Lipid metabolism disorders, lymphocytes cells death, and renal toxicity induced by very low levels of deoxynivalenol and fumonisin b1 alone or in combination following 7 days oral administration to mice.

SCOPE: In our previous study focused on in vitro interactive effect of Fusarium mycotoxins deoxynivalenol (DON) and fumonisin B1 (FB1), we reported that these toxins tested at low level and in association could lead to additive or synergistic cytotoxic effect. The aim of the present study is to confirm those findings by in vivo study. MATERIALS AND METHODS: Swiss mice were orally administered with low doses of DON (45 µg/kg bw/day), FB1 (110 µg/kg bw/day), and their mixture (DON + FB1) for 7 days. RESULTS: As results, no death or abnormal symptoms were observed in all groups. The significant of loss of weight was observed in females group treated with FB1 and its association with DON. Serum chemistry examinations revealed that disorders in lipid metabolism, renal filtration perturb and a rhabdomyolysis. DON has been found as particular inducer of kidney cell deoxyribonucleic acid (DNA) methylation and blood lymphocytes cell death as measured by lymphocytes DNA fragmentation. Female mice were more sensitive and the mixture of DON and FB1 led to additive or more than additive effect particularly for their target kidney which showed different pattern of toxicity. CONCLUSION: Based on the results of this study, the no-observed-adverse effect level (NOAEL) o both DON and FB1 should be low than 45 µg/kg bw/day and 110 µg/kg bw/day, respectively in mice.

Domoic acid induces direct DNA damage and apoptosis in Caco-2 cells: recent advances.

Domoic acid (DA) is a neurotoxin produced by sea-water phytoplankton. Shellfish feeding on the phytoplankton can bioconcentrate DA, leading to a potentially serious health hazard for people consuming the contaminated shellfish. DA is the principal toxin responsible for amnesic shellfish poisoning (ASP). The toxic mechanism of DA is believed to be mediated at the level of the mitochondria, where uncoupling of oxidative phosphorylation decreases membrane permeability, causing cell swelling and ultimately lysis. Literature is poor concerning data on the possible genotoxicity and cytotoxicity of DA. In the present study, we have evaluated the cytotoxicity and genotoxicity of DA on a human colorectal adenocarcinoma cell line (Caco-2). Our results clearly demonstrate that DA decreased cell viability (IC(50) about 70 ng/mL), induced direct DNA damage from 15 ng/mL, and apoptosis in Caco-2 cells at 100 ng/mL. This apoptosis is likely bax-dependent and occurred only at high concentrations of DA, while lower concentrations upregulated both bax and bcl-2 at an apparent constant ratio until a sudden decrease of bcl-2 at 100 ng/mL and increase of bax.

Anticancer effect of a new benzophenanthridine isolated from Zanthoxylum madagascariense (Rutaceline).

Fractionation of the cyclohexane extract from the stem bark powder of Zanthoxylum madagascariense led to the isolation of a new benzophenanthridine-type alkaloid, hydrochloride of 2,3-methylendioxy-8-hydroxy- 7-methoxy-benzo[C]phenanthridine (Rutaceline), characterized on the basis of its spectral data. Rutaceline was evaluated for its antiproliferative capacity on the human colorectal adenocarcinoma (Caco-2) and the African green monkey kidney (Vero) cell lines. The 50% inhibition of cell growth (IC50) obtained after 24 h incubation was similar for both cells lines (110-115 microg/ml, i.e. 269-281 microM), but at 48 h the IC50 value for the Caco-2 cells was lower than for the Vero cells (20 microg/lml, i.e. 49 microM versus 90 microg/ml, i.e. 220 microM) indicating a higher cell growth inhibitory effect on the colon adenocarcinoma cells. At the respective IC50 concentrations, Rutaceline did not significantly induce apoptosis but induced cell cycle arrest in the GO/G1 phase, as well as a decrease of cells in S phase. Rutaceline also induced DNA fragmentation in both cell lines, as revealed by agarose gel electrophoresis, and a dose-dependent clastogenic effect in both cell lines as revealed by the Comet assay.

