Culture conditions for zinc- and pH-regulated gene expression studies in Aspergillus fumigatus.

In Aspergillus fumigatus, the regulation of zinc homeostasis is strongly influenced by environmental pH. Thus, the study of zinc-regulated gene expression in A. fumigatus requires controlling variations in culture pH, as this may affect zinc availability. However, depending on the nitrogen source, the pH of the culture can change dramatically over time. In addition, due to the ubiquitous distribution of zinc and that it is an essential micronutrient required in minute amounts for optimal fungal growth, neither buffering of the culture media to prevent pH variations nor the use of chelating agents is advisable if mycelium is to be used for expression analyses. In this work, the growth of A. fumigatus in several culture media was examined in order to determine the conditions yielding mycelia suitable for gene expression analyses in acid and neutral media, regardless of zinc availability. Our results showed that a zinc-limiting synthetic basal medium could be readily converted into a zinc-replete one and subsequently into acid or neutral medium by using, respectively, ammonium or nitrate as nitrogen source.     

In vivo gene regulation in Salmonella spp. by a salicylate-dependent control circuit

Systems allowing tightly regulated expression of prokaryotic genes in vivo are important for performing functional studies of bacterial genes in host-pathogen interactions and establishing bacteria-based therapies. We integrated a regulatory control circuit activated by acetyl salicylic acid (ASA) in attenuated Salmonella enterica that carries an expression module with a gene of interest under control of the XylS2-dependent Pm promoter. This resulted in 20-150-fold induction ex vivo. The regulatory circuit was also efficiently induced by ASA when the bacteria resided in eukaryotic cells, both in vitro and in vivo. To validate the circuit, we administered Salmonella spp., carrying an expression module encoding the 5-fluorocytosine-converting enzyme cytosine deaminase in the bacterial chromosome or in a plasmid, to mice with tumors. Induction with ASA before 5-fluorocytosine administration resulted in a significant reduction of tumor growth. These results demonstrate the usefulness of the regulatory control circuit to selectively switch on gene expression during bacterial infection.     

Neuronal assembly detection and cell membership specification by principal component analysis

In 1949, Donald Hebb postulated that assemblies of synchronously activated neurons are the elementary units of information processing in the brain. Despite being one of the most influential theories in neuroscience, Hebb's cell assembly hypothesis only started to become testable in the past two decades due to technological advances. However, while the technology for the simultaneous recording of large neuronal populations undergoes fast development, there is still a paucity of analytical methods that can properly detect and track the activity of cell assemblies. Here we describe a principal component-based method that is able to (1) identify all cell assemblies present in the neuronal population investigated, (2) determine the number of neurons involved in ensemble activity, (3) specify the precise identity of the neurons pertaining to each cell assembly, and (4) unravel the time course of the individual activity of multiple assemblies. Application of the method to multielectrode recordings of awake and behaving rats revealed that assemblies detected in the cerebral cortex and hippocampus typically contain overlapping neurons. The results indicate that the PCA method presented here is able to properly detect, track and specify neuronal assemblies, irrespective of overlapping membership.     

Membrane proteomic signatures of karyotypically normal and abnormal human embryonic stem cell lines and derivatives

Cultured human embryonic stem cells (hESCs) and derived derivatives contain heterogeneous cell populations with varying degrees of differentiation and karyotypic stability. The inability to isolate homogenous population presents a challenge toward cell-based applications and therapies. A proteomics approach was utilized to discover novel membrane proteins able to distinguish between the hESC lines BG01, WA09, and abBG02 (trisomy 12, 14, 17 and an extra copy of the X chromosome), along with WA09-derived human neural progenitor (hNP) cells. Membrane protein signatures were developed using sucrose-gradient isolation, 1-D gel electrophoresis followed by in-gel digestion and analysis by reverse phase chromatography coupled to ion trap-FT-ICR. At a ≤1.0% false discovery rate, 1918 proteins were identified; 775 were annotated as membrane proteins and 720 predicted to contain transmembrane spanning regions. Flow cytometry was used to validate cell surface expression of selected proteins. Junctional adhesion molecule 1 expression was shared by BG01, BG02 and abBG02 hESC lines. Dysferlin expression was specific to the WA09 hESC line and not the derived neural or mesenchymal progenitors. Ciliary neurotrophic factor receptor distinguished WA09-derived human neural progenitor cells from the parent hESC population, and WA09-derived mesenchymal progenitor cells. This study expands the current membrane protein data set for hESCs.     

Uncoupling of lipolysis from cyclic AMP by procaine: a tool for studying the mechanism of action of antilipolytic agents.

The initial rate of net glycerol release in norepinephrine-stimulated adipose tissue fragments was inhibited (40-78%) by procaine-HCl (1-5mM), whereas basal (unstimulated) lipolysis was unaffected. A dose-related inhibition of norepinephrine-induced lipolysis by procaine-HCl (0.1-1 mM) also occurred in adipocytes. Procaine-induced antilipolysis was associated with an augmented rather than a reduced hormone-stimulated increment in intracellular cyclic AMP. The dissociation of lipolysis from cyclic AMP accumulation has been termed the uncoupling effect of procaine. This effect of procaine was employed to define the precise mechanism of action of the antilipolytic drug clofibrate (Atromid-S) which inhibits lipolysis by reducing cyclic AMP. A reduction in cyclic AMP by clofibrate was demonstrated in norepinephrine-stimulated cells exposed to procaine (uncoupled system). Thus, the inhibitory effect of clofibrate on cyclic AMP could not be attributed to accumulation of products of lipolysis. Because neither procaine-HCl nor clofibrate had any effect on the low Km 3':5'-cyclic-AMP phosphodiesterase (EC 3.1.4.17) activity in hormone stimulated cells, the clofibrate-induced reduction in cyclic AMP was attributed to its direct action on adipocyte adenylate cyclase.     

