Biosynthesis of glyantrypine by Aspergillus clavatus

The biosynthesis of glyantrypine from radiolabelled amino acid precursors has been shown experimentally to involve anthranilic acid, tryptophan and glycine. Low values for percentage incorporation of radiolabel into glyantrypine were partly influenced by a complex array of other novel alkaloids shown by the radiolabelling experiments to be related to glyantrypine. Interpretation of radiolabel incorporation from [14C-carboxyl]-anthranilic acid into microbial metabolites seen to contain an anthranilyl moiety in various biosynthetic arrangements is discussed. The possibility of diversion of anthranilic acid from the kynurenine pathway to glyantrypine biosynthesis is recognised.     

Development of membrane-potential responsiveness by myeloid leukemia cells during neutrophilic differentiation

The ability of a myeloid leukemia cell line (HL-60) to undergo membrane electrical potential changes was followed during neutrophilic differentiation induced by 2 compounds. Membrane-potential changes were induced with 12-O-tetradecanoylphorbol 13-acetate (TPA) or formyl-methionyl-leucyl-phenylalanine (FMLP) and were monitored by flow cytometry. The magnitude of the membrane-potential response to TPA increased in a more uniform manner as the population of cells matured than did acquisition of mature morphology or ability to undergo the respiratory burst in response to TPA. The response to TPA and FMLP of HL-60 cells, maximally induced to differentiate by dimethylsulfoxide, closely resembled that of neutrophils. Thus, HL-60 cells may be a useful tool in the study of the relation between membrane depolarization and subsequent cellular activation.     

Honeydew amino acids in relation to sugars and their role in the establishment of ant-attendance hierarchy in eight species of aphids feeding on tansy (Tanacetum vulgare)

Abstract. The ratio of the concentration of honeydew total amino acids to total sugars in the honeydew of eight species of aphids, all feeding on tansy, Tanacetum vulgare (L.), was determined and correlated with honeydew production and ant‐attendance. The honeydew of the five ant‐attended aphid species [Metopeurum fuscoviride (Stroyan), Trama troglodytes (v. Hayd), Aphis vandergooti (Börner), Brachycardus cardui (L.), Aphis fabae (Scopoli)] was rich in total amino acids, ranging from 12.9 to 20.8 nmol µL^?1 compared with the unattended aphid Macrosiphoniella tanacetaria (Kalt.) with only 3 nmol µL^?1. Asparagine, glutamine, glutamic acid and serine (all nonessential amino acids) were the predominant amino acids in the honeydew of all species. The total concentration of amino acids in the phloem sap of tansy was much higher (78.7 nmol µL^?1) then in the honeydew samples, and the predominant amino acids were glutamate (34.3%) and threonine (17.7%). A somewhat unexpected result was the finding that those aphid species with the highest total amino acid concentration in the honeydew always had the highest concentration of sugars. The lowest amino acid–sugar combined value was 104–28.8 nmol µL^?1 in the non ant‐attended species M. tanacetaria, and the highest value was an average of 270–89.9 nmol µL^?1 for the three most intensely attended aphid species M. fuscoviride, A. vandergooti and T. troglodytes. There is no evidence that any single amino acid or group of amino acids in the honeydew acted as an attractant for ant‐attendance in these eight aphid species. The richness of the honeydew (rate of secretion × total concentration of sugars), along with the presence of the attractant sugar melezitose, comprised the critical factors determining the extent of ant‐attendance of the aphids feeding on T. vulgare. The high total amino acid concentration in sugar‐rich honeydews can be explained by the high flow‐through of nutrients in aphids that are particularly well attended by ants.     

