Constitutive expression of fluorescent protein by Aspergillus var. niger and Aspergillus carbonarius to monitor fungal colonization in maize plants

Aspergillus niger and Aspergillus carbonarius are two species in the Aspergillus section Nigri (black-spored aspergilli) frequently associated with peanut (Arachis hypogea), maize (Zea mays), and other plants as pathogens. These infections are symptomless and as such are major concerns since some black aspergilli produce important mycotoxins, ochratoxins A, and the fumonisins. To facilitate the study of the black aspergilli–maize interactions with maize during the early stages of infections, we developed a method that used the enhanced yellow fluorescent protein (eYFP) and the monomeric red fluorescent protein (mRFP₁) to transform A. niger and A. carbonarius, respectively. The results were constitutive expressions of the fluorescent genes that were stable in the cytoplasms of hyphae and conidia under natural environmental conditions. The hyphal in planta distribution in 21-day-old seedlings of maize were similar wild type and transformants of A. niger and A. carbonarius. The in planta studies indicated that both wild type and transformants internally colonized leaf, stem and root tissues of maize seedlings, without any visible disease symptoms. Yellow and red fluorescent strains were capable of invading epidermal cells of maize roots intercellularly within the first 3 days after inoculation, but intracellular hyphal growth was more evident after 7 days of inoculation. We also tested the capacity of fluorescent transformants to produce ochratoxin A and the results with A. carbonarius showed that this transgenic strain produced similar concentrations of this secondary metabolite. This is the first report on the in planta expression of fluorescent proteins that should be useful to study the internal plant colonization patterns of two ochratoxigenic species in the Aspergillus section Nigri.     

Kinetics improvement of high-grade sulphides bioleaching by effects separation

Abstract: The kinetics of bioleaching of sulphide concentrates by Thiobacillus ferrooxidans can be improved by performing separately the two operations relating the two effects involved in the indirect contact mechanism, (1) chemical leaching and (2) biological oxidation of the Fe2+ produced in the chemical stage (IBES: Indirect Bioleaching with Effects Separation). This scheme permits the separate enhancement of each stage. On the one hand, the chemical attack of the ferric iron to sulphides can be performed at a higher temperature. On the other hand, the biological oxidation stage is more effective when biofilm models are applied. At the same time, inhibitory phenomena produced as a result of the bacterial breaking caused by the abrasion of the solid particles on microorganisms are prevented. In this work the 1BES process has been applied to a Spanish copper-zinc and a copper sulphide concentrate. The objective has been to selectively dissolve zinc, which may be recovered by solvent exraction, the remaining residue being a copper concentrate suitable for pyrometallurgical treatment. The attributes of moderate temperature, atmospheric pressure, low cost of the leaching agent, flexibility and effectiveness make the process very attractive from an economic point of view.     

Stromal cells provide the matrix for migration of early lymphoid progenitors through the thymic cortex

During steady state lymphopoiesis in the postnatal thymus, migration of precursors outward from the deep cortex toward the capsule is required for normal differentiation. Such migration requires, at a minimum, expression of adhesive receptors on the migrating lymphoid cells, as well as a stable matrix of their ligands persisting throughout the region of migration. In this study, we address the nature of this adhesive matrix. Although some precursor stages bound efficiently to extracellular matrix ligands, a specific requirement for the cell surface ligand VCAM-1 was also found. In situ analysis revealed that early precursors are found in intimate contact with a matrix formed by stromal cells in the cortex, a proportion of which expresses VCAM-1. In vivo administration of an anti-VCAM-1 Ab resulted in decreased thymic size and altered distribution of early precursors within the cortex. These results indicate that precursors migrating outward through the cortex may use a cellular, rather than extracellular, matrix for adhesion, and suggest that the VCAM-1(+) subset of cortical stroma may play a crucial role in supporting the migration of early precursors in the steady state thymus.     

Unusual presentation of choriocarcinoma

Background  

Choriocarcinoma is an aggressive neoplasm arising in the body of the uterus. The disease normally spreads to lung and brain.     

Construction of a tightly regulated plasmid vector for Streptococcus pneumoniae: controlled expression of the green fluorescent protein

We have constructed a regulated plasmid vector for Streptococcus pneumoniae, based on the streptococcal broad-host-range replicon pLS1. As a reporter gene, we subcloned the gfp gene from Aequorea victoria, encoding the green fluorescent protein. This gene was placed under the control of the inducible P(M) promoter of the S. pneumoniae malMP operon which, in turn, is regulated by the product of the pneumococcal malR gene. Binding of MalR protein to the P(M) promoter is inactivated by growing the cells in maltose-containing media. Highly regulated gene expression was achieved by cloning in the same plasmid the P(M)-gfp cassette and the malR gene, thus providing the MalR regulator in cis. Pneumococcal cells harboring this vector gave a linear response of GFP synthesis in a maltose-dependent mode without detectable background levels in the absence of the inducer.     

