Heterologous protein secretion directed by a repressible acid phosphatase system of Aspergillus niger.

A new expression-secretion system of Aspergillus niger which directs the secretion of heterologous proteins is described. The promoter and signal peptide-encoding region of the phosphate-repressible aphA gene of A. niger, when fused to the coding region of the human interferon alpha 2 (hIFN alpha 2)-encoding gene (hIFN alpha 2), drives the expression of this gene and the secretion of the hIFN alpha 2 protein. Synthesis of hIFN alpha 2 in either A. niger or A. nidulans transformants carrying these constructs was regulated by inorganic phosphate (Pi) present in the medium, so that derepression of heterologous protein expression can be attained by lowering Pi concentration.

Characterization of an Aspergillus nidulans genomic DNA fragment conferring phosphate-non-repressible acid-phosphatase activity.

A clone from an Aspergillus nidulans library was identified by its ability to confer enhanced staining for acid phosphatase (APase) activity upon phosphatase-deficient A. nidulans mutants. This APase activity is not repressed by high phosphate concentrations in the medium. The 2.9-kb nucleotide sequence in the region of the clone responsible for the effect reveals two potential protein-coding genes with a common N terminus. One corresponds to an open reading frame (ORF) with no introns, encoding 330 amino acids (aa). The other, shorter gene encoding 113 or 117 aa has the first 65 or 69 codons in common with the long ORF; then, after a single 165-nt intron with a fungal consensus lariat sequence and splice junctions, there are a further 48 codons in a different reading frame. Both correspond in sense direction, and the shorter gene in length, with the only detectable transcript in this region, but both differ from all known APase sequences. The possible identity of these ORFs with the pacG gene is discussed.

Evaluation of transgenic canola plants under field conditions.

Eleven independent transgenic canola (Brassica napus ssp. oleifera L. cv. Westar and Regent) lines were evaluated in the field. The plants carried a neomycin phosphotransferase (NPTII) gene for kanamycin resistance that was introduced via Agrobacterium-mediated transformation. NPTII enzyme assays, Southern blot by hybridizations and progeny analysis, confirmed the stable, heritable integration and expression of the introduced NPTII gene. A number of agronomic characteristics evaluated under field conditions, including maturity yield, and oil and protein content, were all statistically comparable between the transformed and nontransforemd platns. These results indicate that canola can be genetically engineered successfully, and that the Agrobacterium-based transformation system employed does not induced any adverse effects on the intrinsic agronomic and qualitative traits critical to the agricultural industry.

The antiviral action of lignans.

A total of 18 purified lignans was evaluated for antiviral activity against murine cytomegalovirus (CMV) and Sindbis virus, by means of different treatment regimens. Podophyllotoxin and alpha-peltatin were the most potent compounds, and they apparently inhibited murine CMV at an essential early step in the replication cycle after the adsorption of virus to the cells. On the other hand, justicidin B and the diphyllin derivatives were much more effective against Sindbis virus, and 12 of the lignans had no demonstrable effect at all, despite their known activities in other bioassays.

A phosphate-repressible acid phosphatase gene from Aspergillus niger: its cloning, sequencing and transcriptional analysis.

The cloning and sequencing of an Aspergillus niger gene encoding a secreted form of phosphate-repressible acid phosphatase by complementation of a pacA (phosphate-repressible acid phosphatase) mutant of Aspergillus nidulans is described. The gene contains two introns, 201 and 265 nt in length, and codes for a 1.6-kb transcript. Both phosphate concentration and pH of the growth medium affect the level of expression of the gene in A. niger. Similar regulation is observed in A. nidulans transformants. A putative signal peptide, resembling known signal sequences of yeast, is identified.

Studies on the pharmacological activity of Amazonian Euphorbiaceae.

Plant material from 34 Amazonian species of the family Euphorbiaceae were collected and extracts prepared. Sixteen of these species have a documented use as a medicinal agent. The extracts were tested for their ability to inhibit the growth of the bacteria, Escherichia coli and Staphylococcus aureus; the yeasts, Saccharomyces cerevisiae and Candida albicans; the dermatophytic fungi, Microsporum canis, Microsporum fulvum, Microsporum gypseum and Trichophyton gallinae; the viruses, Sindbis virus and murine cytomegalovirus; and tumours induced on potato discs by Agrobacterium tumefaciens. They were also examined for their toxicity to brine shrimp, Artemia salina. The results are discussed with respect to ethnobotanical information available for some of the species.

