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Incidence and mortality rates of invasive aspergillosis clearly indicate the need of novel antifungals to treat patients suffering from this disease. Fungal proteins playing a crucial role in pathogenesis and with no orthologue in human cells are considered as primary therapeutic targets for the development of new antifungals with a high therapeutic index, one of the major drawbacks of the standard antifungal therapy, so far. In this work, we have analyzed the role in pathogenesis of the key enzymes of the Aspergillus fumigatus glyxoxylate cycle, isocitrate lyase and malate synthase, two possible candidates to primary therapeutic targets in this fungus. Deletion strains lacking isocitrate lyase (DeltaacuD strains) or malate synthase (DeltaacuE mutants) were constructed in this work. The Neurospora crassa pyr-4 gene was used as the replacing marker in gene deletion experiments. The pathogenicities of DeltaacuD and DeltaacuE mutants were tested in neutropenic mice and compared with those of two reference wild-type isolates A. fumigatus 237 and A. fumigatus 293. Interestingly, virulence and cytological studies clearly indicated the dispensability of the A. fumigatus glyoxylate cycle for pathogenicity. In addition, these results suggested the suitability of the pyr-4 gene as a valuable replacing marker for virulence studies in this fungus, a fact that was further confirmed by gene expression analyses. Finally, growth tests were performed to investigate the germination and growth of the DeltaacuD and DeltaacuE strains in nutrient deprivation environments, resembling the conditions that A. fumigatus conidia face after phagocytosis. Results obtained in this work strongly suggest that the ability to grow on lipids (triglycerides) of A. fumigatus isocitrate lyase and malate synthase deletion strains accounts for their fully virulent phenotype.

A search for suppressors of the carnitine/acylcarnitine translocase (CACT) deficiency in Aspergillus nidulans permitted the identification of the suaE7 mutation, mapping at a new translational suppressor (suaE) gene. The suaE gene is essential in A. nidulans and encodes the eukaryotic release factor 1 (eRF1). The suaE7 mutation suppresses two acuH alleles (acuH13 and acuH31), both carrying nonsense mutations in the CACT encoding gene that involve the replacement of a CAG (Gln) codon with a premature TAG stop codon. In contrast, the suaE7 gene does not suppress the acuH20 amber nonsense mutation involving a TGG-->TAG change. The phenotype associated to the suaE7 mutation strictly resembles that of mutants at the suaA and suaC genes, two translational suppressor genes previously identified, suggesting that their gene products might functionally interact in translation termination. Sequencing of the suaE7 gene allowed the identification of a mutation in the domain 2 of the omnipotent class-1 eukaryotic release factor involving the Gly265Ser substitution in the A. nidulans eRF1. This mutation creates a structural context unfavourable for normal eRF binding that allows the misreading of stop codons by natural suppressor tRNAs, such as the tRNAs(Gln). Structural analysis using molecular modelling of A. nidulans eRF1 domain 2 bearing the G265S substitution and computer simulation results suggest that this mutation might impair the necessary conformational changes in the eRF1 to optimally recognize the stop codon and simultaneously interact with the peptidyl transferase centre of the 60S ribosomal subunit.

Deficiency of the carnitine/acylcarnitine translocase (CACT), the most severe disorder of fatty acid beta-oxidation, is usually lethal in both humans and animals, precluding the development of animal models of the disease. In contrast, CACT deficiency is conditionally lethal in the fungus Aspergillus nidulans, since loss-of-function mutations in acuH, the translocase structural gene, do not prevent growth on carbon sources other than ketogenic compounds, such as fatty acids. Here, we describe the molecular characterization of extant acuH alleles and the development of a fungal model for CACT deficiency based on the ability of human CACT to fully complement, when expressed at physiological levels, the growth defect of an A. nidulans DeltaacuH strain on acetate and long-chain fatty acids. By using growth tests and in vitro assays this model enabled us to carry out a functional characterization of human CACT mutations showing that it may be useful for distinguishing potentially pathogenic human CACT missense mutations from neutral, single residue substitution-causing polymorphisms.

