Increasing the species diversity in the Aspergillus section Nidulantes: Six novel species mainly from the indoor environment.

Aspergillus section Nidulantes encompasses almost 80 homothallic and anamorphic species, mostly isolated from soil, plant material, or the indoor environment. Some species are clinically relevant or produce mycotoxins. This study reevaluated the species boundaries within several clades of section Nidulantes. Five data sets were assembled, each containing presumptive new species and their closest relatives, and phylogenetic and phenotypic analyses were performed. We tested the hypotheses that the newly isolated or reexamined strains constitute separate species (splitting approach) or should be treated as part of broadly defined species (lumping approach). Four DNA sequence loci were amplified, internal transcribed spacer (ITS) and large subunit (LSU) regions of the rDNA and partial sequences of the ß-tubulin (benA), calmodulin (CaM), and RNA polymerase II second largest subunit (RPB2) genes. The latter three loci were used for the phylogenetic analysis and served as input for single-locus (GMYC, bGMYC, PTP, and bPTP) and multilocus (STACEY and BP&P) species delimitation analyses. The phenotypic analysis comprised macro- and micromorphology (including scanning electron microscopy) and comparison of cardinal growth temperatures. The phylogenetic analysis supported the splitting hypothesis in all cases, and based on the combined approach, we propose six new species, four that are homothallic and two anamorphic. Four new species were isolated from the indoor environment (Jamaica, Trinidad and Tobago, USA), one originated from soil (Australia), and one from a kangaroo rat cheek pouch (USA).

Hydroxylated anthraquinones produced by Geosmithia species.

Geosmithia fungi are little known symbionts of bark beetles. Secondary metabolites of lilac colored species G. lavendula and other nine Geosmithia species were investigated in order to elucidate their possible role in the interactions of the fungi with environment. Hydroxylated anthraquinones (yellow, orange, and red pigments), were found to be the most abundant compounds produced into the medium during the submerged cultivation. Three main compounds were identified as 1,3,6,8-tetrahydroxyanthraquinone (1), rhodolamprometrin (1-acetyl-2,4,5,7-tetrahydroxyanthraquinone; 2), and 1-acetyl-2,4,5,7,8-pentahydroxyanthraquinone (3). Compounds 2 and 3 (representing the majority of produced metabolites) inhibited the growth of G+-bacteria Staphylococcus aureus and Bacillus subtilis with minimum inhibitory concentration of 64-512 microg/mL. Anti-inflammatory activity detected as inhibition of cyclooxygenase-2 was found only for compound 3 at 1 and 10 microg/mL. Compound 2 interfered with the morphology, compound 3 with cell-cycle dynamics of adherent mammalian cell lines.

Production of (+)-globulol needle crystals on the surface mycelium of Quambalaria cyanescens.

The structure of unique colorless needle crystals growing from the surface mycelium of the basidiomycete Quambalaria cyanescens and identified as (+)-globulol was followed by mass spectrometry, X-ray diffraction, and polarimetry. The mechanism of (+)-globulol fragmentation is proposed based on collision induced dissociation mass spectrometry. X-Ray analysis revealed that crystal packing is governed by hydrogen bond O-H.....O connecting the molecules into an infinite helix along a 3-fold screw axis propagating along the longest dimension of the needle crystal (c-axis of the unit cell). The X-ray diffraction data correspond well with the proposed structure determined by mass spectrometry.

Oligosaccharides produced by submerged cultures of Claviceps africana and Claviceps sorghi.

Oligosaccharides produced by submerged cultures of C. africana and C. sorghi were isolated by semipreparative HPLC. Structure of 6-O-beta-D-fructofuranosyl-D-glucopyranose (blastose), 1,6-bis-O-(beta-D-fructofuranosyl)-alpha-D-glucopyranoside (neokestose) and two sugar alcohols, 1-O-beta-D-fructofuranosyl-D-mannitol (fructosylmannitol) and 1,6-bis-O-(beta-D-fructofuranosyl)-D-mannitol (bisfructosylmannitol) was determined by NMR spectrometry. MALDI TOF MS analysis revealed molecular ions [M+Na]+ that indicate the presence of other tetra- and pentasaccharides (m/z = 689.4 and 851.5, respectively) and corresponding sugar alcohol (m/z = 691.4). Rapid conversion of sucrose into series of oligosaccharides and corresponding sugar alcohols was observed in all tested strains.