Preliminary survey of ochratoxin A in millet, maize, rice and peanuts in Côte d'Ivoire from 1998 to 2002.

In a preliminary study, samples of millet (n =33) maize (n=41), rice (n=10) and peanuts (n=10) from Côte d'Ivoire were analysed for ochratoxin A (OTA) by HPLC with fluorimetric detection, followed by confirmation by cleavage of the OTA molecule using carboxypeptidase with HPLC separation and fluorimetric quantification of the released ochratoxin alpha (OTh). With the exception of four samples of peanuts, all samples showed OTA contamination, ranging from 3 to 1738 microg/kg. All cereals were contaminated and the OTA concentrations were in the range of 17-204 microg/kg for millet, 3-1738 microg/kg for maize, 9-92 microg/kg for rice and 0.6-64 microg/kg for peanuts, depending on the year of harvest. Most of the samples would not be accepted according to the EU regulatory limits for this mycotoxin. Following this survey, research for other mycotoxins and the evaluation of the exposure of the population is underway.

Characterization and expression of the cDNA encoding a new kind of phospholipid transfer protein, the phosphatidylglycerol/phosphatidylinositol transfer protein from Aspergillus oryzae: evidence of a putative membrane targeted phospholipid transfer protein in fungi.

The full-length cDNA of a phospholipid transfer protein (PLTP) was isolated from Aspergillus oryzae by a RACE-PCR procedure using degenerated primer pool selected from the N-terminal sequence of the purified phosphatidylinositol/phosphatidylglycerol transfer protein (PG/PI-TP). The cDNA encodes a 173 amino acid protein of 18823 Da. The deduced amino acid sequence from position 38 to 67 is 100% identical to the N-terminal sequence (first 30 amino acids) of the purified PG/PI-TP. This amino acid sequence is preceded by a leader peptide of 37 amino acids which is predicted to be composed of a signal peptide of 21 amino acids followed by an extra-sequence of 16 amino acids, or a membrane anchor protein signal (amino acid 5-29). This strongly suggests that the PG/PI-TP is a targeted protein. The deduced mature protein is 138 amino acids long with a predicted molecular mass of 14933 Da. Comparison of the deduced PG/PI-TP sequence with other polypeptide sequences available in databases revealed a homology with a protein deduced from an open reading frame coding for an unknown protein in Saccharomyces cerevisiae (36% identity and 57% similarity). Apart from this homology, the PG/PI-TP is unique and specific to the filamentous fungi on the basis of comparison of PLTP protein sequences. Northern blot analysis of RNA isolated from A. oryzae cultures grown on glucose or glucose supplemented with phospholipids suggests that the PG/PI-TP is transcribed by only one RNA species and allows us to show that expression of the protein is regulated at the messenger RNA level.

Haematopoiesis radioprotection in Balb/c mice by an aqueous mycelium extract from the Basidiomycete Pleurotus ostreatus mushroom.

The study examined the radioprotective activity of an aqueous extract from Pleurotus ostreatus mycelium administered to Balb/c mice. Male mice were whole-body irradiated on day 0 ((60)Co, at 0.43 Gy/min) and divided into two groups. The extract was administered intraperitoneally to one group (100 mg/kg) on days - 10 to - 6 and - 2 to +1 with respect to the irradiation. The irradiated-control group was injected with saline solution; non-irradiated mice were used as negative controls. The radioprotective effect was evident by increases in bone marrow cellularity (5.1 × 10(6)/femur vs. 1.1 × 10(6)/femur in saline-control mice, p < 0.05), leucocyte counts (10.5 × 10(9)/L vs. 4.5 × 10(9)/L, p < 0.05), and spleen cellularity (11.2 × 10(7)/spleen vs. 6.2 × 10(7)/spleen, p < 0.05). The extract stimulated macrophage phagocytic activity as judged by a faster rate of carbon clearance in terms of absorbance ratios (1.62 vs. 2.01, p < 0.05). Therefore, this extract may be a candidate therapeutic agent with radioprotective activity for haematopoiesis damage, particularly to cells involved in immune function.

