Analytical determination of apparent stability constants using a copper ion selective electrode.

Copper(II) complexes of di-, tri- and tetra peptides with previously published protonation constants were re-investigated using pH and copper ion selective electrode (ISE) potentiometry in conjunction with a modified version of HYPERQUAD computer program. The purpose was to demonstrate the suitability of the ISE approach for the determination of apparent stability constants for copper(II) complexes with ligands for which proton stability constants were not available. The interactions of Cu(2+) with oligopeptides were also analysed using surface enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-ToF-MS). The results provide an insight into the metal complex species formed, their apparent stabilities under selected conditions and the effect of the relative positions of certain amino acids within the peptide sequence.

Review paper: Implications of the "cancer stem cell" hypothesis on murine models of colon cancer and colitis-associated cancer.

The use of murine models to investigate human diseases has been an invaluable tool. In the areas of inflammation and oncogenesis, such models have provided unique insights into pathogenesis and mechanisms to evaluate potential therapy. As such, one facet of these disease processes links inflammation and cancer. Inflammation is associated with at least 15% of the world's malignancies. One example of this relationship is documented in the association between colitis and colorectal cancer. To date, the precise molecular events linking inflammation and cancer remain unclear. A new paradigm that may bridge these processes includes the cancer stem cell hypothesis. In this review, murine models of colitis, colon cancer, and colitis-associated cancer are discussed in reference to the potential of this paradigm to clarify the relationship of these devastating diseases.

TamA interacts with LeuB, the homologue of Saccharomyces cerevisiae Leu3p, to regulate gdhA expression in Aspergillus nidulans.

Previous studies have shown that expression of the gdhA gene, encoding NADP-linked glutamate dehydrogenase (NADP-GDH), in Aspergillus nidulans is regulated by the major nitrogen regulatory protein AreA and its co-activator TamA. We show here that loss of TamA function has a more severe effect on the levels of gdhA expression than loss of AreA function. Using TamA as the bait in a yeast two-hybrid screen, we have identified a second protein that interacts with TamA. Sequencing analysis and functional studies have shown that this protein, designated LeuB, is a transcriptional activator with similar function to the homologous Leu3p of Saccharomyces cerevisiae. Inactivation of leuB revealed that this gene is involved in the regulation of gdhA, and an areA; leuB double mutant was shown to have similar NADP-GDH levels to a tamA single mutant. The requirement for TamA function to promote gdhA expression is likely to be due to its dual interaction with AreA and LeuB.

Role of glutamine synthetase in nitrogen metabolite repression in Aspergillus nidulans.

Glutamine synthetase (GS), EC 6.3.1.2, is a central enzyme in the assimilation of nitrogen and the biosynthesis of glutamine. We have isolated the Aspergillus nidulans glnA gene encoding GS and have shown that glnA encodes a highly expressed but not highly regulated mRNA. Inactivation of glnA results in an absolute glutamine requirement, indicating that GS is responsible for the synthesis of this essential amino acid. Even when supplemented with high levels of glutamine, strains lacking a functional glnA gene have an inhibited morphology, and a wide range of compounds have been shown to interfere with repair of the glutamine auxotrophy. Heterologous expression of the prokaryotic Anabaena glnA gene from the A. nidulans alcA promoter allowed full complementation of the A. nidulans glnADelta mutation. However, the A. nidulans fluG gene, which encodes a protein with similarity to prokaryotic GS, did not replace A. nidulans glnA function when similarly expressed. Our studies with the glnADelta mutant confirm that glutamine, and not GS, is the key effector of nitrogen metabolite repression. Additionally, ammonium and its immediate product glutamate may also act directly to signal nitrogen sufficiency.

A single gene produces mitochondrial, cytoplasmic, and peroxisomal NADP-dependent isocitrate dehydrogenase in Aspergillus nidulans.

