogillin and PAP-I did not interact with the dye, whereas 40% of alpha sarcin and 70-90% of briodin, RTA and gelonin were recovered in the bound fraction. In all cases, the major fraction showed a higher toxicity level in protein synthesis inhibition assays. The unbound alpha sarcin, conjugated with the anti-ovarian carcinoma monoclonal antibody MOv17, showed on OVCA 432 a cytotoxicity which was 900 times higher than that exerted by the alpha sarcin alone.

[Purification and characterization of beta-N-acetyl hexosaminidase from Aspergillus tamarii].

A beta-N-acetylhexosaminidase from mycelium-free culture filtrate of Asp tamarii S215 was purified to PAGE homogenous by ammonium sulfate and polyethylene glycol fractionation precipitation followed by Sephadex G-50 desalt, DEAE-Sephadex A-25 chromatography, hydroxyapatite chromatography and DEAE-Sephadex A-25 rechromatography with 170-fold purification and 24.7% recovery. The ratio of the beta-GlcNAcase and beta-GalNAcase was 2.5 and remained constant throughout the purification. The Mr estimated with concentration gradient PAGE was 140,000 and subunit Mr determined with SDS-PAGE was 72,000, the number of subunit were 2. The pI was 4.2 determined by PAGIEF. The optimum pH was 5.5-6.5 and 5.0-6.0 for beta-GlcNAcase and beta-GalNAcase respectively with stable pH range 5.5-8.3 for both. The optimum temperature was 60 degrees C for beta-GlcNAcase and beta-GalNAcase. The residual activity of beta-GlcNAcase was 52.7% after treated at 50 degrees C for 8 h and it was 44.9% for beta-GalNAcase. The residual activities of both were down to 1% after treated at 62 degrees C for 10 min. The activity was slightly activated by Mn2+ or Fe2+, while strongly inhibited by Hg2+ and slightly by Ag+, Cu2+, Pb2+, Cd2+ or Zn2+. Analyses of amino acids composition showed that the beta-HexNAcase contained about 24.2% acidic amino acids and 14.9% basic amino acids and only 0.6% methionine.

Improved methods for the preparation of high molecular weight DNA from large and small scale cultures of filamentous fungi.

Improved methods are described for the isolation of pure, high molecular weight DNA from small and large scale cultures of filamentous fungi. The methods depend on the extraction of DNA under conditions which prevent nuclease activity and contamination by carbohydrate. The small scale method depends on enzymatic digestion of the wall whereas the large scale method uses partial damage followed by autolysis. High yields of DNA are obtained by both methods and the DNA is suitable for restriction analysis. Southern Blotting, RFLP analysis, dot blotting and the production of gene libraries. The small scale method can be used for the simultaneous analysis of multiple cultures.

[Biological properties of an alcoholic extract of Aspergillus terreus]. / Proprietà biologiche di un estratto alcoolico di Aspergillus terreus.

The following effects of Aspergillus terreus alcoholic extract are investigated: antiblastic and antiviral effect, and the ability of modification of interferon production and lymphocyte blastic transformation. In particular the extract showed more evident antiproliferative effect on transformed or EBV immortalised cells and lower effect on normal cells. Moreover doses from 12.5 +/- micrograms/ml depressed significatively the in vitro interferon production and lymphocyte blastic transformation induced by PHA on human cells. Mengo and Semliki Forest viruses replication was reduced, although temporarily, in the presence of 50 +/- micrograms/ml of the extract. Eventually the study of some fractions separated on chromatographic column demonstrated the contemporary presence of fractions able to inhibit or stimulate K-562 erythroleukemic cells replication.

Toxicity of echinulin from Aspergillus chevalieri in rabbits.

Rabbits were injected intraperitoneally with purified echinulin, a product of Aspergillus chevalieri. After 2 h, the rabbits were bled and enzyme analyses were carried out on the supernates of liver homogenates and citrated plasma. Elevated levels of total plasma lactate dehydrogenase, cardiac derived isozyme, glutamic-oxaloacetic and glutamic-pyruvic transaminase activities in animals receiving toxin were observed. These levels were statistically significant compared to the vehicle control. A significant increase in liver lactate dehydrogenase of toxin-treated rabbits was also observed. Light-microscopic examination of lung and liver showed a significant degree of damage. The increase in plasma enzyme levels is indicative of damage to these organs.

Binding of fibrinogen to the pathogenic Aspergillus species.

The binding of human fibrinogen to the pathogenic aspergilli was investigated in vitro by different procedures using either fibrinogen in solution or fixed, insolubilized fibrinogen. Binding of fibrinogen was detected at the surface of hyphae and conidia by an immunofluorescence assay. Ultrastructural localization of the binding sites was visualized with fibrinogen-sensitized gold particles. The labelling was restricted to the outer cell wall layer of the 'smooth' walled conidia. Quantitative analysis of the binding carried out with 125I-labelled human fibrinogen on 33 species belonging to different groups (opportunistic fungi, strictly saprophytic or phytopathogenic fungi and dermatophytes or related species) clearly demonstrated that among all the fungi tested, only the pathogenic aspergilli significantly bound fibrinogen. The average amount of fibrinogen bound to individual conidia was also quantified. Binding was greater to Aspergillus niger (5-fold), Aspergillus fumigatus (2.5-fold) and Aspergillus flavus conidia (2.3-fold) than to Aspergillus terreus conidia. The results suggest that fibrinogen binding could contribute to the pathogenesis of aspergillosis.