Catalytic properties of thioredoxin immobilized on superparamagnetic nanoparticles

Thioredoxin (Trx1), a very important protein for regulating intracellular redox reactions, was immobilized on iron oxide superparamagnetic nanoparticles previously coated with 3-aminopropyltriethoxysilane (APTS) via covalent coupling using the EDC (1-ethyl-3-{3-dimethylaminopropyl}carbodiimide) method. The system was extensively characterized by atomic force microscopy, vibrational and magnetic techniques. In addition, gold nanoparticles were also employed to probe the exposed groups in the immobilized enzyme based on the SERS (surface enhanced Raman scattering) effect, confirming the accessibility of the cysteines residues at the catalytic site. For the single coated superparamagnetic nanoparticle, by monitoring the enzyme activity with the Ellman reagent, DTNB = 5,5′-dithio-bis(2-15 nitrobenzoic acid), an inhibitory effect was observed after the first catalytic cycle. The inhibiting effect disappeared after the application of an additional silicate coating before the APTS treatment, reflecting a possible influence of unprotected iron-oxide sites in the redox kinetics. In contrast, the doubly coated system exhibited a normal in-vitro kinetic activity, allowing a good enzyme recovery and recyclability.     

Evaluation of myelin sheath and collagen reorganization pattern in a model of peripheral nerve regeneration using an integrated histochemical approach

Peripheral nerves are complex histological structures that can be affected by a variety of conditions with different degree of axonal degeneration and demyelination. For the study of peripheral nerve regeneration in pathology and tissue engineering, it is necessary to evaluate the regeneration, remyelination and extracellular matrix reorganization of the neural tissue. Currently, different histochemical techniques must be used in parallel, and a correlation among their findings should be further performed. In this work, we describe a new histochemical method for myelin and collagen fibers based on luxol fast blue and picrosirius methods, for the evaluation of the morphology, the myelin sheath and the collagen fiber reorganization using a model of peripheral nerve regeneration. Whole brain, normal sciatic nerve and regenerating peripheral nerve samples were fixed in 10% neutral buffered formalin and paraffin-embedded, for the performance of the hematoxylin-eosin stain, the Luxol fast blue method and the new histochemical method for myelin and collagen. The results of this technique revealed that this new histochemical method allowed us to properly evaluate histological patterns, and simultaneously observe the histochemical reaction for myelin sheath and collagen fibers in normal tissue, and during the regeneration process. In conclusion, this new method combines morphological and histochemical properties that allowed us to determine with high accuracy the degree of remyelination and collagen fibers reorganization. For all these reasons, we hypothesize that this new histochemical method could be useful in pathology and tissue engineering.     

Oxygen-evolving extrinsic proteins (PsbO,P,Q,R): bioinformatic and functional analysis

The water-splitting and oxygen-evolving (OE) reaction is carried out by a large multisubunit protein complex, Photosystem II (PSII), that has two distinct regions: a membrane intrinsic-region that includes most of the PSII subunits and a lumenal extrinsic-region that is in close association to the manganese catalytic center. The recently determined PSII 3D structures from cyanobacteria provide a considerable amount of new knowledge about the OE architecture (K.N. Ferreira, T.M. Iverson, K. Maghlaoui, J. Barber, S. Iwata, Architecture of the photosynthetic oxygen-evolving center, Science 303 (2004) 1831-1838; B. Loll, J. Kern, W. Saenger, A. Zouni, J. Biesiadka, Towards complete cofactor arrangement in the 3.0 A resolution structure of photosystem II, Nature 438 (2005) 1040-1044). Most of the intrinsic core PSII polypeptides have been well conserved through evolution from ancient cyanobacteria to modern plants, keeping the essence of PSII light driven reactions from prokaryotes to eukaryotes; but what is striking is the large number of changes that have occurred in the oxygen-evolving extrinsic proteins (OEEp) associated to PSII lumenal side. For unknown reasons plant PSII has required the "invention" of three OEEps: PsbP (23 kDa), PsbQ (16 kDa) and PsbR (10 kDa); associated to the ubiquitous OEEp PsbO (33 kDa). This set of proteins seems to be required in plants for the full activity and stability of the OE center in vivo, but their specific function is not clear. In this paper, bioinformatics and functional data show that the OEEps present in plants and green algae are very distinct from their prokaryotic counterparts. Moreover, clear differences are found for PsbQ from higher plants and green algae; and a relationship has been found between PsbR and the Mn cluster.     

Una nueva especie de <Emphasis Type="Italic">Pseudomiltemia</Emphasis> (Rubiaceae) de Chiapas,México

Pseudomiltemia davidsonii is described and illustrated as a new endemic species from the Sierra Madre of Chiapas, Mexico. It is a shrub or small tree characterized by having flowers axillary and solitary or in monochasia and with large yellow corollas. Pseudomiltemia davidsonii differs from P. filisepala, the only other species in this genus, by its opposite leaves with longer petioles, inflorescences with shorter peduncles 5–13 mm long, smaller bracts, longer pedicels 6–15 mm long, and narrower fruits 3.5–4.5 mm in diameter.