Small structural changes of the imidazopyridine diacylglycerol acyltransferase 2 (DGAT2) inhibitors produce an improved safety profile.

Small molecule DGAT2 inhibitors have shown promise for the treatment of metabolic diseases in preclinical models. Herein, we report the first toxicological evaluation of imidazopyridine-based DGAT2 inhibitors and show that the arteriopathy associated with imidazopyridine 1 can be mitigated with small structural modifications, and is thus not mechanism related.

Identification of growth phenotype-related genes in Aspergillus oryzae by heterologous macroarray and suppression subtractive hybridization.

Aspergillus oryzae requires polarized growth for colonization of solid substrates, and this growth phenotype differs from that seen in liquid medium. Various experimental approaches were used to identify genes that are differentially expressed when A. oryzae is grown on wheat kernels and in a wheat-based liquid medium. Hybridization of A. oryzae RNAs to a macroarray bearing cDNAs isolated from a library representing at least 16% of the total number of A. niger genes identified 14 differentially expressed cDNA clones, showing that heterologous macroarray analysis with an A. niger cDNA library can be used to identify regulated gene transcripts in the related species A. oryzae. Moreover, Northern analysis with a selection of eight probes for A. niger genes encoding proteins involved in morphological development and cell wall biosynthesis identified five more differentially expressed genes. A suppression subtractive hybridization procedure revealed another 12 differentially expressed genes. The results presented show that, of the 29 identified genes which are expressed at higher levels during growth on wheat kernels, six encode proteins that are functionally related to polarized growth, four encode products known to be involved in morphogenesis, three code for proteins related to cell wall composition, and nine of the cDNA clones encode novel proteins. These findings pinpoint genes associated with the changes in cellular morphogenesis seen in A. oryzae grown on wheat kernels as opposed to wheat-based liquid medium.

Differential effects of naturally occurring isothiocyanates on the activities of cytochrome P450 2E1 and the mutant P450 2E1 T303A.

The effects of benzyl (BITC) and phenethyl isothiocyanate (PEITC) on the activity of a P450 2E1 mutant where the conserved threonine at position 303 was replaced with an alanine residue (P450 2E1 T303A) were examined. PEITC inactivated the mutant enzyme with a K(I) of 1.6 microM. PEITC also inactivated the wild-type P450 2E1 as efficiently with a K(I) of 2.7 microM. The inactivation was entirely dependent on NADPH and followed pseudo-first-order kinetics. Previously we reported the mechanism-based inactivation of wild-type P450 2E1 by BITC with a K(I) of 13 microM. In contrast to the wild-type enzyme, the P450 2E1 T303A mutant was not inactivated by BITC but it was inhibited in a competitive manner with a K(i) of 3 microM. The binding constants determined by spectral binding studies were similar for both enzymes. The binding of BITC produced characteristic Type I spectral changes in the wild-type and mutant enzyme. A radiolabeled BITC metabolite bound to P450 2E1 and to P450 2E1 T303A when both enzymes were incubated with [(14)C]BITC and NADPH. Whole protein electrospray ion trap mass spectrometry indicated that a mass consistent with one molecule of benzylisocyanate and oxygen was adducted to the wild-type enzyme. The mass adducted to the T303A mutant was consistent with the addition of one hydroxylated BITC or of one benzylisocyanate moiety and one sulfur molecule. Analysis of the metabolites of BITC indicated that each enzyme produced similar metabolites but that the mutant enzyme generated significantly higher amounts of benzaldehyde and benzoic acid when compared to the wild-type enzyme.

Expression of the Avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum is regulated by the global nitrogen response factor NRF1.

Here we describe the role of the Cladosporium fulvum nitrogen response factor 1 (Nrf1) gene in regulation of the expression of avirulence gene Avr9 and virulence on tomato. The Nrf1 gene, which was isolated by a polymerase chain reaction-based strategy, is predicted to encode a protein of 918 amino acid residues. The protein contains a putative zinc finger DNA-binding domain that shares 98% amino acid identity with the zinc finger of the major nitrogen regulatory proteins AREA and NIT2 of Aspergillus nidulans and Neurospora crassa, respectively. Functional equivalence of Nrf1 to areA was demonstrated by complementation of an A. nidulans areA loss-of-function mutant with Nrf1. Nrf1-deficient transformants of C. fulvum obtained by homologous recombination were unable to utilize nitrate and nitrite as a nitrogen source. In contrast to what was observed in the C. fulvum wild-type, the Avr9 gene was no longer induced under nitrogen-starvation conditions in Nrf1-deficient strains. On susceptible tomato plants, the Nrf1-deficient strains were as virulent as wild-type strains of C. fulvum, although the expression of the Avr9 gene was strongly reduced. In addition, Nrf1-deficient strains were still avirulent on tomato plants containing the functional Cf-9 resistance gene, indicating that in planta, apparently sufficient quantities of stable AVR9 elicitor are produced. Our results suggest that the NRF1 protein is a major regulator of the Avr9 gene.