A study of the biodegradation of several chiral polychlorinated biphenyls (PCBs, IUPAC numbers 45, 88, 91, 95, 136, 144, 149, and 176) by a naturally occurring soil bacterium as well as the stereoselectivity of such process has been performed using Electrokinetic Chromatography with cyclodextrins (CD-EKC). In order to achieve the analysis of chiral PCBs, a CD-EKC system based on 50 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.5) containing 2 M urea, 20 mM carboxymethylated ccyclodextrin (CM-c-CD), and 10 mM b-cyclodextrin (b-CD) was used. First, the percentage degradation of PCBs by the microorganism used in this work was measured for different incubation times (22, 46, 71, 93, 165, 213, and 262 hours). The results showed a high degree of biodegradation (from 61% to 94%) for the eight PCBs studied after 262 hours of incubation with the microorganism, degradation being favoured for the least chlorinated congeners. Secondly, measurement of the atropisomeric ratio of the PCB atropisomers during the biodegradation process revealed no significant variation of the atropisomeric ratios with the degradation time, indicating a nonstereoselective degradation of PCBs by the microorganism, The authors thank the Comunidad Autónoma de Madrid (Spain) for projects 07 M/0640/1997 and 07 M/0049/1998.

In Aspergillus nidulans, activity of the glyoxylate cycle enzyme isocitrate lyase is finely regulated. Isocitrate lyase is induced by growth on C2 compounds and long-chain fatty acids and repressed by glucose. In addition, activity of isocitrate lyase is subject to a second mechanism of catabolite control, glucose-induced inactivation. Here, we demonstrate that the catabolite inactivation of A. nidulans isocitrate lyase, a process that takes place during glucose adaptation of cells grown under gluconeogenic conditions, occurs by proteolysis of the enzyme. Ultrastructural analyses were carried out in order to investigate the cellular processes that govern the catabolite inactivation of this peroxisomal enzyme. Addition of glucose to oleate-induced cells triggered the specific engulfment and sequestration of peroxisomes by the vacuoles. Sequestration of various peroxisomes by a single vacuole was a frequently observed phenomenon. Results obtained by immunoelectron microscopy using antibodies against A. nidulans isocitrate lyase showed that degradation of this peroxisomal enzyme occurred inside the vacuole. In addition, ultrastructural studies demonstrated that microautophagy was the autophagic pathway involved in degradation of redundant peroxisomes during glucose adaptation of oleate-induced cells of A. nidulans.

The Aspergillus nidulans acuH gene, required for growth on acetate and long-chain fatty acids, was cloned by complementation of the acuH13 mutation. Northern blotting analysis showed that transcription of the acuH gene occurs in acetate-grown mycelium and at higher levels in oleate-grown mycelium, but not during growth on glucose minimal medium. The acuH gene encodes a protein of 326 amino acids that belongs to the mitochondrial carrier family. The ACUH protein contains three related segments of approximately 100 amino acids in length, each segment comprising two hydrophobic domains that are probably folded into two transmembrane alpha-helices linked by an extensive polar region. Sequence comparisons suggest that the acuH gene of A. nidulans encodes the homologue of the carnitine/acylcarnitine carrier of rat and man. The uncharacterised proteins YOR100C of Saccharomyces cerevisiae, COLT of Drosophila melanogaster, and DIF-1 of Caenorhabditis elegans also seem to be homologues of ACUH. In addition to the motifs present in all members of the mitochondrial carrier family, we propose the highly conserved motif R(A,S)(V,F)PANAA(T,C)F within the sixth hydrophobic domain of these proteins as the characteristic feature of the carnitine carrier subfamily. The proposed function of the ACUH protein is the transport of acetylcarnitine molecules from the cytosol to the mitochondrial matrix, a process required during growth on acetate or on long-chain fatty acids.

Some commercial soybean products—soybean protein isolate, soybean flour, textured soybean, whole soybeans, and soybean dairy-like products (liquid and powdered milks, shake, yogurt, and infant formulas)—have been analysed for their content in solids, ash, pH, acidity, protein, fat, phosphorus, and some metal ions (calcium, copper, iron, potassium and zinc). The differences found in the protein, phosphorus, and metal ion content and other chemical properties of these products are discussed, taking into account the procedures used to produce the above derivatives.