Phylogenetic relationships and rate of early diversification of Australian Sphenomorphus group scincids (Scincoidea, Squamata)

The Australian Sphenomorphus group is a morphologically and ecologically diverse clade of lygosomine scincids, collectively comprising more than one‐half of the Australian scincid fauna. A previous phylogenetic analysis of mitochondrial 12S and 16S rRNA, and ND4 and adjacent tRNA sequences for a series of Australian Sphenomorphus group scincids recovered several well‐supported, major clades, although these were generally separated by relatively short branches associated with low support values. Applying a recently described methodology for inferring lineage‐level polytomies, I employ ATP synthetase‐β subunit intron sequences and the existing mitochondrial (mt)DNA data set (with sequences for additional taxa) to assess the hypothesis that the poorly resolved basal relationships within the Australian Sphenomorphus group are a consequence of the major clades having originated essentially simultaneously. Phylogenetic analyses of the separate mtDNA and intron sequence data reveal a number of congruent clades, including Anomalopus, Calyptotis, Ctenotus, Lerista, the Eulamprus quoyii group, the Glaphyromorphus crassicaudis group (including Glaphyromorphus cracens, Glaphyromorphus darwiniensis, and Glaphyromorphus fuscicaudis), Glaphyromorphus gracilipes + Hemiergis, Coeranoscincus reticulatus + Ophioscincus truncatus + Saiphos, and Eulamprus amplus + Eulamprus tenuis + Gnypetoscincus + Nangura. The relationships among these clades indicated by the two data sets, however, are generally incongruent. Although this may be partially ascribed to error in estimating phylogenetic relationships due to insufficient data, some incongruence is evident when uncertainty in inferred relationships is allowed for. Moreover, the congruent clades are typically separated by very short branches, several having a length insignificantly different from zero. These results suggest that initial diversification of Australian Sphenomorphus group scincids was rapid relative to the substitution rates of the mtDNA and intron fragments considered, if not essentially simultaneous. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 92 , 347–366.     

Characterization of Aspergillus niger catalase

Aspergillus niger catalase has been characterized by a variety of physical techniques including gel filtration, sedimentation rate and equilibrium methods and photon correlation spectroscopy. The catalase has a sedimentation coefficient (S₂₀⁰) of 14.2 ± 0.08 S and diffusion coefficient (D₂₀⁰) of 4.14 ± 0.35 × 10⁻⁷ cm² s⁻¹. The average molecular weight of the catalase from all available data including current sedimentation equilibrium measurements and two previous literature values is 345 000. The frictional ratio of the molecule assuming a hydration parameter similar to that of bovine liver catalase (.3 g H₂O g⁻¹) is 1.103, suggesting that Aspergillus niger catalase has an asymmetric structure with an axial ratio of approximately 3 (the Stokes radius is 5.83 ± 0.49 nm). The titration curve and amino acid analysis indicate that in the native conformation only 23% of the ionizable amino acid residues are titratable between pH 3 and 10.5. Denaturation with sodium n-dodecylsulphate increases the number of titratable groups to 46%. The ratio of anionic to cationic amino acid residues in Aspergillus niger catalase is 2.46 and the isoelectric point is 6.5. The optimum pH for catalytic activity is approximately 7.     

Tyrosine Hydroxylase Is Activated and Phosphorylated on Different Sites in Rat Pheochromocytoma PC12 Cells Treated with Phorbol Ester and Forskolin

Abstract: Incubation of rat pheochromocytoma PC12 cells with 4β-phorbol-12β-myristate-13α-acetate (PMA), an activator of Ca²⁺/phospholipid-dependent protein kinase (protein kinase C), or forskolin, an activator of adenylate cyclase, is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase. Neither the activation nor increased phosphorylation of tyrosine hydroxylase produced by PMA is dependent on extracellular Ca²⁺. Both activation and phosphorylation of the enzyme by PMA are inhibited by pretreatment of the cells with trifluo-perazine (TFP). Treatment of PC 12 cells with l-oleoyl-2-acetylglycerol also leads to increases in the phosphorylation and enzymatic activity of tyrosine hydroxylase; 1, 2-diolein and 1, 3-diolein are ineffective. The effects of forskolin on the activation and phosphorylation of the enzyme are independent of Ca²⁺ and are not inhibited by TIT⁵. Forskolin elicits an increase in cyclic AMP levels in PC 12 cells. The increases in both cyclic AMP content and the enzymatic activity and phosphorylation of tyrosine hydroxylase following exposure of PC 12 cells to different concentrations of forskolin are closely correlated. In contrast, cyclic AMP levels do not increase in cells treated with PMA. Tryptic digestion of the phosphorylated enzyme isolated from untreated cells yields four phosphopeptides separable by HPLC. Incubation of the cells in the presence of the Ca²⁺ ionophore ionomycin increases the phosphorylation of three of these tryptic peptides. However, in cells treated with either PMA or forskolin, there is an increase in the phosphorylation of only one of these peptides derived from tyrosine hydroxylase. The peptide phosphorylated in PMA-treated cells is different from that phosphorylated in forskolin-treated cells. The latter peptide is identical to the peptide phosphorylated in dibutyryl cyclic AMP-treated cells. These results indicate that tyrosine hydroxylase is activated and phosphorylated on different sites in PC 12 cells exposed to PMA and forskolin and that phosphorylation of either of these sites is associated with activation of tyrosine hydroxylase. The results further suggest that cyclic AMP-dependent and Ca²⁺/ phospholipid-dependent protein kinases may play a role in the regulation of tyrosine hydroxylase in PC 12 cells.     

