Experimental aflatoxin production in Manchego-type cheese

Manchego-type cheese, a typical Spanish cheese, was inoculated in various ways with an aflatoxigenic organism, Aspergillus parasiticus NRRL 2999, to study the production of aflatoxin. When the original milk was contaminated with a spore suspension, aflatoxin was not detected in paraffin-covered cheeses although it was present in the top layer of non-paraffin-covered cheeses after ripening at 15°C for 60 d. When the cheese surface was inoculated, no aflatoxins were detected in paraffin-covered cheeses after ripening for 60 d although they were found when the cheeses were ripened for 30 d. In non-paraffin-covered cheeses aflatoxins were detected only in the top layer and in the second 10 mm layer when cheeses were incubated after the normal ripening at 28°C for 30 d. When the centre of the cheese was inoculated, no aflatoxins were detected although Aspergillus grew slightly along the inoculation area. When cheese portions were inoculated, fungal growth was evident after incubation at 28° and 15°C for 6 d but there was no growth at 10°C after 50 d. At 28°C aflatoxins were detected at a concentration of 132 μg/g after 13 d, the highest level obtained. In cheese paste at 28° and 15°C, growth was intense, but the level of aflatoxins detected was lower than in cheese portions. At 10°C the growth was heavy, but aflatoxins were not detected.     

Phylogenetic utility of ycf1 in orchids: a plastid gene more variable than matK

Plastid DNA sequences have been widely used by systematists for reconstructing plant phylogenies. The utility of any DNA region for phylogenetic analysis is determined by ease of amplification and sequencing, confidence of assessment in phylogenetic character alignment, and by variability across broad taxon sampling. Often, a compromise must be made between using relatively highly conserved coding regions or highly variable introns and intergenic spacers. Analyses of a combination of these types of DNA regions yield phylogenetic structure at various levels of a tree (i.e., along the spine and at the tips of the branches). Here, we demonstrate the phylogenetic utility of a heretofore unused portion of a plastid protein-coding gene, hypothetical chloroplast open reading frame 1 (ycf1), in orchids. All portions of ycf1 examined are highly variable, yet alignable across Orchidaceae, and are phylogenetically informative at the level of species. In Orchidaceae, ycf1 is more variable than matK both in total number of parsimony informative characters and in percent variability. The nrITS region is more variable than ycf1, but is more difficult to align. Although we only demonstrate the phylogenetic utility of ycf1 in orchids, it is likely to be similarly useful among other plant taxa.     

Rit contributes to nerve growth factor-induced neuronal differentiation via activation of B-Raf-extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades

Rit is one of the original members of a novel Ras GTPase subfamily that uses distinct effector pathways to transform NIH 3T3 cells and induce pheochromocytoma cell (PC6) differentiation. In this study, we find that stimulation of PC6 cells by growth factors, including nerve growth factor (NGF), results in rapid and prolonged Rit activation. Ectopic expression of active Rit promotes PC6 neurite outgrowth that is morphologically distinct from that promoted by oncogenic Ras (evidenced by increased neurite branching) and stimulates activation of both the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase signaling pathways. Furthermore, Rit-induced differentiation is dependent upon both MAP kinase cascades, since MEK inhibition blocked Rit-induced neurite outgrowth, while p38 blockade inhibited neurite elongation and branching but not neurite initiation. Surprisingly, while Rit was unable to stimulate ERK activity in NIH 3T3 cells, it potently activated ERK in PC6 cells. This cell type specificity is explained by the finding that Rit was unable to activate C-Raf, while it bound and stimulated the neuronal Raf isoform, B-Raf. Importantly, selective down-regulation of Rit gene expression in PC6 cells significantly altered NGF-dependent MAP kinase cascade responses, inhibiting both p38 and ERK kinase activation. Moreover, the ability of NGF to promote neuronal differentiation was attenuated by Rit knockdown. Thus, Rit is implicated in a novel pathway of neuronal development and regeneration by coupling specific trophic factor signals to sustained activation of the B-Raf/ERK and p38 MAP kinase cascades.     

Hematological,biochemical, respiratory,cardiovascular and electroneurophysiological parameters in African green monkeys (Cercopithecus aethiops sabaeus). Its use in non‐clinical toxicological studies

Background The purpose of this study is to better characterize the hematological, biochemical, respiratory, cardiovascular and electroneurophysiological parameters in young adult Cercopithecus aethiops sabaeus of both sexes. The rhesus and cynomolgus monkeys are widely used as experimental primate models. However, only few articles have been published testing toxicological effects of pharmaceuticals on African green monkey. Methods The present study was carried out with the recompilation of all parameters recorded before the first drug administration in five sub‐chronic or chronic toxicological studies performed on 66 Cercopithecus aethiops sabaeus, born in Cuba. Results This study provides hematological, biochemical, respiratory, cardiovascular and electroneurophysiological data for both choosing animals to be included into experiments and monitoring these parameters during the study. Conclusions We conclude that this study provides valuable integrated data for determining the health status, including electroneurophysiological parameters, data not previously reported for this species, of the African green monkey.     