Summary: The role of actin in apical growth and enzyme secretion in the filamentous fungusAspergillus nidulanswas studied by treating the hyphae with cytochalasin A (CA), which inhibits actin polymerization. Indirect immunofluorescence microscopy revealed actin at the tips of main hyphae and branches, and at the sites of developing septa. CA inhibited the growth of the fungus and changed the growth pattern of hyphal tips from cylindrical tubes to spherical beads. The regions with swellings showed no actin fluorescence, and neither was actin seen in association with septa. After 4 h exposure, hyphae were able to resume the normal tip growth pattern in the presence of CA for a short period of time and new cylindrical hyphae, with actin fluorescence at the apex, emerged from the swollen tips. Later, the tips of the hyphae swelled again, which led to a beaded apperance. We also studied the effect of CA on the secretion of α- and β-galactosidase. α-Galactosidase is secreted into the culture medium, whereas β-galactosidase remains in the mycelium, with part of its activity bound to the cell wall. WhenA. nidulansmycelium was incubated in the presence of CA, a reduction in the secretion of α-galactosidase into the culture medium and a decrease in the α- and β-galactosidase activities bound to the cell wall was detected. However, the CA dose used for the hyphae did not modify the secretion of the enzymes from protoplasts. Results described here provide evidence that a polymerized actin cytoskeleton is required for normal apical growth, hyphal tip shape and polarized enzyme secretion inA. nidulans.Cytochalasin-induced disruptions of the actin cytoskeleton could result in the alterations of apical growth and inhibition of enzyme secretion observed by blocking secretory vesicle transport to the apex.

Some newly isolated microorganisms could solubilize different kinds of Spanish coals. Certain fungi and bacteria could solubilize lignite when growing in a mineral medium. However, to solubilize higher rank coals (hard coal and subbituminous coal) microorganisms require a complete medium for growth. In order to get some insight into the mechanisms of biosolubilization, cell-free culture filtrates of fungi were tested on their capacity to solubilize different Spanish coals. Two kinds of cell-free culture filtrates were used: a non-coal induced cell-free culture filtrate (NCI) and a coal induced cell-free culture filtrate (CI). Either of them was able to solubilize hard coal, subbituminous coal and most efficiently lignite. In most of the culture filtrates the measured pH values were approximately neutral, even acidic in some cases. The agent(s) for coal solubilization that was present in NCI was heat stable (100°C) but the active substance(s) in CI was thermosensitive. Microorganisms were observed by electron microscopy to grow on the coal surface and cover it. A fibrilar extracellular polymer which could facilitate not only the adhesion to the coal particle but microbial attack too, was produced by the microorganisms.

In order to facilitate the purification of isocitrate lyase from Aspergillus nidulans , the isocitrate lyase overexpressing strain JCB4a was derived. Isocitrate lyase was purified to homogeneity by the criterion of polyacrylamide gel electrophoresis and anti-isocitrate lyase polyclonal antibodies were raised. Stabilization of purified enzyme, when stored at – 20 °C, required the addition of 1 mM dithiothreitol (DTT) plus 1 mM ethylenediaminetetraacetate (EDTA). Aspergillus nidulans isocitrate lyase is a multimeric enzyme with a native molecular weight of 240 kDa and composed of four monomers of 59 kDa. The enzyme required 5 mM Mg and 1 mM DTT or cysteine for full activity. EDTA at 1 mM replaced the requirement of a thiol compound for activity. The K m for threo -D s -isocitrate was 0·050 mM, and the enzyme activity was inhibited by succinate, itaconate and structural analogs of glyoxylate as well as by fructose-1,6-bisphosphate.

Aspergillus nidulans is able to grow on oleic acid as sole carbon source. Characterization of the oleate-induced beta-oxidation pathway showed the presence of the two enzyme activities involved in the first step of this catabolic system: acyl-CoA oxidase and acyl-CoA dehydrogenase. After isopicnic centrifugation in a linear sucrose gradient, microbodies (peroxisomes) housing the beta-oxidation enzymes, isocitrate lyase and catalase were clearly resolved from the mitochondrial fraction, which contained fumarase. Growth on oleic acid was associated with the development of many microbodies that were scattered throughout the cytoplasm of the cells. These microbodies (peroxisomes) were round to elongated, made up 6% of the cytoplasmic volume, and were characterized by the presence of catalase. The beta-oxidation pathway was also induced in acetate-grown cells, although at lower levels; these cells lacked acyl-CoA oxidase activity. Nevertheless, growth on acetate did not cause a massive proliferation of microbodies in A. nidulans.