Cloning and expression in phospholipid containing cultures of the gene encoding the specific phosphatidylglycerol/phosphatidylinositol transfer protein from Aspergillus oryzae: evidence that the pg/pi-tp is tandemly arranged with the putative 3-ketoacyl-CoA thiolase gene.

The phosphatidylglycerol/phosphatidylinositol transfer protein (PG/PI-TP) is a new and original phospholipid transfer protein (PLTP) isolated from the Deuteromycete, Aspergillus oryzae. We have isolated a genomic clone of the A. oryzae pg/pi-tp using a probe derived from the corresponding cDNA and sequenced the complete gene. The DNA sequence analysis revealed that pg/pi-tp gene is composed of three exons encoding a 18,823 Da protein of 175 amino acids as previously described and of two introns as deduced by cDNA and genomic sequence alignment. The isolated pg/pi-tp gene do not show similarity with other PLTP genes or the deduced PG/PI-TP protein with proteins already known. Comparison of the encoded PG/PI-TP with other deduced proteins from recent genomic or cDNA sequence from databases revealed that the PG/PI-TP was close to two encoded proteins deduced from the cDNA database of Aspergillus nidulans (54% identity and 68% similarity) and the second from Neurospora crassa (53% identity and 76% similarity). Therefore, we suggested that both proteins might belong to the PLTP family. Southern blot analysis of A. oryzae genomic DNA show that the PG/PI-TP was encoded by a single gene. Expression of pg/pi-tp was performed in phospholipid containing cultures with increasing carbon source concentrations in order to study the regulation of the PLTPs in the filamentous fungus cell. This was done to know if a high density culture could yield a high amount of biomass with high phospholipid transfer activity. Results showed that phospholipids as compared to glucose in standard cultures stimulated mycelial growth and global phospholipid transfer activity, but not the pg/pi-tp transcript accumulation. However, high concentration of both carbon sources yielded an inhibition of the expression of the pg/pi-tp gene and of the global phospholipid transfer activity. In conclusion, both carbon sources are not suitable to increase the PLTP production in high density cultures for biotechnological applications. Finally, using the gene walking sequencing method it is demonstrated that the pg/pi-tp is tandemly arranged on opposite DNA strands in a tail-to-tail orientation with a putative gene encoding the 3-ketoacyl-CoA thiolase (EC 2.3.1.16). Unlike the pg/pi-tp gene, this thiolase gene show a putative 'beta-oxidation box' and encodes a putative 44,150 Da protein of 321 amino acids composed of a putative N-terminal PTS2 (Peroxisomal Targeting Signal) consensus sequence for the peroxisome targeting. Comparison of the amino acid sequence of the A. oryzae thiolase to that of the Yarrowia lipolytica showed a 50% identity and a 69% similarity.

Cloning and analysis of Pycnoporus cinnabarinus cellobiose dehydrogenase.

We have cloned and sequenced a gene encoding cellobiose dehydrogenase (CDH) from Pycnoporus cinnabarinus (Pci). PCR primers that may recognize a homologous cdh were designed using regions of complete conservation of amino acid sequence within the known sequences of Trametes versicolor (Tv) and Phanerochaete chrysosporium (Pc) CDH. Upstream primers hybridized to regions encoding the heme domain, whereas downstream primers recognized highly conserved regions within the flavin domain. Eight different primer pairs yielded three PCR products close in size to the control amplification, which used cloned Tv cdh as template. The PCR products that were close to the control size were cloned, and one of these, a 1.8-kb product, was completely sequenced. The PCR product was highly homologous to both Tv and Pch cdh, and contained eight putative introns. The cloned product was used to isolate a full-length clone encoding CDH from a Pci genomic library. Pci cdh encoded a protein with 83% identity with Tv CDH and 74% identity with Pch CDH. Northern blot analysis revealed that Pci cdh was transcribed as a single mRNA species and was expressed in the presence of cellulose as the carbon source.