Physiological dendrogram of Claviceps spp. based on sucrose metabolism in submerged cultures and its comparison with phylogenetic tree.

Sixteen isolates of Claviceps spp. were analyzed for the production of polysaccharides, oligosaccharides, and sucrose metabolism under conditions of submerged fermentation. Physiological markers calculated by the Verhulst-Pearl law were used for hierarchical cluster analysis. Low correlation was found between physiologically based dendrogram and phylogenetic analysis constructed from an alignment of rDNA sequences. To confirm the intraspecific uniformity of physiological markers three isolates of C. africana from different hosts and locations were included. The influence of genotype, physiological variability, environmental location and habitat on metabolite production is discussed.

The effect of retinoids and butyrate on the expression of CRX and IRBP in retinoblastoma cells.

We sought to determine whether differentiation agents such as retinoids and butyrate regulate transcript levels of interphotoreceptor retinoid binding protein (IRBP) and cone rod homeobox (CRX), a homeodomain transcription factor that regulates IRBP promoter activity. WERI-Rb1 retinoblastoma cells were treated with all-trans retinol, all-trans retinoic acid, or butyrate. IRBP and CRX mRNA levels were determined by quantitative RT-PCR. Butyrate at low concentrations increased both mRNA levels but suppressed them at higher concentrations. Retinoic acid had minimal effects. Retinol increased CRX mRNA over four fold. IRBP and CRX transcript levels are sensitive to butyrate and CRX expression is sensitive to retinol.

Endogenous CRX expression and IRBP promoter activity in retinoblastoma cells.

PURPOSE: To determine whether antisense oligonucleotides (AODNs) targeted against CRX, a photoreceptor-specific trans-acting factor, suppress CRX expression and interphotoreceptor retinoid binding protein (IRBP) promoter activity. METHODS: Cultures of human retinoblastoma cells were transfected with chloramphenicol acetyltransferase (CAT) reporter plasmids containing a mouse IRBP promoter and AODNs directed against CRX. RT-PCR using primers specific to CRX, OTX2, GAPDH, or RNase H was conducted on total RNA isolated from retinoblastoma cells at various times following transfection with AODNs. RESULTS: Transfection of retinoblastoma cells with IRBP promoter CAT constructs alone produced high activity. Co-transfection with AODNs suppressed IRBP promoter activity in a concentration-dependent manner, with half-maximal effect produced at about 2 nM AODN concentration. Transfection with CAT constructs containing an SV40 promoter produced high activity that was unaffected by co-transfection with AODNs. RT-PCR products were obtained for all target sequences. CRX RT-PCR product from cells transfected with AODNs was greatly diminished following transfection with an AODN whereas control transcripts, including that of OTX2, were relatively unaffected. CONCLUSIONS: The CRX-specific AODNs specifically and potently suppressed CRX expression and IRBP promoter activity, as measured by RT-PCR and transient transfection assays, respectively. Little or no effect was seen on controls. These data suggest that endogenous CRX is required for IRBP promoter activity in retinoblastoma cells.

Evidence of a tissue-restricting DNA regulatory element in the mouse IRBP promoter.

The expression of interphotoreceptor retinoid binding protein (IRBP) is limited to photoreceptor cells of the retina and pinealocytes of the pineal gland. We sought to define cis-elements of the mouse IRBP 5' flanking region that are required for this restricted activity. In vitro transient transfections of retinoblastoma and neuroblastoma cells and in vivo experiments with transgenic Xenopus laevis indicate that -1783/+101 and -156/+101 IRBP gene fragments directed expression predominantly to the retina and pineal, with minor neuronal expression elsewhere. In contrast, a -70/+101 fragment was less restrictive in controlling expression, exhibiting activity not only in retina, but also in forebrain, hindbrain, spinal cord, and motor neurons innervating gills.