The effect of the antimicrotubular drug methyl benzimidazol-2-yl carbamate (MBC) on the production and secretion of acid phosphatase, α-galactosidase and β-galactosidase in Aspergillus nidulans was studied. A wild type and two benomyl resistant mutant ( benA10 and benC28 ) strains were used. All the strains secreted acid phosphatase and α-galactosidase into the culture medium, whereas β-galactosidase remained in the mycelium with a portion of its activity bound to the cell wall. When the wild type strain was incubated in the presence of a sublethal dose of MBC, a decrease of the activity of the enzymes studied was found. A reduction in the secretion of acid phosphatase and α-galactosidase into the culture medium was also observed. In addition, a decrease in the percentage of α- and β-galactosidase activities bound to the cell wall was detected. The MBC dose used for the wild type strain did not modify either the total enzyme activities or the secretion of the enzymes studied in the benomyl resistant mutant benA and benC strains. However, when those strains were grown in the presence of a sublethal dose of MBC, a decrease in the total enzyme activities as well as a reduction in acid phosphatase and α-galactosidase secretion was found. In addition, alterations in the percentage of enzyme activities bound to the cell wall were observed in both mutant strains. Results described in this work, clearly suggest that microtubules are involved in the polarized secretion of enzymes in A. nidulans .

The ability of Acinetobacter calcoaceticus MM5 to degrade heating oil was studied. Certain culture conditions relevant to the degradation of this petroleum derivative were also analyzed. Degradation of heating oil by MM5 was followed by gas chromatography during a 30-day incubation period. A. calcoaceticus MM5 degraded essentially the n-alkane components of the fuel, and left the branched hydrocarbons pristane and phytane undegraded. The degradation of heating oil was favoured by aeration of the cultures. There was an absolute nutritional requirement for nitrogen and phosphorus sources for hydrocarbon degradation. Strain MM5 degraded heating and diesel oil to a similar extent. The presence of commercial additives in the heating oil did not modify the degradation of this fuel. Infrared spectroscopy of remaining heating oil after incubation with strain MM5 showed the appearance of two new bands, corresponding to compounds with carbonyl groups derived from the metabolism of hydrocarbons.

A bacterial strain was isolated from a sample of contaminated heating oil and identified as a strain of Acinetobacter calcoaceticus, named MM5. The bacterial isolate was able to grow on petroleum derivatives and brought about an emulsification of those compounds. A bioemulsifier was extracted from the culture medium of MM5 strain and partially characterized. This compound was able to emulsify petroleum fuels and both aliphatic and aromatic pure hydrocarbons and was stable over a wide range of temperatures. Studies developed by light, scanning electron and transmission electron microscopy showed that, during the growth on petroleum derivatives, the microorganisms were orientated on the surface of drops enclosed in a skin or membranous polymer produced by the bacteria. These droplets may represent the hydrocarbon/water emulsion of the liquid culture. The growth of A. calcoaceticus MM5 on media containing both hydrocarbon and water-soluble substrates as carbon sources also results in the formation of a film, consisting of amorphous and membranous layers. The bacteria were connected to the biofilm and showed intercellular contacts through cell-surface appendages, forming a complex network. The importance of the biofilms for bacterial adhesion to oil droplets and for its nourishment is discussed.

Twenty aerobic bacterial strains were isolated from altered heating oil. Among them the strain catalogued as MM5 and identified as Acinetobacter calcoaceticus is able to grow on hydrocarbon substrates. When strain MM5 was grown on heating oil, crude oil and tetradecane, increases of protein concentration and of caprilate-lipase and acetate-esterase enzymatic activities were observed in the culture filtrate, with a simultaneous pH drop. A strong emulsification of petroleum by-products was also noticed. Degradation of heating oil was followed by gas chromatography and infrared spectroscopy. Presence of available nitrogen and phosphorus sources were essential for hydrocarbon biodegradation. Intracellular electron transparent inclusions were observed by transmission electron microscopy when strain MM5 cells were grown on hydrocarbons. Light and scanning electron microscopy showed bacteria interconnected by an extracellular polymer and attached to hydrocarbon droplets and to sheets of polymeric material. A bioemulsifier was extracted from the cell-free culture supernatants of strain MM5 grown on tetradecane. The emulsifier is a high molecular weight product that comprises proteins, sugars and fatty acids and which is resistant to high temperature. Strain MM5 should be helpful for the design of strategies for the bioremediation of hydrocarbon contaminated sites.