Iron Specifically Protects Corn Protoplasts from T-Toxin of Cochliobolus heterostrophus.

Ferric ion reduced the damaging effects of T-toxin, a series of linear beta-polyketols produced by the pathogenic fungus Cochliobolus heterostrophus, on leaf mesophyll protoplasts from susceptible T-cytoplasm corn. Of nine metals tested, only ferric and ferrous ions had this effect. Despite the presence of 12 available oxygen atoms in each T-toxin molecule, there was no evidence for the formation of an aqueous Fe(2+)- or Fe(3+)-T-toxin complex. The protective effect of iron was eliminated by a molar excess of EDTA. Iron had no effect on the sensitivity of T-cytoplasm mitochondria to T-toxin, even at a 1000-fold molar excess, nor did it protect roots of T-cytoplasm corn seedlings from inhibition by T-toxin. The mechanism by which iron specifically protects protoplasts from T-toxin is not understood, but time lapse experiments suggest that iron acts on some intracellular site to modify T-toxin sensitivity and not on a transport system at the cell surface.

A cloned tryptophan-synthesis gene from the ascomycete Cochliobolus heterostrophus functions in Escherichia coli, yeast and Aspergillus nidulans.

A gene (TRP1) in the tryptophan biosynthetic pathway of the fungal plant pathogen Cochliobolus heterostrophus was isolated by complementation of an Escherichia coli trpF mutant which lacked phosphoribosylanthranilate isomerase (PRAI) activity. The cloned gene also complemented an E. coli trpC mutant lacking indoleglycerolphosphate synthase (IGPS) activity, a yeast trp1 mutant missing PRAI activity and an Aspergillus nidulans trpC mutant. It functioned in E. coli and A. nidulans without apparent rearrangement but in yeast only after the 5' end of the gene was deleted. The gene was subcloned on a 4.65-kb DNA fragment and the PRAI domain was localized to a 2.9-kb region. It showed homology to the A. nidulans trpC and Neurospora crassa trp-1 genes. There was one predominant transcript of C. heterostrophus TRP1, the same size (2.6-kb) as one of the two functional transcripts produced by A. nidulans trpC. The constitutive activity of the C. heterostrophus TRP1 gene was high whereas that of the A. nidulans trpC gene was low.

An ethnopharmacological examination of Virola elongata bark: a South American arrow poison.

The use of the resin of Virola elongata as an arrow poison was investigated. Aqueous and methanolic extracts of the dried bark were not observed to have toxic effects when administered intraperitoneally to mice. In an attempt to determine if the hallucinogenic indole alkaloid constituents of the bark, which form the basis for the alternate use of this material as a ceremonial snuff, could also be responsible for its use as an arrow poison, alkaloidal and non-alkaloidal extracts were compared with respect to their behavioral effects on mice. The non-alkaloidal extract was more effective in producing an observable alteration in behavior. This consisted of a marked reduction in spontaneous locomotor activity. The extract was fractionated and 13 of the major constituents assayed for their ability to reduce spontaneous locomotor activity. Most of this biological activity of the extract was attributable to the presence of the bis-tetrahydrofuran lignans, epi-sesartemin, sesartemin, epi-yangambin and yangambin. Each of these compounds was also observed to reduce isolation induced aggression when administered to mice.

Justicia pectoralis: a study of the basis for its use as a hallucinogenic snuff ingredient.

The use of Justicia pectoralis var. stenophylla as a Virola snuff admixture and also as the sole ingredient of a snuff was investigated. Extracts of the plant did not contain alkaloids, although the ubiquitous compound, betaine, was isolated because of its reaction with alkaloid reagents. Nor did extracts have any significant effect upon the gross behavioral effects, or increased spontaneous motor activity, elicited in mice by 5-methoxy-N,N-dimethyl-tryptamine (5-MeODMT), the primary psychotropic constituent of the Virola resin snuff. Coumarin and umbelliferone were identified because they are major constituents of the plant and because of their ability to relax smooth muscle.