Cloning and sequencing of a gene encoding cellobiose dehydrogenase from Trametes versicolor.

Cellobiose dehydrogenase (CDH) is an enzyme produced under lignocellulose-degrading conditions by Trametes versicolor strain 52J (Tv) and several other wood-degrading fungi, including Phanerochaete chrysosporium (Pc). In order to understand better the nature and properties of this enzyme, we isolated a genomic clone of Tv cdh using heterologous probes derived from the sequence of Pc cdh. DNA sequence analysis revealed that Tv cdh consists of 3091 bp of coding sequence interrupted by 14 introns. Southern blotting showed that the gene was present in a single copy in the strain of Tv analyzed. Tv cdh was shown by Northern blot analysis to be expressed as a single transcript under cellulolytic conditions. RT-PCR of poly(A)+ RNA isolated under cellulolytic conditions was used to generate a near full-length cDNA copy of the cdh mRNA. The deduced protein encoded by Tv cdh consists of 768 amino acids (aa), including a predicted 19 aa signal peptide. The protein had 73% identity to the corresponding protein from Pc, which is the only other CDH-encoding gene that has been cloned. Based upon its deduced primary structure and alignment to similar sequences, Tv CDH shares a general structural organization with Pc CDH and other hemoflavoenzymes. Amino acid residues H-109 and M-61 in the N-terminal heme domain are hypothesized to function in heme binding; the C-terminal flavin domain contained a consensus sequence for flavin binding between residues 217-222. Although the protein is known to bind to cellulose, no obvious homology to bacterial or fungal cellulose binding domains was observed. However, a strong homology was observed to a region of Pc CDH that is hypothesized to be involved in cellulose binding.

Selection of Pycnoporus cinnabarinus strains for laccase production.

A comparison of Pycnoporus cinnabarinus strains for laccase production was carried out. A dikaryotic strain, I-937 strain, producing a high level of laccase (9500 U l(-1)) was selected. The study of the life cycle in vitro of this dikaryotic strain led to isolation of monokaryons. Forty-eight monokaryotic strains were isolated and screened for laccase production. One of these strains, ss3, produced a higher level of laccase than the parental strain I-937. The maximum production reached 29000 U l(-1) in medium supplemented with ferulic acid.

Cloning and sequencing of a phenol hydroxylase gene of Pseudomonas pseudoalcaligenes strain MH1: a bacterium able to mineralize various aromatic compounds.

The phenol-degrading strain Pseudomonas pseudoalcaligenes MH1, identified by the rRNA approach, was isolated from wastewater enrichment culture. It utilized phenol up to 1.5 g/L as the sole source of carbon and energy. In addition, cresols (o-, m-, p-), 4-hydroxybenzoic acid, syringic acid, and vanillic acid were metabolized as sole substrates by phenol-grown cells of strain MH1. Using primers, designed on the basis of the sequence of the dmp operon of P. putida strain CF600, a gene encoding phenol hydroxylase, which catalyzes the hydroxylation of phenol to catechol, was detected in strain MH1. The whole phenol hydroxylase operon of strain MH1 was amplified in a polymerase chain reaction fragment of 5.207 kb that was cloned and sequenced. The total sequence revealed a cluster of six ATG starting open reading frames (ORFs). Analysis of the regulatory signals showed a putative promoter region, 40 bp upstream from the transcriptional start of ORF1, which have a strong homology to a set of positively controlled promoters. Comparison of the MH1 phenol hydroxylase gene sequence with those of other Pseudomonas strains revealed higher homology except in the 5' region. Thus, the deduced amino acid sequence of the first subunit of phenol hydroxylase of P. pseudoalcaligenes strain MH1 exhibited a difference at the N-terminal region (the first 10 amino acids) compared with that of known phenol hydroxylase of Pseudomonas strains.

Isolation of a new laccase isoform from the white-rot fungi Pycnoporus cinnabarinus strain ss3.