The role of microtubules (MTs) in the protein secretion process in Cladosporium cucumerinum has been studied using the antimicrotubular drug methyl benzimidazol-2-yl carbamate (MBC). Sublethal doses of this compound increased the level of acid and alkaline phosphatase enzymes in the culture filtrate, although fungal growth was reduced. The incorporation of [35S]methionine into the extracellular proteins was not modified by MBC. However, alterations in the electrophoretic pattern of proteins were observed in the presence of the fungicide. Distribution of MTs in hyphae was studied by indirect immunofluorescence microscopy; in untreated control samples, the fluorescence was especially abundant in the apical dome of the hyphae; following incubation of the fungus in MBC, a decrease in tubulin-positive staining in this zone was observed.

A screening procedure has been set up for isolating microorganisms capable of liquefying coal. Spanish coals were used in the tests, namely a brown lignite from Galicia, sub-bituminous coal from Teruel and hard coal from Minas Figaredo S.A. (Asturias). Among the isolated strains several microorganisms proved capable of liquefying untreated lignite. When lignites were pretreated a more intense and rapid liquefaction was achieved, chelating agents being among the best pretreatments. None of the isolated microorganisms could satisfactorily liquefy sub-bituminous and hard coals in solid media. On the other hand, some fungi grew specifically on the untreated coals, engulfing them and in many cases a soft slurry was obtained. Several of the isolated microorganisms were able to solubilize all three untreated Spanish coals in liquid media. Coal solubilization was measured spectrophotometrically at 300, 400 and 450 nm.

Aspergillus nidulans released α-galactosidase into the culture medium during the exponential growth on either lactose or galactose as the only carbon source. This enzyme is a glycoprotein. Its treatment with endoglycosidases produces a reduction in its molecular mass but the resulting enzyme conserved some of their carbohydrate components in addition to its enzymatic activity. Mycelia of A. nidulans growing in the presence of tunicamycin synthesized an underglycosylated α-galactosidase which was not released into the culture media but remained bound to the cell-wall. Tunicamycin did not prevent the synthesis and secretion of α-galactosidase by protoplasts. N-linked oligosaccharide chains seem not to be essential for the synthesis and secretion of α-galactosidase of A. nidulans, but they could be necessary for proper targeting at the extracellular level.

The role of microtubules (MTs) in the protein secretion process in Aspergillus nidulans has been studied using two antimicrotubular drugs, methyl benzimadazol-2-yl carbamate (MBC) and griseofulvin, and two strains of A. nidulans: a wild-type and a benomyl resistant-mutant (ben A10). Sublethal doses of MBC and griseofulvin reduced the total amount of proteins secreted by the wildtype strain and modified the electrophoretic pattern of the extracellular proteins, inhibiting the secretion of certain proteins and favouring the secretion of several others that were not secreted in the control cultures. Some of these modifications were the same with both antimicrotubular compounds. MBC did not alter either the amount of proteins secreted or the electrophoretic patterns of proteins in the A. nidulans benomyl-resistant strain, demonstrating that its action is mediated via the microtubules. Our results suggest the involvement of MTs in the polarized secretion process of A. nidulans.

Cladosporium cucumerinum produced multiple isozymes of extracellular acid phosphatase, in response to phosphate concentration in the medium and related to growth phase. Production of the largest, a 174 kDa molecular form, was not affected by culture conditions and this isozyme could be a constitutive enzyme. Another form, with a mol. wt of 59 kDa, was subject to phosphate repression and was secreted to the culture medium in the early phase of fungal growth. Two minor forms, which exhibited mol. wt of 389 and 562 kDa, were detected in culture filtrates at the end of exponential growth. A single 174 kDa molecular form of acid phosphatase was detected in mycelial extracts. Furthermore, two forms of alkaline phosphatase were determined in culture filtrates and mycelial extracts in response to the phosphate level in the medium. A repressible alkaline phosphatase, with a mol. wt of 174 kDa and a form which was promoted by phosphate, with a mol. wt of 98 kDa were also found.