Effects of benzyl isothiocyanate on rat and human cytochromes P450: identification of metabolites formed by P450 2B1.

Naturally occurring isothiocyanates, such as benzyl isothiocyanate (BITC), are potent and selective inhibitors of carcinogenesis induced by a variety of chemical carcinogens. These effects appear to be mediated through favorable modification of both phase I and II enzymes involved in carcinogen metabolism. The inactivation of rat and human cytochromes P450 (P450s) in microsomes and the reconstituted system by BITC was investigated. BITC is a mechanism-based inactivator of rat P450s 1A1, 1A2, 2B1, and 2E1, as well as human P450s 2B6 and 2D6. BITC was most effective in inactivating P450s 2B1, 2B6, 1A1, and 2E1, whereas the activities of human P450 2C9 and rat P450 3A2 were not altered. The concentrations required for half-maximal inactivation (K(I)) of P450s 1A1, 1A2, 2B1, and 2E1 were 35, 28, 16, and 18 microM, respectively. The corresponding values for k(inact) were 0.26, 0.09, 0.18, and 0.05 min(-1), respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of P450 2B1 inactivated by [(14)C]BITC indicated specific and covalent modification of the P450 apoprotein by a metabolite of BITC. High-performance liquid chromatography analysis of the BITC metabolites revealed that benzylamine was the major metabolite and there were lesser amounts of benzoic acid, benzaldehyde, N,N'-di-benzylurea, and N,N'-di-benzylthiourea. Presumably, BITC was metabolized to the reactive benzyl isocyanate intermediate that covalently modified the P450 apoprotein or hydrolyzed to form benzylamine. BITC was an efficient inactivator of P450 2B1 with a partition ratio of approximately 11:1. This irreversible inactivation of P450s by BITC could contribute significantly to its chemopreventative action.

Inactivation of cytochrome P450 2B1 by benzyl isothiocyanate, a chemopreventative agent from cruciferous vegetables.

A series of arylalkyl isothiocyanates were evaluated for their ability to inactivate purified cytochrome P450 2B1 in a reconstituted system. Benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) occur naturally in several cruciferous vegetables, and the inhibition of cytochrome P450 (P450) enzymes has been implicated in their chemopreventative abilities. The naturally occurring isothiocyanates BITC and PEITC inactivated P450 2B1 in a time- and concentration-dependent manner, whereas the synthetic isothiocyanates phenylpropyl and phenylhexyl isothiocyanate did not result in inactivation, but were potent competitive inhibitors of P450 2B1 activity. The kinetics of inactivation of P450 2B1 by BITC were characterized. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of P450 2B1 was inactivated in a mechanism-based manner. The loss of O-deethylation activity followed pseudo-first-order kinetics, was saturable, and required NADPH. The BITC concentration required for half-maximal inactivation (K(I)) was 5.8 microM, and the maximal rate constant for inactivation was 0.66 min(-)(1) at 23 degrees C. BITC was a very efficient inactivator of P450 2B1 with a partition ratio of approximately 9. The mechanism of BITC-mediated inactivation of P450 2B1 was also investigated. More than 80% of the catalytic activity was lost within 12 min with a concomitant loss of approximately 45% in the ability of the reduced enzyme to bind CO. The magnitude of the UV/visible absorption spectrum of the inactivated protein did not decrease significantly, and subsequent HPLC analysis indicated no apparent modification of the heme. HPLC and protein precipitation analyses indicated that the P450 apoprotein was covalently modified by a metabolite of BITC. Determination of the binding stoichiometry indicated that 0.90 +/- 0. 16 mol of radiolabeled metabolite was bound per mole of enzyme that was inactivated, suggesting the modification of a single amino acid residue per molecule of enzyme that was inactivated. The results reported here indicate that BITC is a mechanism-based inactivator of P450 2B1 and that inactivation occurs primarily through protein modification.