Two extracellular laccase isoforms (Lac I and Lac II) produced by the white-rot fungus Pycnoporus cinnabarinus from the monokaryotic strain ss3 were purified from ferulic-acid-induced liquid culture medium using ammonium sulphate precipitation, followed by anion-exchange chromatography on a Mono Q column. Strain ss3 is the first generation of the parental strain P. cinnabarinus I-937. The new isolated isoform, Lac II, consists of an 86,000 molecular weight protein as determined by SDS polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of both isoforms were determined, and compared to known laccase protein sequences of other organisms.

Spatial and temporal accumulation of mRNAs encoding two common lignin peroxidases in Phanerochaete chrysosporium.

Accumulation of peroxidases and their mRNAs was localized in colonies of Phanerochaete chrysosporium sandwiched between perforated polycarbonate membranes. Northern (RNA) blot analyses of colonial rings and in situ hybridizations with specific probes for manganese(II)-dependent peroxidase (MnP-1) and lignin peroxidase (LiP H8) mRNAs indicated that the expression of MnP-1 and Lip H8 genes started simultaneously in the central area of 3-day-old colonies. With time the signals for both transcripts spread to more-peripheral areas while decreasing in intensity. Furthermore, the appearance of MnP protein, as detected with specific immune serum, immediately followed accumulation of the MnP-1 mRNA transcript. However, LiP protein could be detected only some time after accumulation of LiP H8 mRNA.

Localization of growth and secretion of proteins in Aspergillus niger.

Hyphal growth and secretion of proteins in Aspergillus niger were studied using a new method of culturing the fungus between perforated membranes which allows visualization of both parameters. At the colony level the sites of occurrence of growth and general protein secretion were correlated. In 4-d-old colonies both growth and secretion were localized at the periphery of the colony, whereas in a 5-d-old colony growth and secretion also occurred in a more central zone of the colony where conidiophore differentiation was observed. However, in both cases glucoamylase secretion was mainly detected at the periphery of the colonies. At the hyphal level immunogold labelling showed glucoamylase secretion at the tips of leading hyphae only. Microautoradiography after labelling with N-acetylglucosamine showed that these hyphae were probably all growing. Glucoamylase secretion could not be demonstrated immediately after a temperature shock which stopped growth. These results indicate that glucoamylase secretion is located at the tips of growing hyphae only.

In situ localization of the secretion of lignin peroxidases in colonies of Phanerochaete chrysosporium using a sandwiched mode of culture.

Protein secretion and growth were investigated in Phanerochaete chrysosporium by using cultures sandwiched between perforated polycarbonate membranes. Labelling of colonies with radioactive N-acetylglucosamine and L-methionine indicated a close correlation between growth and general protein secretion, even in a central area of the colony secreting the idiophase enzymes lignin peroxidase (LiP) and manganese-dependent lignin peroxidase (MnP). Comparison of the sites of release into the medium of newly synthesized proteins and immuno-detected lignin peroxidases suggested that diffusion of the enzymes from the walls was a limiting step in the release of peroxidases into the medium. Microautoradiography of colonies exposed to N-acetyl[3H]glucosamine revealed the apical growth of thin hyphae and branches (4 to 5 microns diameter on average) in the central secreting area. These secondary hyphae showed peroxidase activity and reacted with lignin peroxidase antibodies. Although it was not possible to directly visualize secretion at hyphal tips, the results suggest that peroxidases (LiP and MnP) are initially secreted at the apex of secondary growing hyphae and later slowly released into the surrounding medium.

Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I-937 and expression in Pichia pastoris.