NADP-dependent isocitrate dehydrogenase enzymes catalyze the decarboxylation of isocitrate to 2-oxoglutarate accompanied by the production of NADPH. In mammals two different genes encode mitochondrial and cytoplasmic/peroxisomal located enzymes, whereas in Saccharomyces cerevisiae three separate genes specify compartment specific enzymes. We have identified a single gene, idpA, in the filamentous fungus Aspergillus nidulans that specifies a protein with a high degree of identity to mammalian and S. cerevisiae enzymes. Northern blot analysis and reverse transcription-polymerase chain reaction revealed the presence of two idpA transcripts and two transcription start points were identified by sequencing cDNA clones and by 5'-rapid amplification of cDNA ends. The shorter transcript was found to be inducible by acetate and by fatty acids while the longer transcript was present in higher amounts during growth in glucose containing media. The longer transcript is predicted to encode a polypeptide containing an N-terminal mitochondrial targeting sequence as well as a C-terminal tripeptide (ARL) as a potential peroxisomal targeting signal. The shorter transcript is predicted to encode a polypeptide lacking the mitochondrial targeting signal but retaining the C-terminal sequence. Immunoblotting using antibody raised against S. cerevisiae Idp1p detected two polypeptides consistent with these predictions. The functions of the predicted targeting sequences were confirmed by microscopic analysis of transformants containing fluorescent protein fusion constructs. Using anti-Idp1p antibodies, protein localization to mitochondria and peroxisomes was observed during growth on glucose whereas cytoplasmic and peroxisomal localization was found upon acetate or fatty acid induction. Therefore, we have established that by the use of two transcription start points a single gene is sufficient to specify localization of NADP-dependent isocitrate dehydrogenase to three different cellular compartments in A. nidulans.

Functional analysis of TamA, a coactivator of nitrogen-regulated gene expression in Aspergillus nidulans.

The tam A gene of Aspergillus nidulans encodes a 739-amino acid protein with similarity to Uga35p/Dal81p/DurLp of Saccharomyces cerevisiae. It has been proposed that TamA functions as a co-activator of AreA, the major nitrogen regulatory protein in A. nidulans. Because AreA functions as a transcriptional activator under nitrogen-limiting conditions, we investigated whether TamA was also present in the nucleus. We found that a GFP-TamA fusion protein was predominantly localised to the nucleus in the presence and absence of ammonium, and that AreA was not required for this distribution. As the predicted DNA-binding domain of TamA is not essential for function, we have used a number of approaches to further define functionally important regions. We have cloned the tamA gene of A. oryzae and compared its functional and sequence characteristics with those of A. nidulans tamA and S. cerevisiae UGA35/DAL81/DURL. The Aspergillus homologues are highly conserved and functionally interchangeable, whereas the S. cerevisiae gene does not complement a tamA mutant when expressed in A. nidulans. Uga35p/Dal81p/DurLp was also found to be unable to recruit AreA. The sequence changes in a number of tamA mutant alleles were determined, and altered versions of TamA were tested for tamA complementation and interaction with AreA. Changes in most regions of TamA appeared to destroy its function, suggesting that the overall conformation of the protein may be critical for its activity.

The kinetics and mechanisms of the reaction of iron(III) with gallic acid, gallic acid methyl ester and catechin.

The kinetics and mechanisms of the reactions of a number of pyrogallol-based ligands with iron(III) have been investigated in aqueous solution at 25 degrees C and ionic strength 0.5 M NaClO(4). Mechanisms have been proposed which account satisfactorily for the kinetic data. These are generally consistent with a mechanism in which the 1:1 complex that is formed initially when the metal reacts with the ligand subsequently decays through an electron transfer reaction. There was also some evidence for the formation of a 1:2 ligand-to-metal complex at higher pH values. The kinetics of complex formation were investigated with either the ligand or metal in pseudo-first-order excess. Rate constants for k(1) of 2.83(+/-0.09)x10(3), 1.75(+/-0.045)x10(3) and 3300(+/-200) M(-1) s(-1) and k(-1) of 20(+/-6.0), 35(+/-13) and 25+/-7.6 M(-1) s(-1) have been evaluated for the reaction of Fe(OH)(2+) with gallic acid, gallic acid methyl ester and catechin, respectively. The stability constant of each [Fe(L)](+) complex has been calculated from the kinetic data. The iron(III) assisted decomposition of the initial iron(III) complex formed was investigated. Analysis of the kinetic data yielded both the equilibrium constants for protonation of the iron(III) complexes initially formed together with the rate constants for the intramolecular electron transfers for gallic acid and gallic acid methyl ester. All of the suggested mechanisms and calculated rate constants are supported by calculations carried out using global analysis of time-dependent spectra.