Aspergillus and other human respiratory disease agents in turkey confinement houses.

The atmosphere of a turkey confinement house on a large Minnesota farm was examined over the course of a year in order to determine levels of airborne contaminants and to evaluate the hazard potential posed by the contaminants to farm workers. Air concentrations of total and respirable dust, ammonia, carbon monoxide, hydrogen sulfide, nitrogen dioxide, methane, and Aspergillus (a fungal respiratory disease agent) were evaluated. Inter- and intra-seasonal variations in confinement house contaminant concentrations were observed. The highest concentrations of dust, ammonia and Aspergillus occurred during the winter months when dust levels averaged 9.3 mg/m3 and ammonia levels averaged 35 parts per million (ppm). Aspergillus levels were lower than expected, never exceeding 73 colony forming units per cubic meter (cfu/m3). Ammonia levels were found to be particularly high during tilling of the confinement house when concentrations greater than 100 ppm were reached. Concentrations of carbon monoxide, hydrogen sulfide, nitrogen dioxide and methane were below detectable levels.

Standardized high-performance liquid chromatography of 182 mycotoxins and other fungal metabolites based on alkylphenone retention indices and UV-VIS spectra (diode array detection).

A general standardized method for the analysis of mycotoxins and other fungal secondary metabolites has been developed, based on high-performance liquid chromatography (HPLC) with an alkylphenone retention index and photodiode-array detection combined with thin-layer chromatography (TLC) in two different eluents. Each fungal secondary metabolite is characterized by its bracketed alkylphenone retention time index, its UV-VIS absorption maxima and its retardation factors relative to griseofulvin in two TLC eluents. This system is effective for the comparison of chemotaxonomic data in different laboratories and for a precise identification of fungi based on organic solvent extracts of fungal cultures. All important groups of mycotoxins and other fungal secondary metabolites could be detected in the HPLC system described and data are listed for 182 metabolites. The fungal secondary metabolites separated and characterized include aflatoxin B1, B2, G1 and G2, ochratoxin A, citrinin, penicillin acid, viomellein, penitrem A, patulin, sterigmatocystin, alternariol, tenuazonic acid, trichothecenes, roquefortines, fusarin C, zearalenone, PR-toxin, citreoviridin, viridicatumtoxin, verruculogen, rugulosin, cyclopiazonic acid, penicillin G and many other alkaloids, polyketides and terpenes.

Averufanin is an aflatoxin B1 precursor between averantin and averufin in the biosynthetic pathway.

Wild-type Aspergillus parasiticus produces, in addition to the colorless aflatoxins, a number of pigmented secondary metabolites. Examination of these pigments demonstrated that a major component was an anthraquinone, averufanin. Radiolabeling studies with [14C]averufanin showed that 23% of the label was incorporated into aflatoxin B1 by the wild type and that 31% of the label was incorporated into O-methylsterigmatocystin by a non-aflatoxin-producing isolate. In similar studies with blocked mutants of A. parasiticus the 14C label from averufanin was accumulated in averufin (72%) and versicolorin A (54%) but not averantin. The results demonstrate that averufanin is a biosynthetic precursor of aflatoxin B1 between averantin and averufin.

[Antiproliferative effect of an extract of Aspergillus terreus]. / Effetto antiproliferativo di un estratto di Aspergillus terreus.

We have studied antiproliferative effect of Aspergillus terreus extract on various cell lines: I-407 normal human cells, L-929 normal murine cells, K-562 erythroleukemic human cells, IAD lymphoblastoid human cells and B 95-8 monkey lymphoblastoid cells. K-562 and lymphoblastoid cells showed higher sensitivity to the extract. Moreover, "a cytostatic dose" of the extract on K-562 cells (12.5 micrograms/ml showed a reduction of 3H-thymidine (but not of 14C-uracil) incorporation, leading us to hypothesize a prevalent action on DNA synthesis.

Anti-inflammatory activity of 6MFA, a compound obtained from fungus Aspergillus ochraceus.

6MFA, an interferon-inducing substance obtained from fungus, Aspergillus ochraceus, has shown anti-inflammatory activity both in acute and chronic animal models of inflammation. It was found that 6MFA was equally effective in inhibiting both exudative as well as granulative phase of inflammation. The compound suppressed also cellular migration during inflammatory process and potentiated significantly the anti-inflammatory activity of indomethacin. The compound was devoid of analgesic or antipyretic activity. The probable mechanism of action of this compound is not fully understood. However, the possibility of triggering the induction of endogenous anti-inflammatory substance(s) along with interferon(s), or interaction of induced interferon(s) directly or indirectly with the prostaglandin system has been attributed.