Extra-hepatic expression of serum albumin mRNA in mouse retina.

PURPOSE: In some mammals, serum albumin protein exists in the interphotoreceptor space (IPS), the space between photoreceptor cells and the retinal pigment epithelium. Serum albumin is synthesized largely in the liver, though low levels of extra-hepatic expression have been documented in several other tissues, including fetal rat kidney, pancreas, lung, and heart. The purpose of this study was to investigate whether serum albumin protein and mRNA are found in mouse retina. METHODS: Using albumin rabbit antibodies and HRP goat anti-(rabbit IgG), we performed immunoassays on mouse IPS washes to detect the presence of serum albumin protein. Protein extracts from IPS washes were subjected to Affigel Blue chromatography. This resin has an affinity for serum albumin. Reverse transcription-polymerase chain reaction (RT-PCR) of retina total RNA was performed to search for albumin mRNA. Also, real-time reverse transcription polymerase chain reaction (RT-RT-PCR) was employed to look at the levels of expression in different age groups. RESULTS: A constituent of the IPS washes specifically bound and eluted from Affigel Blue column, suggesting that the washes contained serum albumin. SDS PAGE revealed that the size of the constituent was 67 kDa, the size of serum albumin. This 67 kDa band reacted with mouse serum antibody. An RT-PCR amplified fragment of serum albumin mRNA from retina displayed the expected size. The sequence of this fragment is identical to authentic serum albumin cDNA sequence. RPE and choroid were negative for serum albumin mRNA. However, rd1(-)/rd1(-) retina was positive, suggesting that at least some serum albumin is synthesized in the inner layers of the retina. RT-RT-PCR showed that serum albumin mRNA levels in whole retina reached a maximum at about postnatal day 15 and gradually decreased to about one-sixth of maximum at 12 months age. CONCLUSIONS: Serum albumin protein and mRNA were found in mouse IPS and retina, suggesting that the protein is synthesized in the retina. The previously demonstrated ability of serum albumin to bind fatty acids and retinoids and its presence in the mouse IPS suggest a role for serum albumin in transporting retinoids in the retina or IPS, especially at young ages when concentrations appear greatest.

Structural characterization and comparison of promoter activity of mouse and bovine interphotoreceptor retinoid-binding protein (IRBP) gene 5' flanking regions in WERI, Y79, chick retina cells, and transgenic mice.

PURPOSE: To determine the sequences of the mouse and bovine interphotoreceptor retinoid-binding protein (IRBP) 5' flanking regions and whether these 5' flanking regions contain functional IRBP promoter activity in multiple cell types using both quantitative and statistical analyses. METHODS: We sequenced the bovine and mouse 5' flanking regions of the IRBP gene and compared these sequences to the human gene sequence. To test for functional activity of this region, we used the same DNA construct, p1783, in four different cell types. Mobility shift, DNase footprints, and southwestern blots were used to determine where nuclear protein complexes bind the IRBP 5' flanking region. RESULTS: The 5' flanking regions of the bovine, human, and mouse IRBP genes exhibit sequence similarity in regions immediately adjacent to the start of transcription (roughly 350 bases in length) and also over a 220 base sequence about 1.25 to 1.50 kb upstream of the transcription start site. Two different statistical approaches showed that the IRBP 5' flanking region possesses promoter activity in four different cell types. By using mobility shift, DNase I-protection experiments, and southwestern blotting, a region of about 45 bases at position -300 was identified that specifically binds a protein from the nuclei of bovine retina and Y79 cells. CONCLUSIONS: Specific DNA binding events are an essential part of IRBP promoter activity. The conservation of sequences far upstream of the transcription start suggest that unknown physiological processes remain to be understood in IRBP transcriptional regulation.