AoHapB, AoHapC and AoHapE, subunits of the Aspergillus oryzae CCAAT-binding complex, are functionally interchangeable with the corresponding subunits in Aspergillus nidulans.

Two genes, AohapB and AohapE, encoding subunits of the Aspergillus oryzae CCAAT-binding complex were cloned and sequenced. The polypeptides encoded by AohapB and AohapE were expressed in Escherichia coli and used to reconstitute a DNA-binding complex with recombinant AoHapC. The DNA-binding activity was observed only in the presence of all three subunits, indicating that AoHapB, AoHapE and AoHapC are essential for CCAAT-binding. Furthermore, introduction of the AohapB, AohapC and AohapE genes into the A. nidulans hapB delta, hapC delta and hapE delta strains, respectively, revealed that the A. oryzae Hap subunits are functionally interchangeable with the corresponding subunits in A. nidulans.

The kinetics and mechanisms of the reactions of aluminium(III) with gallic acid, gallic acid methyl ester and adrenaline.

The kinetics and mechanisms of the reactions of gallic acid, gallic acid methyl ester and adrenaline with aluminium(III) have been investigated in aqueous solution at 25 degrees C and an ionic strength of 0.5 M. A mechanism has been proposed which accounts satisfactorily for the kinetic data. This is consistent with a mechanism in which complex formation takes place almost exclusively by reaction of [Al(H2O)5OH]2+ with the ligands. [Al(H2O)5OH]2+ reacts with gallic acid, gallic acid methyl ester and adrenaline with rate constants of 1145, 1330 and 316 M(-1) s(-1) respectively. These data together with the equilibrium data enable the rate constants for reaction of [Al(H2O)6]3+ with both gallic acid and gallic acid methyl ester to be calculated. In view of the dissociative nature of water exchange on [Al(H2O)6]3+ and [Al(H2O)5(OH)]2+ the complex formation rate constants are discussed in terms of the Eigen-Wilkins-Tamm mechanism. The overall mechanisms have been validated using global analysis. The results are compared with previously published data on the complex formation reactions of aluminium(III). In addition, the rate constants and mechanisms for replacement of maltol by gallic acid methyl ester and diethylenetriaminepentaacetic acid (dtpa) have been investigated.

The CDC42 homolog of the dimorphic fungus Penicillium marneffei is required for correct cell polarization during growth but not development.

The opportunistic human pathogenic fungus Penicillium marneffei is dimorphic and is thereby capable of growth either as filamentous multinucleate hyphae or as uninucleate yeast cells which divide by fission. The dimorphic switch is temperature dependent and requires regulated changes in morphology and cell shape. Cdc42p is a Rho family GTPase which in Saccharomyces cerevisiae is required for changes in polarized growth during mating and pseudohyphal development. Cdc42p homologs in higher organisms are also associated with changes in cell shape and polarity. We have cloned a highly conserved CDC42 homolog from P. marneffei named cflA. By the generation of dominant-negative and dominant-activated cflA transformants, we have shown that CflA initiates polarized growth and extension of the germ tube and subsequently maintains polarized growth in the vegetative mycelium. CflA is also required for polarization and determination of correct cell shape during yeast-like growth, and active CflA is required for the separation of yeast cells. However, correct cflA function is not required for dimorphic switching and does not appear to play a role during the generation of specialized structures during asexual development. In contrast, heterologous expression of cflA alleles in Aspergillus nidulans prevented conidiation.

The Aspergillus nidulans multimeric CCAAT binding complex AnCF is negatively autoregulated via its hapB subunit gene.