Feline panleukopenia virus revisited: molecular characteristics and pathological lesions associated with three recent isolates.

The low incidence of clinical signs or pathological lesions compatible with feline panleukopenia in cats has created the perception among practitioners that the disease has disappeared since the emergence of canine parvovirus type 2 in the late 1970s. Three parvoviruses that were recently isolated from a domestic cat and 2 cheetahs in cell culture or detected by means of the polymerase chain reaction were shown to be typical feline parvoviruses. Phylogenetic comparison with other FPV isolates did not reveal a particular African cluster.

Transcription of the avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum is regulated by a GATA-type transcription factor in Aspergillus nidulans.

The avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum is highly induced during infection of tomato plants. Expression of the Avr9 gene can also be induced in vitro when cells are grown on synthetic liquid medium containing little or no nitrogen. The Avr9 promoter contains six copies of the sequence TAGATA and six additional copies of the core sequence GATA within 0.4 kb upstream of the translation start site. In the filamentous fungi Aspergillus nidulans and Neurospora crassa, these promoter sequences have been identified as the binding sites for a wide-domain GATA-type regulator (AREA in A. nidulans and NIT2 in N. crassa) involved in nitrogen utilization. Quantification of GUS activity of A. nidulans transformants containing a single copy of the fully active Avr9 promoter-uidA (GUS) reporter gene fusion in different areA backgrounds, following starvation for nitrogen, showed that induction of the Avr9 promoter is regulated similarly in A. nidulans and C. fulvum. This suggests that AREA can regulate the Avr9 promoter and that C. fulvum contains an AREA-like regulator that can bind to these specific sequence motifs. Comparison of the induction profiles of Avr9 and niaD showed that Avr9 expression is independent of NIRA, as is niaD expression upon nitrogen starvation. Studies with Avr9 promoter-uidA fusions in which all or most of these sequences had been deleted, showed that Avr9 promoter activity is dependent on the presence of these specific cis-regulatory elements, suggesting that they do indeed function in transcriptional regulation of the Avr9 gene.

Feline herpesvirus infection in a group of semi-captive cheetahs.

Clinical disease caused by feline herpesvirus type-1 in wild felid species is similar to that in domestic cats. Herpesviruses are endemic in free-ranging lions in South Africa but actual clinical disease due to them has not been reported in free-ranging felids. The first reports of feline herpesvirus infection associated with clinical disease in wild felids came from Australia and the USA in 1970. Subsequent reports of clinical disease in cheetahs and other wild felid species were limited to captive animals. This report deals with clinical disease in a group of semi-captive cheetahs in which 18 animals were affected, and included 12 adult males, 4 adult females and 2 subadults. No mortalities occurred in this group, the most common clinical signs being sneezing, nasal discharge and loss of appetite.

Cloning, sequencing, disruption and phenotypic analysis of uvsC, an Aspergillus nidulans homologue of yeast RAD51.

We have cloned the uvsC gene of Aspergillus nidulans by complementation of the A. nidulans uvsC114 mutant. The predicted protein UVSC shows 67.4% sequence identity to the Saccharomyces cerevisiae Rad51 protein and 27.4% sequence identity to the Escherichia coli RecA protein. Transcription of uvsC is induced by methyl-methane sulphonate (MMS), as is transcription of RAD51 of yeast. Similar levels of uvsC transcription were observed after MMS induction in a uvsC+ strain and the uvsC114 mutant. The coding sequence of the uvsC114 allele has a deletion of 6 bp, which results in deletion of two amino acids and replacement of one amino acid in the translation product. In order to gain more insight into the biological function of the uvsC gene, a uvsC null mutant was constructed, in which the entire uvsC coding sequence was replaced by a selectable marker gene. Meiotic and mitotic phenotypes of a uvsC+ strain, the uvsC114 mutant and the uvsC null mutant were compared. The uvsC null mutant was more sensitive to both UV and MMS than the uvsC114 mutant. The uvsC114 mutant arrested in meiotic prophase-I. The uvsC null mutant arrested at an earlier stage, before the onset of meiosis. One possible interpretation of these meiotic phenotypes is that the A. nidulans homologue of Rad51 of yeast has a role both in the specialized processes preceding meiosis and in meiotic prophase I.