We have isolated and characterised a novel aerobic bacterial strain, designated MS3-02, belonging to the genus Janibacter sp. The capability of this new strain to degrade polychlorinated biphenyls (PCBs) in a commercial mixture (Aroclor 1242) in liquid medium and in soil (sterile and non sterile soil), under laboratory scale, has been evaluated. MS3-02 was isolated from the soil around of an incinerator, located in the east of Madrid (Spain). Gas-chromatographic analysis showed that MS3-02 was able to reduce most peaks observed in the chromatogram between 70% and 100% after seven days of incubation in a culture mineral medium containing yeast extract, but without the addition of biphenyl. The presence of biphenyl in the culture medium decreased the rate of PCB degradation by this bacterium. Comparing the performance of the MS3-02 in liquid culture medium and in soil, degradation was less efficient in sterile soil and still less efficient in non sterile soil. Under the best conditions (sterile soil and 20 weeks of incubation) MS3-02 was able to reduce, between 50% and 100%, nine of the main gas-chromatographic peaks in Aroclor 1242.

The location of the Aspergillus nidulans carnitine/acyl-carnitine carrier (ACUH) was studied. ACUH with a His-tag at its N-terminus was over-expressed in Escherichia coli and purified by Ni(2+) affinity chromatography. The purified protein was utilised to raise polyclonal antibodies which were characterised by Western blotting. For localisation studies A. nidulans T1 strain, that contains the acuH gene under control of the strong promoter alcA(p), was derived. Results obtained demonstrate the exclusively mitochondrial localisation of ACUH and therefore exclude the targeting of the acuH gene product to the peroxisomal membrane.

The development of a homologous transformation system for the opportunistic human pathogenic fungus Aspergillus fumigatus is described. The system is based on the sC gene encoding ATP sulfurylase. Several A. fumigatus sC mutant strains were readily isolated by strong selection for selenate resistance. The coding region plus upstream and downstream regulatory sequences of the A. fumigatus sC gene were cloned by inverse PCR and then sequenced. Sequencing of the sC cDNA revealed the presence of five introns located within the first half of the gene. The A. fumigatus sC gene encodes a protein of 574 amino acids which is highly similar to ATP sulfurylases from the filamentous fungal species Aspergillus nidulans, Aspergillus terreus and Penicillium chrysogenum. By contrast, ATP sulfurylases from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe lack the C-terminal adenosine-5′-phosphosulfate kinase-like domain present in the filamentous fungal orthologues. A 3.8-kb DNA fragment amplified by PCR and containing the sC gene plus 5′ and 3′ flanking regions was cloned into pUC19 to give the vector pSCFUM. Transformation of two different sC mutant isolates with the plasmid pSCFUM established the functionality of this new homologous transformation system. Molecular analysis of sC + transformants showed that up to 44% of transformed clones contained one or more copies of the entire plasmid integrated at the sC locus. This result also demonstrates the utility of the sC marker for targeting specific genetic constructs to the A. fumigatus sC locus, facilitating studies of gene regulation and function.

Several fundamental aspects of microbial coal liquefaction/solubilization were studied. The liquefied/solubilized products from coal by microorganisms were analysed. The liquid products analysed by IR titration and UV/visible spectrometry showed some alterations with regard to the original coal. Humic acids extracted from the liquefied lignite showed a reduction in the average molecular weight and a increase in the condensation index, probably due to depolymerization caused by microorganisms. The mechanisms implicated in coal biosolubilization by two fungal strains, M2 (Trichoderma sp.) and M4 (Penicillium sp.) were also studied. Extracellular peroxidase, esterase and phenoloxidase enzymes appear to be involved in coal solubilization.

In previous work, we have demonstrated that oleate induces a massive proliferation of microbodies (peroxisomes) in Aspergillus nidulans. Although at a lower level, proliferation of peroxisomes also occurrs in cells,growing under conditions that induce penicillin biosynthesis. Here, microbodies in oleate-grown A. nidulans cells were characterized by using several antibodies that recognize peroxisomal enzymes and peroxins in a brood spectrum of eukaryotic organisms such as yeast, and plant, and mammalian cells. Peroxisomes were immunolabeled by anti-SKL and anti-thiolase antibodies, which suggests that A. nidulans conserves both PTS1 and PTS2 import mechanisms. Isocitrate lyase and malate synthase, the two key enzymes of the glyoxylate cycle, were also localized in these organelles. In contrast to reports of Neurospora crassa, our results demonstrate that A. nidulans contains only one type of microbody (peroxisomes) that carry out the glyoxylate cycle and contain 3-ketoacyl-CoA thiolase and proteins with the C-terminal SKL tripeptide.