Cis-acting CCAAT elements are frequently found in eukaryotic promoter regions. Many of them are bound by conserved multimeric complexes. In the fungus Aspergillus nidulans the respective complex was designated AnCF (A. nidulans CCAAT binding factor). AnCF is composed of at least three subunits designated HapB, HapC and HapE. Here, we show that the promoter regions of the hapB genes in both A. nidulans and Aspergillus oryzae contain two inversely oriented, conserved CCAAT boxes (box alpha and box beta). Electrophoretic mobility shift assays (EMSAs) using both nuclear extracts and the purified, reconstituted AnCF complex indicated that AnCF binding in vitro to these boxes occurs in a non-mutually exclusive manner. Western and Northern blot analyses showed that steady-state levels of HapB protein as well as hapB mRNA were elevated in hapC and hapE deletion mutants, suggesting a repressing effect of AnCF on hapB expression. Consistently, in a hapB deletion background the hapB-lacZ expression level was elevated compared with the expression in the wild-type. This was further supported by overexpression of hapB using an inducible alcA-hapB construct. Induction of alcA-hapB expression strongly repressed the expression of a hapB-lacZ gene fusion. However, mutagenesis of box beta led to a fivefold reduced expression of a hapB-lacZ gene fusion compared with the expression derived from a wild-type hapB-lacZ fusion. These results indicate that (i) box beta is an important positive cis-acting element in hapB regulation, (ii) AnCF does not represent the corresponding positive trans-acting factor and (iii) that AnCF is involved in repression of hapB.

An STE12 homolog from the asexual, dimorphic fungus Penicillium marneffei complements the defect in sexual development of an Aspergillus nidulans steA mutant.

Penicillium marneffei is an opportunistic fungal pathogen of humans and the only dimorphic species identified in its genus. At 25 degrees P. marneffei exhibits true filamentous growth, while at 37 degrees P. marneffei undergoes a dimorphic transition to produce uninucleate yeast cells that divide by fission. Members of the STE12 family of regulators are involved in controlling mating and yeast-hyphal transitions in a number of fungi. We have cloned a homolog of the S. cerevisiae STE12 gene from P. marneffei, stlA, which is highly conserved. The stlA gene, along with the A. nidulans steA and Cryptococcus neoformans STE12alpha genes, form a distinct subclass of STE12 homologs that have a C2H2 zinc-finger motif in addition to the homeobox domain that defines STE12 genes. To examine the function of stlA in P. marneffei, we isolated a number of mutants in the P. marneffei-type strain and, in combination with selectable markers, developed a highly efficient DNA-mediated transformation procedure and gene deletion strategy. Deletion of the stlA gene had no detectable effect on vegetative growth, asexual development, or dimorphic switching in P. marneffei. Despite the lack of a detectable function, the P. marneffei stlA gene complemented the sexual defect of an A. nidulans steA mutant. In addition, substitution rate estimates indicate that there is a significant bias against nonsynonymous substitutions. These data suggest that P. marneffei may have a previously unidentified cryptic sexual cycle.

The formamidase gene of Aspergillus nidulans: regulation by nitrogen metabolite repression and transcriptional interference by an overlapping upstream gene.

The ability to utilize formamide as a sole nitrogen source has been found in numerous fungi. We have cloned the fmdS gene encoding a formamidase from Aspergillus nidulans and found that it belongs to a highly conserved family of proteins separate from the major amidase families. The expression of fmdS is primarily regulated via AreA-mediated nitrogen metabolite repression and does not require the addition of exogenous inducer. Consistent with this, deletion analysis of the 5' region of fmdS has confirmed the presence of multiple AreA-binding sites containing a characteristic core GATA sequence. Under carbon starvation conditions the response to nitrogen starvation is eliminated, indicating that the lack of a carbon source may result in inactivation of AreA. Sequence analysis and isolation of cDNAs show that a gene of unknown function lies directly 5' of fmdS with its transcript overlapping the fmdS coding region. Disruption of the 5' gene and analysis of the effects of overexpression of this gene on fmdS expression has shown that expression of this upstream gene interferes with fmdS transcription, resulting in a strong dependence on AreA activation for expression. Therefore the relative position of these two genes is essential for normal regulation of fmdS.

The abaA homologue of Penicillium marneffei participates in two developmental programmes: conidiation and dimorphic growth.