Polarity of meiotic gene conversion is 5' to 3' within the niaD gene of Aspergillus nidulans.

We have examined polarity of meiotic gene conversion in the niiA-niaD gene cluster of Aspergillus nidulans in two-point crosses. The type and position of the mutations represented by the niaD alleles and the correlation between the relative frequency of gene conversion and the physical position of these mutations were determined. We show that polarity of meiotic gene conversion is 5' to 3' (transcribed strand) within the niaD gene. Additional crosses involving a niiA allele and a niaD allele show little polarity of gene conversion, which suggests that the recombination events leading to restoration of the niaD gene are initiated upstream of the coding region of the niaD gene but within the niiA-niaD gene cluster, possibly within the intergenic promoter region.

Isolation and characterization of the glucose-6-phosphate dehydrogenase encoding gene (gsdA) from Aspergillus niger.

Genomic and cDNA clones encoding glucose-6-phosphate dehydrogenase (G6PD) were isolated from the fungus Aspergillus niger. Sequence analysis of the glucose-6-phosphate dehydrogenase gene (gsdA) revealed an open reading frame of 1530 bp, encoding a protein of 58,951 kDa. The gsdA gene is interrupted by nine introns the most proximal of which is exceptionally large (348 bp). The region upstream of the ATG contains several C+T-rich stretches. The two major and one minor transcription start points are all located within these regions. In the upstream region several direct and inverted repeats, but no clear TATA or CCAAT boxes can be found. A. niger strains overproducing G6PD were constructed by cotransformation of gsdA subclones. Overexpression of G6PD was shown to be deleterious for the fungus, especially when cotransformants were grown on media containing ammonia. Attempts to construct a gsdA null mutant by gene disruption were unsuccessful.

The in-planta induced ecp2 gene of the tomato pathogen Cladosporium fulvum is not essential for pathogenicity.

During the colonization of tomato leaves, the fungal pathogen Cladosporium fulvum excretes low-molecular-weight proteins in the intercellular spaces of the host tissue. These proteins are encoded by the ecp genes which are highly expressed in C. fulvum while growing in planta but are not, or are only weakly, expressed in C. fulvum grown in vitro. To investigate the function of the putative pathogenicity gene ecp2, encoding the 17-kDa protein ECP2, we performed two successive disruptions of the gene. In the first of these, the ecp2 gene was interrupted by a hygromycin B resistance gene cassette. In the second gene disruption, the ecp2 gene was completely deleted from the genome, and replaced by a phleomycin resistance gene cassette. Both disruption mutants were still pathogenic on tomato seedlings, indicating that the C. fulvum ecp2 gene is not essential for pathogenicity in tomato.

Purification and characterization of glucose-6-phosphate dehydrogenase from Aspergillus niger and Aspergillus nidulans.

Glucose-6-phosphate dehydrogenase (G6PD; D-glucose 6-phosphate:NADP+ oxidoreductase, EC 1.1.1.49) has been purified from Aspergillus nidulans and Aspergillus niger by a combination of affinity and anion exchange chromatography. A 500-1000-fold purification was obtained and the final enzyme preparations were shown to be pure but not homogeneous. For both fungi the purified enzyme preparation gave two bands on native and denaturing gels. The catalytically active form is a multimer. The molecular mass of the monomers is 60 and 57 kDa for A. nidulans and 55 and 53 kDa for A. niger. Both enzymes exhibited strict specificity towards both substrates glucose 6-phosphate and NADP+. The A. nidulans and A. niger G6PD enzymes catalyse the conversion of glucose 6-phosphate via a random order mechanism. Inhibition studies provided evidence for the physiological role of G6PD as producer of NADPH in both fungi.

Transformation of Mycobacterium aurum and Mycobacterium smegmatis with the broad host-range gram-negative cosmid vector pJRD215.

The transformation of Mycobacterium aurum and Mycobacterium smegmatis with the Gram-negative RSF1010-derived cosmid pJRD215 is described. The plasmid is stably maintained in both species and the antibiotic resistance determinants for kanamycin and streptomycin are expressed. Southern blot analysis shows that rearrangements take place both in M. aurum and in M. smegmatis. The use of pJRD215 in mycobacterial cloning systems is discussed.

Genetic analysis of amdS transformants of Aspergillus niger and their use in chromosome mapping.