Conidia of Aspergillus nidulans were mutagenized with ultraviolet light and were incubated on a special selective medium containing the catalase inhibitor 3-amino-1,2,4-triazole. From approximately 5 x 10(7) viable UV-irradiated conidia tested, 423 stable mutants resistant to 3-amino-1,2,4-triazole were recovered, of which 40 were unable to grow on minimal medium with oleic acid as the sole carbon source. These oleate-nonutilizing (Ole-) mutants did not grow on medium with carbon sources requiring functional peroxisomes (oleate, butyrate, acetate, or ethanol), but grew well on medium with carbon sources supposedly not requiring such organelles (glucose, glycerol, l-glutamate, or l-proline). The Ole- mutants carried mutations in one of five nuclear genes affecting acetate utilization: acuJ, acuH, acuE, acuL, and perA. The perA21 strain (DL21) carried a mutation in a gene that is not allelic with any of the known acu loci and displayed a phenotype resembling that described in the Pim- (peroxisome import defective) mutants of Hansenula polymorpha. Hyphae of the perA21 mutant contained a few small peroxisomes with the bulk of peroxisomal enzymes remaining in the 20,000 x g supernatant, but produced wild-type levels of penicillin.

A method previously developed for the analysis of organohalogenated compounds in dairy products is now validated for polychlorinated biphenyls (PCBs) determination in soybean infant formulas. The results of this study are consistent with those found for PCBs in powdered full-fat milk. The methodology is based on a solid–liquid extraction step enabling a semi-selective extraction of the apolar lipids of the matrix without affecting the efficiency for the recovery of PCBs. Mean recoveries for the spiked coplanar congeners studied were in the 88–114% range, with relative standard deviations (R.S.D.s) lower than 9.8%. The R.S.D.s related to the determination of endogenous PCBs were in the 1.5–10.0% range. The validated methodology was applied to the PCB analysis in different trademarks of soybean infant formulas commercialised in Spain. Toxic tetraequivalents of tetrachlorodibenzo-p-dioxin and daily intake corresponding to each one were calculated and compared with values previously published and with those found in literature for human breast milk in different countries., Authors thank the Comunidad Autónoma of Madrid for financial support (project COR 0035/94).

A rapid separation of the main soybean proteins (7S and 11S globulins) was carried out by reversed-phase high-performance liquid chromatography. For this purpose, a linear binary gradient acetonitrile--water--0.1% trifluoroacetic acid, at a flow-rate of 1 ml/min and 50 degrees C temperature was designed. Under the experimental conditions of this work, it was possible to separate five peaks corresponding to the globulins from a soybean protein isolate in 9 min. The characterization of soybean proteins was accomplished by analyzing the 7S and 11S purified fractions obtained from a soybean protein isolate. The method was applied to the separation of soybean proteins from commercial foodstuffs: soybean flour, textured soybean and soybean milks.

The existence of a second mechanism of catabolite control of isocitrate lyase of Aspergillus nidulans, in addition to the carbon catabolite repression phenomenon recently reported was analysed. Isocitrate lyase was rapidly and specifically inactivated by glucose. The inactivation was irreversible at all stages in the presence of cycloheximide, showing that reactivation depends on de novo protein synthesis. In addition, analysis of glucose-induced inactivation of isocitrate lyase in a creAd-30 strain showed that the creA gene is not involved in this process.