Multiparametric comparison of mesenchymal stromal cells obtained from trabecular bone by using a novel isolation method with those obtained by iliac crest aspiration from the same subjects

Trabecular bone fragments from femoral heads are sometimes used as bone grafts and have been described as a source of mesenchymal progenitor cells. Nevertheless, mesenchymal stromal cells (MSC) from trabecular bone have not been directly compared with MSC obtained under standard conditions from iliac crest aspiration of the same patients. This is the ideal control to avoid inter-individual variation. We have obtained MSC by a novel method (grinding bone fragments with a bone mill without enzymatic digestion) from the femoral heads of 11 patients undergoing hip replacement surgery and compared them with MSC obtained by standard iliac crest aspiration of bone marrow from the same patients. We have shown that trabecular bone MSC obtained by mechanically fragmented femoral heads fulfil the immunophenotypic and multilineage (adipogenic, osteogenic and chondrogenic) differentiation criteria used to define MSC. We have also differentially compared cellular yields, growth kinetics, cell cycle assessment, and colony-forming unit-fibroblast content of MSC from both sources and conclude that these parameters do not significantly differ. Nevertheless, the finding of slight differences, such as a higher expression of the immature marker CD90, a lower expansion time through the different passages, and a higher percentage of cycling cells in the trabecular bone MSC, warrants further studies with the isolation method proposed here in order to gain further knowledge of the status of MSC in this setting. The present study was partially supported by grant HUS01B07 from the Consejería de Educación and by grant SAN196/SA13/07 from the Consejería de Sanidad, Junta de Castilla y León, Spain.     

Internal motional amplitudes and correlated bond rotations in an alpha-helical peptide derived from 13C and 15N NMR relaxation

Peptide GFSKAELAKARAAKRGGY folds in an alpha-helical conformation that is stabilized by formation of a hydrophobic staple motif and an N-terminal capping box (Munoz V. Blanco FJ, Serrano L, 1995, Struct Biol 2:380-385). To investigate backbone and side-chain internal motions within the helix and hydrophobic staple, residues F2, A5, L7, A8, and A10 were selectively 13C- and 15N-enriched and NMR relaxation experiments were performed in water and in water/trifluoroethanol (TFE) solution at four Larmor frequencies (62.5, 125, 150, and 200 MHz for 13C). Relaxation data were analyzed using the model free approach and an anisotropic diffusion model. In water, angular variances of motional vectors range from 10 to 20 degrees and backbone phi,psi bond rotations for helix residues A5, L7, A8, and A10 are correlated indicating the presence of Calpha-H, Calpha-Cbeta, and N-H rocking-type motions along the helix dipole axis. L7 side-chain CbetaH2 and CgammaH motions are also correlated and as motionally restricted as backbone CalphaH, suggesting considerable steric hindrance with neighboring groups. In TFE which stabilizes the fold, internal motional amplitudes are attenuated and rotational correlations are increased. For the side chain of hydrophobic staple residue F2, wobbling-in-a-cone type motions dominate in water, whereas in TFE, the Cbeta-Cgamma bond and phenyl ring fluctuate more simply about the Calpha-Cbeta bond. These data support the Daragan-Mayo model of correlated bond rotations (Daragan VA, Mayo KH, 1996, J Phys Chem 100:8378-8388) and contribute to a general understanding of internal motions in peptides and proteins.     

Inhibitors of alphavirus entry and replication identified with a stable Chikungunya replicon cell line and virus-based assays

Chikungunya virus (CHIKV), an alphavirus, has recently caused epidemic outbreaks and is therefore considered a re-emerging pathogen for which no effective treatment is available. In this study, a CHIKV replicon containing the virus replicase proteins together with puromycin acetyltransferase, EGFP and Renilla luciferase marker genes was constructed. The replicon was transfected into BHK cells to yield a stable cell line. A non-cytopathic phenotype was achieved by a Pro718 to Gly substitution and a five amino acid insertion within non-structural protein 2 (nsP2), obtained through selection for stable growth. Characterization of the replicon cell line by Northern blotting analysis revealed reduced levels of viral RNA synthesis. The CHIKV replicon cell line was validated for antiviral screening in 96-well format and used for a focused screen of 356 compounds (natural compounds and clinically approved drugs). The 5,7-dihydroxyflavones apigenin, chrysin, naringenin and silybin were found to suppress activities of EGFP and Rluc marker genes expressed by the CHIKV replicon. In a concomitant screen against Semliki Forest virus (SFV), their anti-alphaviral activity was confirmed and several additional inhibitors of SFV with IC₅₀ values between 0.4 and 24 µM were identified. Chlorpromazine and five other compounds with a 10H-phenothiazinyl structure were shown to inhibit SFV entry using a novel entry assay based on a temperature-sensitive SFV mutant. These compounds also reduced SFV and Sindbis virus-induced cytopathic effect and inhibited SFV virion production in virus yield experiments. Finally, antiviral effects of selected compounds were confirmed using infectious CHIKV. In summary, the presented approach for discovering alphaviral inhibitors enabled us to identify potential lead structures for the development of alphavirus entry and replication phase inhibitors as well as demonstrated the usefulness of CHIKV replicon and SFV as biosafe surrogate models for anti-CHIKV screening.     