Penicillium marneffei is the only known species of its genus that is dimorphic. At 25 degrees C, P. marneffei exhibits true filamentous growth and undergoes asexual development producing spores borne on complex structures called conidiophores. At 37 degrees C, P. marneffei undergoes a dimorphic transition to produce uninucleate yeast cells that divide by fission. We have cloned a homologue of the Aspergillus nidulans abaA gene encoding an ATTS/TEA DNA-binding domain transcriptional regulator and shown that it is involved in both these developmental programs. Targeted deletion of abaA blocks asexual development at 25 degrees C before spore production, resulting in aberrant conidiophores with reiterated terminal cells. At 37 degrees C, the abaA deletion strain fails to switch correctly from multinucleate filamentous to uninucleate yeast cells. Both the transitional hyphal cells, which produce the yeast cells, and the yeast cells themselves contain multiple nuclei. Expression of the abaA gene is activated during both conidiation and the hyphal-yeast switch. Interestingly, the abaA gene of the filamentous monomorphic fungus A. nidulans can complement both conidiation and dimorphic switching defects in the P. marneffei abaA mutant. In addition, ectopic overexpression of abaA results in anucleate yeast cells and multinucleate vegetative filamentous cells. These data suggest that abaA regulates cell cycle events and morphogenesis in two distinct developmental programmes.

An Aspergillus oryzae CCAAT-binding protein, AoCP, is involved in the high-level expression of the Taka-amylase A gene.

Aspergillus oryzae contains a nuclear protein designated AoCP, which binds specifically to a CCAAT sequence in the promoter region of the A. oryzae Taka-amylase A gene. A gene encoding a homologue of Aspergillus nidulans HAPC, a subunit of the A. nidulans CCAAT binding complex, was isolated from A. oryzae and designated AohapC. AoHAPC comprises 215 amino acids and shows 84% identity to A. nidulans HAPC. Transformation of the A. nidulans hapC deletion strain with the AohapC gene restored the CCAAT binding activity, resulting in both enhancement of taa gene expression and complementation for the poor growth phenotype of this strain. Furthermore, introduction of the AohapC gene also restored the expression of the A. nidulans eglA gene, encoding an endo-beta-1,4-glucanase, in the deletion strain. These results indicate functional interchangeability of the genes from two species.

The TamA protein fused to a DNA-binding domain can recruit AreA, the major nitrogen regulatory protein, to activate gene expression in Aspergillus nidulans.

The areA gene of Aspergillus nidulans encodes a GATA zinc finger transcription factor that activates the expression of a large number of genes subject to nitrogen metabolite repression. The amount and activity of the AreA protein under different nitrogen conditions is modulated by transcriptional, post-transcriptional, and post-translational controls. One of these controls of AreA activity has been proposed to involve the NmrA protein interacting with the DNA-binding domain and the extreme C terminus of AreA to inhibit DNA binding under nitrogen sufficient conditions. In contrast, mutational evidence suggests that the tamA gene has a positive role together with areA in regulating the expression of genes subject to nitrogen metabolite repression. This gene was identified by the selection of mutants resistant to toxic nitrogen source analogues, and a number of nitrogen metabolic activities have been shown to be reduced in these mutants. To investigate the role of this gene we have used constructs encoding the TamA protein fused to the DNA-binding domain of either the FacB or the AmdR regulatory proteins. These hybrid proteins have been shown to activate expression of the genes of acetate or GABA utilization, respectively, as well as the amdS gene. Strong activation was shown to require the AreA protein but was not dependent on AreA binding to DNA. The homologous areA gene of A. oryzae and nit-2 gene of Neurospora crassa can substitute for A. nidulans areA in this interaction. We have shown that the same C-terminal region of AreA and NIT-2 that is involved in the interaction with NmrA is required for the TamA-AreA interaction. However, it is unlikely that TamA requires the same residues as NmrA within the GATA DNA-binding domain of AreA.

AnCF, the CCAAT binding complex of Aspergillus nidulans, is essential for the formation of a DNase I-hypersensitive site in the 5' region of the amdS gene.

The CCAAT sequence in the amdS promoter of Aspergillus nidulans is recognized by AnCF, a complex consisting of the three evolutionary conserved subunits HapB, HapC, and HapE. In this study we have investigated the effect of AnCF on the chromatin structure of the amdS gene. The AnCF complex and the CCAAT sequence were found to be necessary for the formation of a nucleosome-free, DNase I-hypersensitive region in the 5' region of the amdS gene. Deletion of the hapE gene results in loss of the DNase I-hypersensitive site, and the positioning of nucleosomes over the transcriptional start point is lost. Likewise, a point mutation in the CCAAT motif, as well as a 530-bp deletion which removes the CCAAT box, results in the loss of the DNase I-hypersensitive region. The DNase I-hypersensitive region and the nucleosome positioning can be restored by insertion of a 35-bp oligonucleotide carrying the CCAAT motif. A DNase I-hypersensitive region has been found in the CCAAT-containing fmdS gene and was also hapE dependent. These data indicate a critical role for the AnCF complex in establishing an open chromatin structure in A. nidulans.