Accidental oil spills and waste disposal are important sources for environmental pollution. We investigated the biodegradation of alkanes by Pseudomonas aeruginosa ATCC 55925 in relation to a rhamnolipid surfactant produced by the same bacterial strain. Results showed that the linear C11-C21 compounds in a heating oil sample degraded from 6% to 100%, whereas the iso-alkanes tended to be recalcitrant unless they were exposed to the biosurfactant; under such condition total biodegradation was achieved. Only the biodegradation of the commercial C12-C19 alkanes could be demonstrated, ranging from 23% to 100%, depending on the experimental conditions. Pristane (a C19 branched alkane) only biodegraded when present alone with the biosurfactant and when included in an artificial mixture even without the biosurfactant. In all cases the biosurfactant significantly enhanced biodegradation. The electron scanning microscopy showed that cells depicted several adaptations to growth on hydrocarbons, such as biopolymeric spheres with embedded cells distributed over different layers on the spherical surfaces and cells linked to each other by extracellular appendages. Electron transmission microscopy revealed transparent inclusions, which were associated with hydrocarbon based-culture cells. These patterns of hydrocarbon biodegradation and cell adaptations depended on the substrate bioavailability, type and length of hydrocarbon.

alpha-Galactosidases from mycelial extract and culture filtrate of Aspergillus nidulans have been purified to homogeneity and utilised to obtain polyclonal antibodies anti-alpha-galactosidase. The enzymatic characteristics and the cross reactivity of the antibodies suggest that alpha-galactosidases isolated from the two sources were the same enzyme. Thus, A. nidulans synthesized and secreted only one enzymatic form of alpha-galactosidase which is a multimeric enzyme of 370 kDa composed of four monomers of 87 kDa and a pI of 6.3. The optimum temperature of activity was 50 degrees C and the optimum pH 4-5. The enzyme was stable over a wide range of pH but quite unstable to temperature. alpha-Galactosidase of A. nidulans is a very specific enzyme, it is active only on p-nitrophenyl-alpha-D-galactoside (PNPG), melibiose and raffinose. When PNPG was utilised as substrate melibiose, raffinose, galactose and glucose were competitive inhibitors of the activity.

Wall formation inSaccharomyces cerevisiae seems to be the result of two main patterns of wall material deposition: (i) around the whole periphery of the cell in nonbudding ones, and (ii) mainly at the tip of the daughter cell or at the cross wall that separates dividing cells. This interpretation has been obtained following experiments in which RNA or protein synthesis has been inhibited. Under these conditions, glucan formation takes place, and wall thickening is probably due to the accumulation of this polysaccharide. Furthermore, once a pattern of wall deposition has been established, it is not modified by inhibition of RNA or protein synthesis.

Galactanase from Phytophthora infestans and an arabinosidase isoenzyme from Sclerotinia fructigena attacked the cortical cell walls of apple fruits liberating galactose and arabinose residues, respectively. Other arabinosidase isoenzymes from S. fructigena attacked cell walls very slowly. A S. fructigena polygalacturonase isoenzyme liberated half of the uronic acid residues with few associated neutral residues, while a second polygalacturonase isoenzyme released more uronic acid with a substantial proportion of arabinose and galactose and lesser amounts of xylose, rhamnose and glucose; reaction products of this enzyme could be further degraded by the first isoenzyme to give high MW fragments, rich in arabinose with most of the xylose, rhamnose and glucose, and low MW fragments rich in galactose and uronic acid. Endoglucanase from Trichoderma viride released a small proportion of the glucose residues from cell walls together with uronic acid, arabinose, xylose and galactose; more extensive degradation occurred if walls were pre-treated with the second polygalacturonase isoenzyme. Endoglucanase reaction products were separated into a high MW fraction, rich in arabinose, and lower MW fractions rich in galactose and glucose residues. The high MW polygalacturonase and endoglucanase products could be degraded with an arabinosidase isoenzyme to release about 75% of their arabinose. Cell walls from ripe fruit showed similar susceptibility to arabinosidase and galactanase to those from unripe apples. Cell walls from fruit, ripened detached from the tree were more susceptible to degradation by polygalacturonase than walls from unripe fruit or fruit ripened on the tree. Endoglucanase released less carbohydrate from ripe fruit cell walls than from unripe fruit cell walls.

Two antifungal antibiotics, named PA-5 and PA-7 were produced by an actinomycete strain, characterized and identified as Streptoverticillium sp 43/16. These antibiotics were extracted from mycelial cake with methanol and purified using different organic solvents and LH-20 Sephadex column chromatography. PA-5 and PA-7 were characterized as, a pentaene and a heptaene macrolide antibiotic, respectively. These antibiotics exhibit strong antifungal activity against pathogen yeasts and molds.