Laccases are multicopper-containing enzymes which catalyse the oxidation of phenolic and nonphenolic compounds with the concomitant reduction of molecular oxygen. In this study, a full-length cDNA coding for laccase (lac1) from Pycnoporus cinnabarinus I-937 was isolated and characterized. The corresponding open reading frame is 1557 nucleotides long and encodes a protein of 518 amino acids. The cDNA encodes a precursor protein containing a 21 amino-acid signal sequence corresponding to a putative signal peptide. The deduced amino-acid sequence of the encoded protein was similar to that of other laccase proteins, with the residues involved in copper coordination sharing the greatest extent of similarity. The cDNA encoding for laccase was placed under the control of the alcohol oxidase (Aox 1) promoter and expressed in the methylotropic yeast Pichia pastoris. The laccase leader peptide, as well as the Saccharomyces cerevisiae alpha-factor signal peptide, efficiently directed the secretion into the culture medium of laccase in an active form. Moreover, the laccase activity was directly detected in plates. The identity of the recombinant product was further confirmed by protein immunoblotting. The expected molecular mass of the mature protein is 81 kDa. However, the apparent molecular mass of the recombinant protein is 110 k Da, thus suggesting that the protein expressed in P. pastoris may be hyperglycosylated.

Localization of a phosphatidylglycerol/phosphatidylinositol transfer protein in Aspergillus oryzae.

The subcellular localization of the phosphatidylglycerol/phosphatidylinositol transfer protein (PG/PI-TP) of Aspergillus oryzae was investigated using Western blot analysis of the cell protein extracts, a cellular membrane fractionation technique, and transmission electron microscopy. The PG/PI-TP, as detected by Western blot analysis with a specific immune serum, was found to be mainly cytoplasmic and partly associated with intracellular membranes. A fractionation experiment was conducted after homogenization of the filamentous fungus mycelium. The endoplasmic reticulum, Golgi-like vesicles, and the plasma membrane were separated by isopycnic ultracentrifugation on a sucrose gradient, and our data revealed that the immunodetected PG/PI-TP was only associated with the Golgi-like apparatus. All these results were documented by electron microscopy and indicate here for the first time that there exists a specific phospholipid transfer protein in a filamentous fungus that is localized in the cytoplasm and associated with Golgi-like vesicles.

Characterization of Peroxidase Secretion and Subcellular Organization of Phanerochaete chrysosporium INA-12 in the Presence of Various Soybean Phospholipid Fractions.

Stimulation of lignin peroxidase production by exogenous phospholipids depends on the composition of the phospholipid fraction prepared by using the Nattermann process. The fraction composed mainly of negatively charged phospholipids (NAT 89) was the most efficient source for exoprotein secretion by Phanerochaete chrysosporium INA-12. The results of biochemical marker assays and ultrastructural morphology determination by electron microscopy were correlated. Activities of succinate dehydrogenase, a mitochondrial marker, and cytochrome c oxidoreductase, an endoplasmic reticulum (ER) marker, were increased 1.3- and 2.2-fold, respectively, in the presence of NAT 89. Electron microscopy observations suggested that the amount of mitochondria and ER in culture containing phospholipids was increased at the optimum day of lignin peroxidase production. Therefore, phospholipids enhanced energetic metabolism of strain INA-12 and markedly modified fungus physiology. Since ER is involved in enzyme synthesis, we suggest that its increased amount in mycelium cultured with NAT 89 is directly associated with the higher production of lignin peroxidase.

Sequence analysis of the glyceraldehyde-3-phosphate dehydrogenase genes from the basidiomycetes Schizophyllum commune, Phanerochaete chrysosporium and Agaricus bisporus.

GPD genes encoding glyceraldehyde-3-phosphate dehydrogenase were isolated from the homobasidiomycetes Schizophyllum commune, Phanerochaete chrysosporium and Agaricus bisporus. All three species contain one transcriptionally active GPD gene, but A. bisporus also contains an inactive GPD gene (tandemly linked to the active gene). These genes contain 5-9 introns located at conserved positions, differing (except in one case) from intron positions in ascomycetous GPD genes. The predicted amino-acid sequences of the proteins encoded by the three active GPD genes are highly homologous. A comparison with protein sequences from filamentous ascomycetes shows a clear distinction, whereas the GPD genes from ascomycetous yeasts are quite distinct from both the filamentous ascomycetes and basidiomycetes. Promoter regions of ascomycetous GPD genes do not correspond to those of the GPD genes of basidiomycetes which may (partly) explain poor expression in basidiomycetes of introduced genes driven by an ascomycete GPD promoter.