Differential expression level of cytokeratin 8 in cells of the bovine nucleus pulposus complicates the search for specific intervertebral disc cell markers

Introduction  

Development of cell therapies for repairing the intervertebral disc is limited by the lack of a source of healthy human disc cells. Stem cells, particularly mesenchymal stem cells, are seen as a potential source but differentiation strategies are limited by the lack of specific markers that can distinguish disc cells from articular chondrocytes.     

Human histone pre-mRNA assembles histone or canonical mRNA-processing complexes by overlapping 3′-end sequence elements

Human pre-mRNA processing relies on multi-subunit macromolecular complexes, which recognize specific RNA sequence elements essential for assembly and activity. Canonical pre-mRNA processing proceeds via the recognition of a polyadenylation signal (PAS) and a downstream sequence element (DSE), and produces polyadenylated mature mRNAs, while replication-dependent (RD) histone pre-mRNA processing requires association with a stem–loop (SL) motif and a histone downstream element (HDE), and produces cleaved but non-polyadenylated mature mRNAs. H2AC18 mRNA, a specific H2A RD histone pre-mRNA, can be processed to give either a non-polyadenylated mRNA, ending at the histone SL, or a polyadenylated mRNA. Here, we reveal how H2AC18 captures the two human pre-mRNA processing complexes in a mutually exclusive mode by overlapping a canonical PAS (AAUAAA) sequence element with a HDE. Disruption of the PAS sequence on H2AC18 pre-mRNA prevents recruitment of the canonical complex in vitro, without affecting the histone machinery. This shows how the relative position of cis-acting elements in histone pre-mRNAs allows the selective recruitment of distinct human pre-mRNA complexes, thereby expanding the capability to regulate 3′ processing and polyadenylation.     

The PmlI-PmlR quorum-sensing system in Burkholderia pseudomallei plays a key role in virulence and modulates production of the MprA protease

Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infection of humans and animals. The virulence of this pathogen is thought to depend on a number of secreted proteins, including the MprA metalloprotease. We observed that MprA is produced upon entry into the stationary phase, when the cell density is high, and this prompted us to study cell density-dependent regulation in B. pseudomallei. A search of the B. pseudomallei genome led to identification of a quorum-sensing system involving the LuxI-LuxR homologs PmlI-PmlR. PmlI directed the synthesis of an N-acylhomoserine lactone identified as N-decanoylhomoserine lactone. A B. pseudomallei pmlI mutant was significantly less virulent than the parental strain in a murine model of infection by the intraperitoneal, subcutaneous, and intranasal routes. Inactivation of pmlI resulted in overproduction of MprA at the onset of the stationary phase. A wild-type phenotype was restored following complementation with pmlI or addition of cell-free culture supernatant. In contrast, there was no significant difference between the virulence of a B. pseudomallei mprA mutant and the virulence of the wild-type strain. These results suggest that the PmlI-PmlR quorum-sensing system of B. pseudomallei is essential for full virulence in a mouse model and downregulates the production of MprA at a high cell density.     

Thermodynamic dissection of the binding energetics of proline-rich peptides to the Abl-SH3 domain: implications for rational ligand design

The inhibition of the interactions between SH3 domains and their targets is emerging as a promising therapeutic strategy. To date, rational design of potent ligands for these domains has been hindered by the lack of understanding of the origins of the binding energy. We present here a complete thermodynamic analysis of the binding energetics of the p41 proline-rich decapeptide (APSYSPPPPP) to the SH3 domain of the c-Abl oncogene. Isothermal titration calorimetry experiments have revealed a thermodynamic signature for this interaction (very favourable enthalpic contributions opposed by an unfavourable binding entropy) inconsistent with the highly hydrophobic nature of the p41 ligand and the Abl-SH3 binding site. Our structural and thermodynamic analyses have led us to the conclusion, having once ruled out any possible ionization events or conformational changes coupled to the association, that the establishment of a complex hydrogen-bond network mediated by water molecules buried at the binding interface is responsible for the observed thermodynamic behaviour. The origin of the binding energetics for proline-rich ligands to the Abl-SH3 domain is further investigated by a comparative calorimetric analysis of a set of p41-related ligands. The striking effects upon the enthalpic and entropic contributions provoked by conservative substitutions at solvent-exposed positions in the ligand confirm the complexity of the interaction. The implications of these results for rational ligand design are discussed.