Cotransformation of Aspergillus nidulans: a tool for replacing fungal genes

Summary When a non-selected DNA sequence was added during the transformation of amdS320 deletion strains of Aspergillus nidulans with a vector containing the wild-type amdS gene the AmdS⁺ transformants were cotransformed at a high frequency. Cotransformation of an amdS320, trpC801 double mutant strain showed that both the molar ratio of the two vectors and the concentration of the cotransforming vector affected the cotransformation frequency. The maximum frequency obtained was defined by the gene chosen as selection marker for transformation. Cotransformation was used to induce a gene replacement in A. nidulans. An amdS320 strain was transformed to AmdS⁺ and cotransformed with a DNA fragment containing a fusion between a non-functional A. nidulans trpC gene and the Escherichia coli lacZ gene. Ten AmdS⁺, LacZ⁺ transformants with a Trp^– mutant phenotype were selected. All of these strains could be transformed with a functional copy of the A. nidulans trpC gene, but only two strains yielded TrpC⁺ transformants which, with a low frequency, had a LacZ^– phenotype. These latter transformants had also lost the AmdS⁺ phenotype. Southern blotting analysis of DNA from these transformants confirmed the inactivation of the wild-type trpC gene, but revealed that amdS vector sequences were also involved in the gene replacement events.     

Aspergillus myositis in a patient with a myelodysplastic syndrome

We present the case of an elderly man who, while being treated with corticosteroids for a myelodysplastic syndrome, developed myositis of the calf due to Aspergillus fumigatus. Despite therapy with amphotericin B the myositis failed to resolve and he died. At autopsy, a localized necrotizing myositis of the right calf was found with no evidence of disseminated Aspergillus infection. Myositis in the setting of disseminated candidiasis or cryptococcosis has been previously reported. This case is unique in that it is the first reported case of localized fungal myositis and of myositis caused by Aspergillus.     

Effect on aflatoxin production of competition between wild-type and mutant strains of Aspergillus parasiticus

Co-cultivation of a strain of Aspergillus parasiticus, capable of making aflatoxins, with blocked mutant strains, capable of producing none or only a low level of aflatoxins, reduced the net yield of aflatoxins more than that expected based on spore recovery. Yields of aflatoxins were 8-fold less for a norsolorinic acid-producing strain, 14-fold less for an averantin-producing strain, 6-fold less for an averufin-producing strain, and 21-fold less for a versicolorin A-producing strain when co-cultured in equal amounts with a wild-type strain of Aspergillus parasiticus. Even when the wild-type strain was initially present in 100-fold excess, with two of the mutant strains, reduced aflatoxin production was still observed.     

Effect of roasting on ochratoxin A level in green coffee beans inoculated with Aspergillus ochraceus

The heat stability of ochratoxin A in green coffee beans inoculated with Aspergillus ochraceus was studied. Heat treatment (roasting) at 200 °C for 10 or 20 min reduced the levels of ochratoxin A by only 0–12% in the dried whole beans. Almost all of the ochratoxin A was infused into the coffee decoction when the roasted samples were ground and extracted with boiling water. Therefore, the reduction of ochratoxin A concentration of contaminated coffee beans by roasting under these conditions is ineffective.