Extension of the Messapia x dicoccoides linkage map of Triticum turgidum (L.) Thell

A set of recombinant inbred lines (RIL) derived from a cross between the cultivar Messapia of durum wheat (Triticum turgidum var. durum) and the accession MG4343 of T. turgidum var. dicoccoides was analysed to increase the number of assigned markers and the resolution of the previously constructed genetic linkage map. An updated map of the durum wheat genome consisting of 458 loci was constructed. These loci include 261 Restriction Fragment Length Polymorphisms (RFLPs), 91 microsatellites (Simple Sequence Repeats, SSRs), 87 Amplified Fragment Length Polymorphisms (AFLPs), two ribosomal genes, and nine biochemical (seven seed storage proteins and two isozymes) and eight morphological markers. The loci were mapped on all 14 chromosomes of the A and B genomes, and covered a total distance of 3038.4 cM with an average distance of 6.7 cM between adjacent markers. The molecular markers were evenly distributed between the A and the B genomes (240 and 218 markers, respectively). An additional forty loci (8.8%) could not be assigned to a specific linkage group. A fraction (16.4%) of the markers significantly deviated from the expected Mendelian ratios; clusters of loci showing distorted segregation were found on the 1B, 2A, 2B, 3A, 4A, 7A and 7B chromosomes. The genetic lengths of the chromosomes range from 148.8 cM (chromosome 6B) to 318.0 cM (chromosome 2B) and approximately concur with their physical lengths. Chromosome 2B has the largest number of markers (47), while the chromosomes with the fewest markers are 3A and 6B (23). There are two gaps larger than 40 cM on chromosomes 2A and 3B. The durum wheat map was compared with the published maps of bread and durum wheats; the order of most common RFLP and SSR markers on the 14 chromosomes of the A and B genomes were nearly identical. A core-map can be extracted from the high-density Messapia x dicoccoides map and a subset of uniformly distributed markers can be used to detect and map quantitative trait loci.     

Explaining geographical variation in the isotope composition of mouse lemurs (Microcebus)

Aim We sought to quantify geographical variation in the stable isotope values of mouse lemurs (Microcebus) and to determine whether this variation reflects trophic differences among populations or baseline isotopic differences among habitats. If the latter pattern is demonstrated, then Microcebus can become a proxy for tracking baseline habitat isotopic variability. Establishing such a baseline is crucial for identifying niche partitioning in modern and ancient communities. Location We studied five species of Microcebus from eight distinct habitats across Madagascar. Methods We compared isotopic variation in C₃ plants and Microcebus fur within and among localities. We predicted that carbon and nitrogen isotope values of Microcebus should: (1) vary as a function of abiotic variables such as rainfall and temperature, and (2) covary with isotopic values in plants. We checked for trophic differences among Microcebus populations by comparing the average difference between mouse lemur and plant isotope values for each locality. We then used multiple regression models to explain spatial isotope variation in mouse lemurs, testing a suite of explanatory abiotic variables. Results We found substantial isotopic variation geographically. Ranges for mean isotope values were similar for both Microcebus and plants across localities (carbon 3.5–4.0‰; nitrogen 10.5–11.0‰). Mean mouse lemur and plant isotope values were lowest in cool, moist localities and highest in hot, dry localities. Rainfall explained 58% of the variation in Microcebus carbon isotope values, and mean plant nitrogen isotope values explained 99.7% of the variation in Microcebus nitrogen isotope values. Average differences between mouse lemur and plant isotope values (carbon 5.0‰; nitrogen 5.9‰) were similar across localities. Main conclusions Isotopic data suggest that trophic differences among Microcebus populations were small. Carbon isotope values in mouse lemurs were negatively correlated with rainfall. Nitrogen isotope values in Microcebus and plants covaried. Such findings suggest that nitrogen isotope values for Microcebus are a particularly good proxy for tracking baseline isotopic differences among habitats. Our results will facilitate future comparative research on modern mouse lemur communities, and ecological interpretations of extinct Holocene communities.     

Characterization of an RNase with two catalytic centers. Human RNase6 catalytic and phosphate-binding site arrangement favors the endonuclease cleavage of polymeric substrates

Background

Human RNase6 is a small cationic antimicrobial protein that belongs to the vertebrate RNaseA superfamily. All members share a common catalytic mechanism, which involves a conserved catalytic triad, constituted by two histidines and a lysine (His15/His122/Lys38 in RNase6 corresponding to His12/His119/Lys41 in RNaseA). Recently, our first crystal structure of human RNase6 identified an additional His pair (His36/His39) and suggested the presence of a secondary active site.