HAP-Like CCAAT-binding complexes in filamentous fungi: implications for biotechnology.

Regulatory CCAAT boxes are found frequently in eukaryotic promoter regions. They are bound by different CCAAT-binding factors. Until now, a single CCAAT-binding complex has been reported in fungi. It is also found in higher eukaryotes and is highly conserved among eukaryotic organisms. This multimeric protein complex is designated HAP, AnCF, CBF, or NF-Y. The complex consists of at least three subunits. In fungi, only the HAP complex of Saccharomyces cerevisiae had been known for a long time. The recent cloning of genes encoding the components of the corresponding complex (AnCF/PENR1) of Aspergillus nidulans and characterization of CCAAT-regulated genes in A. nidulans, as well as other filamentous fungi, led to a deeper insight into the role of this transcription complex, in particular in aerobically growing fungi. An overview of the function of HAP-like complexes in gene regulation in filamentous fungi is presented. Some of the genes that have been found to be regulated by HAP-like complexes encode enzymes of biotechnological interest, like taka-amylase, xylanases, cellobiohydrolase, and penicillin biosynthesis enzymes. The importance of HAP-like complexes in controlling the expression of biotechnologically important genes is discussed.

The Aspergillus nidulans gltA gene encoding glutamate synthase is required for ammonium assimilation in the absence of NADP-glutamate dehydrogenase.

Mutants of Aspergillus nidulans lacking NADP-glutamate dehydrogenase activity grow more poorly than wild-type strains on ammonium as a sole nitrogen source. The leaky growth of these mutants is indicative of an alternative pathway of ammonium assimilation and glutamate biosynthesis. We have PCR-amplified a portion of the A. nidulans gene encoding glutamate synthase and used this sequence to inactivate the genomic copy. This gene, designated gltA, was found to be dispensable for growth on ammonium in the presence of NADP-glutamate dehydrogenase activity. However, a strain carrying the gltA inactivation together with an NADP-glutamate dehydrogenase structural gene mutation (gdhA) was unable to grow on ammonium or on nitrogen sources metabolized via ammonium. The gltA gene was located to linkage group V of the A. nidulans genetic map.

AnCF, the CCAAT binding complex of Aspergillus nidulans, contains products of the hapB, hapC, and hapE genes and is required for activation by the pathway-specific regulatory gene amdR.

CCAAT binding factors (CBFs) positively regulating the expression of the amdS gene (encoding acetamidase) and two penicillin biosynthesis genes (ipnA and aatA) have been previously found in Aspergillus nidulans. The factors were called AnCF and PENR1, respectively. Deletion of the hapC gene, encoding a protein with significant similarity to Hap3p of Saccharomyces cerevisiae, eliminated both AnCF and PENR1 binding activities. We now report the isolation of the genes hapB and hapE, which encode proteins with central regions of high similarity to Hap2p and Hap5p of S. cerevisiae and to the CBF-B and CBF-C proteins of mammals. An additional fungus-specific domain present in HapE was revealed by comparisons with the homologs from S. cerevisiae, Neurospora crassa, and Schizosaccharomyces pombe. The HapB, HapC, and HapE proteins have been shown to be necessary and sufficient for the formation of a CCAAT binding complex in vitro. Strains with deletions of each of the hapB, hapC, and hapE genes have identical phenotypes of slow growth, poor conidiation, and reduced expression of amdS. Furthermore, induction of amdS by omega amino acids, which is mediated by the AmdR pathway-specific activator, is abolished in the hap deletion mutants, as is growth on gamma-aminobutyric acid as a sole nitrogen or carbon source. AmdR and AnCF bind to overlapping sites in the promoters of the amdS and gatA genes. It is known that AnCF can bind independently of AmdR. We suggest that AnCF binding is required for AmdR binding in vivo.