Examination of naturally occurring polyacetylenes and alpha-terthienyl for their ability to induce cytogenetic damage.

alpha-Terthienyl and 5 polyacetylenes were examined for chromosome damaging activity using Syrian hamster cells. None of these naturally occurring compounds induced sister chromatid exchanges and neither alpha-terthienyl nor phenylheptatriyne induced chromosome aberrations.

Induction of sister-chromatid exchanges in Chinese hamster ovary cells by thiol and hydrazine compoudns.

Cysteine, cysteamine and glutathione all induce sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells when applied to cell cultures at concentrations between 10(-4) and 10(-2) M. Acute exposure of cells th thiol compound for a period of 2--3 h resulted in a unique dose--response relationship in each instance. This consisted of two peak SCE frequencies, one at either extreme of the concentration range. Each peak corresponded to a 2--3-fold increase over the spontaneous level. A chronic exposure of 24 h, in contrast, resulted in a dose--response relationship consisting of a single peak SCE frequency (representing a 4--5-fold increase over the spontaneous level) at a concentration of approx. 4 x 10(-4) M. The effect of Cu2+ ions included in the medium at a concentration of 10(-5) M was to increase the toxicity and, at some concentrations, the SCE levels occurring after either acute or chronic exposure to thiols. Hydrazine and its derivatives, dimethylhydrazine and isonicotinic acid hydrazide (isoniazid), as well as hydrogen peroxide, also induce SCEs in CHO cells. A 2--3-fold increase over the spontaneous level was observed, depending upon the particular treatment protocol applied. SCE yields after 3 h treatment with dimethylhydrazine and isoniazid were increased if Mn2+, but not Cu2+, was included in the tissue culture medium at a concentration of 10(-5) M. SCE yields after a 24-h treatment with dimethylhydrazine in which Mn2+ was present in, and absent from, the medium were similar. Catalase was observed to reduce the SCE levels resulting from treatment with hydrogen peroxide, dimethylhydrazine and isoniazid. The effect of catalase upon SCEs induced by dimethylhydrazine and isoniazid in the presence of Mn2+ was more evident than when Mn2+ was not included in the culture medium. The significance of these results with respect to the possible active chemical species produced and the mutagenic/carcinogenic risk associated with thiol and hydraizine compounds is discussed.

Effects of arginine deprivation upon chromosome aberrations, sces and survival of cho cells treated with mutagenic agents.

Arginine deprivation sensitizes CHO cells to the clastogenic activity of the mutagenic agents UV light, N-methyl-N'-nitro-N-nitrosoguanidine, mitomycin C and 4-nitroquinoline-1-oxide. Cells were allowed to undergo proliferative arrest by deprivation of the amino acid arginine, treated with mutagenic agent and refed with complete medium. The resulting mitotic cells displayed more chromosome aberrations than did mitotic cells in proliferating cell cultures which had been treated similarly. This effect was observed at each dose tested (representing a 300-fold range in concentration). Survival of arginine-deprived cells exposed to UV light was also markedly reduced in comparison to the response of proliferating cells. Sister-chromatid exchange levels induced by MNNG, in contrast, were similar in arginine-deprived and proliferating cells.

Sister chromatid exchanges induced in cultured mammalian cells by chromate.

Chromate compounds induced sister chromatoid exchanges (SCEs) and chromosome aberrations in cultured mammalian cells. Similar increases in SCE frequency were observed in human fibroblasts exposed to the compounds K2Cr2O7 and K2CrO4. Marked increases in SCE frequency in cells exposed to chromate for a 48-h period were detected at concentrations between 10(-7) and 10(-6) M. Chromosome aberrations (primarily chromatid breaks) were also produced in human cells exposed to K2CrO4 at concentrations between 8 . 10(-7) and 3 . 10(-6) M. K2CrO4, but not the trivalent compound CrCl3, induced SCEs in Chinese hamster ovary (CHO) cells at low concentrations.