The Aspergillus nidulans gene coding for acetamidase (amdS) was introduced into A. niger by transformation. Twelve Amd+ transformants were analysed genetically. The amdS inserts were located in seven different linkage groups. In each transformant the plasmid was integrated in only a single chromosome. Our (non-transformed) A. niger strains do not grow on acetamide and are more resistant to fluoroacetamide than the transformants. Diploids hemizygous for the amdS insert have the Amd+ phenotype. We exploited the opportunity for two-way selection in A. niger: transformants can be isolated based on the Amd+ phenotype, whereas counter-selection can be performed using resistance to fluoroacetamide. On this basis we studied the phenotypic stability of the heterologous amdS gene in A. niger transformants as well as in diploids. Furthermore, we mapped the plasmid insert of transformant AT1 to the right arm of chromosome VI between pabA1 and cnxA1, providing evidence for a single transformational insert. The results also show that the amdS transformants of A. niger can be used to localize non-selectable recessive markers and that the method meets the prerequisites for efficient mitotic mapping. We suggest the use of amdS transformants for mitotic gene mapping in other fungi.

Tryptophan auxotrophic mutants in Aspergillus niger: inactivation of the trpC gene by cotransformation mutagenesis.

Aspergillus niger tryptophan auxotrophic mutants have been isolated after UV irradiation of conidiospores. The mutants belong to two different complementation groups, trpA and trpB, which complement each other in heterokaryons. Neither of the mutations could be complemented with the cloned A. niger trpC gene. To obtain A. niger trpC mutants in a direct way, gene inactivation by cotransformation was performed. For this purpose an in-frame gene fusion between the A. niger trpC and Escherichia coli lacZ genes was constructed and shown to be functionally expressed after introduction into A. niger by cotransformation with the pyrA gene as selective marker. Among the beta-galactosidase expressing cotransformants, obtained with either circular or linearized vectors, no trpC mutants were detected, even after enrichment. Such mutants, however, could be obtained by cotransformation of A. niger with specific fragments of the fusion gene. Biochemical analysis of the cotransformants indicated that in nearly all cases the fusion gene had replaced the wild-type trpC gene. Genetic analysis showed that the trpC mutation is not linked to any of the A. niger loci described so far. The trpC mutants can be complemented by the cloned A. niger trpC gene as well as by the A. nidulans trpC gene.

Cotransformation of Aspergillus nidulans: a tool for replacing fungal genes.

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.

Transformation of Aspergillus niger using the homologous orotidine-5'-phosphate-decarboxylase gene.

A homologous transformation system for the filamentous fungus Aspergillus niger has been developed, based on the orotidine-5'-phosphate-decarboxylase gene. A. niger Pyr- mutants have been selected from 5-fluoro-orotic acid resistant mutants. These mutants were found to comprise two complementation groups, pyrA and pyrB. The A. niger OMP-decarboxylase gene was isolated from a gene library by heterologous hybridization with the Neurospora crassa pyr4 gene. The cloned gene is capable to transform A. nidulans pyrG mutants at high frequencies. Transformation of A. niger pyrA mutants occurs with moderate frequencies (about 50 transformants/micrograms DNA) whereas the pyrB mutants cannot be complemented with the cloned OMP-decarboxylase gene. Analysis of the DNA of the A. niger PyrA+ transformants showed that transformation resulted in integration of the vector DNA into the genome by homologous recombination. Both gene replacements and integration of one or more copies of the complete vector have been observed.

Genetic analysis of Aspergillus nidulans AmdS+ transformants.

To correlate the genetic background of the Aspergillus nidulans amdS deletion strain MH1277 with the integrational behaviour of transforming vectors, classical genetic methods were used to construct AmdS- strains in which whole chromosomes had been exchanged with those of a master strain. Progeny strains were transformed to the AmdS+ phenotype with vector p3SR2. From Southern analysis it was concluded that transformants from all constructions contained tandemly repeated, multiple copy inserts of vector DNA as found for MH1277-derived AmdS+ transformants. AmdS+ transformants of MH1277 were analysed genetically to prove that the transformant phenotype is genome linked and that transformation by integration can take place on various chromosomes. In one case the AmdS+ property showed linkage to both chromosomes II and IV, due to a chromosomal translocation. Sexual analysis of two transformants with AmdS+ insertions on the same chromosome revealed a considerable instability of the AmdS+ phenotype in one of the strains upon selfing. Due to this instability no decisive answer could be given for the degree of linkage between the AmdS+ insertions in these transformants.