Guarana (Paullinia cupana var. sorbilis), an anciently consumed stimulant from the Amazon rain forest: the seeded-fruit transcriptome

Guarana (Paullinia cupana var. sorbilis) is a plant native to the central Amazon basin. Roasted seed extracts have been used as medicinal beverages since pre-Colombian times, due to their reputation as stimulants, aphrodisiacs, tonics, as well as protectors of the gastrointestinal tract. Guarana plants are commercially cultivated exclusively in Brazil to supply the national carbonated soft-drink industry and natural product stores around the world. In this report, we describe and discuss the annotation of 15,387 ESTs from guarana seeded-fruits, highlighting sequences from the flavonoid and purine alkaloid pathways, and those related to biotic stress avoidance. This is the largest set of sequences registered for the Sapindaceae family.     

Molecular architecture of a multifunctional MCM complex

DNA replication is strictly regulated through a sequence of steps that involve many macromolecular protein complexes. One of them is the replicative helicase, which is required for initiation and elongation phases. A MCM helicase found as a prophage in the genome of Bacillus cereus is fused with a primase domain constituting an integrative arrangement of two essential activities for replication. We have isolated this helicase-primase complex (BcMCM) showing that it can bind DNA and displays not only helicase and primase but also DNA polymerase activity. Using single-particle electron microscopy and 3D reconstruction, we obtained structures of BcMCM using ATPγS or ADP in the absence and presence of DNA. The complex depicts the typical hexameric ring shape. The dissection of the unwinding mechanism using site-directed mutagenesis in the Walker A, Walker B, arginine finger and the helicase channels, suggests that the BcMCM complex unwinds DNA following the extrusion model similarly to the E1 helicase from papillomavirus.     

Resolving structure and mechanical properties at the nanoscale of viruses with frequency modulation atomic force microscopy

Structural Biology (SB) techniques are particularly successful in solving virus structures. Taking advantage of the symmetries, a heavy averaging on the data of a large number of specimens, results in an accurate determination of the structure of the sample. However, these techniques do not provide true single molecule information of viruses in physiological conditions. To answer many fundamental questions about the quickly expanding physical virology it is important to develop techniques with the capability to reach nanometer scale resolution on both structure and physical properties of individual molecules in physiological conditions. Atomic force microscopy (AFM) fulfills these requirements providing images of individual virus particles under physiological conditions, along with the characterization of a variety of properties including local adhesion and elasticity. Using conventional AFM modes is easy to obtain molecular resolved images on flat samples, such as the purple membrane, or large viruses as the Giant Mimivirus. On the contrary, small virus particles (25-50 nm) cannot be easily imaged. In this work we present Frequency Modulation atomic force microscopy (FM-AFM) working in physiological conditions as an accurate and powerful technique to study virus particles. Our interpretation of the so called "dissipation channel" in terms of mechanical properties allows us to provide maps where the local stiffness of the virus particles are resolved with nanometer resolution. FM-AFM can be considered as a non invasive technique since, as we demonstrate in our experiments, we are able to sense forces down to 20 pN. The methodology reported here is of general interest since it can be applied to a large number of biological samples. In particular, the importance of mechanical interactions is a hot topic in different aspects of biotechnology ranging from protein folding to stem cells differentiation where conventional AFM modes are already being used.     

Operon fusions of Mu d1(Ap,lac) to thel-proline biosynthetic genes ofescherichia coli K-12

Operon fusions to the promoter of either theproA,proB, orproC genes of the proline biosynthetic pathway were obtained by the use of the Mu d1(Ap,lac) bacteriophage. These fusions were further stabilized by transformation with plasmid pGW600 containing the wildtype Mu repressor gene or by transduction with phage pSG1. The level of -galactosidase in the fusion strains was not affected by the presence of exogenously addedl-proline or high concentrations of NaCl in the growth medium. A Tn5 insertion nearproBA increased -galactosidase expression 140- to 200-fold in strains carrying theproA-lac andproB-lac fusions, but the level of this enzyme was unaltered in strains carrying theproC-lac fusion. The Tn5 insertion increased intracellular proline concentrations 8- to 10-fold, suggesting that mechanisms other than allosteric inhibition may regulate proline biosynthesis, but did not confer osmotolerance to cells growing in a medium with a high concentration of salt.     

Effects of sulpiride on mRNA levels of steroid 5alpha-reductase isozymes in prostate of adult rats

Prolactin (PRL) is implicated in prostate growth and in the development and regulation of benign prostatic hypertrophy (BPH) and prostate cancer (PCa). PRL may exert its effects on prostate in synergism with androgens. The most active androgen in the prostate is the 5alpha-dihydrotestosterone (DHT) obtained from testosterone by the 5alpha-reductase (5alpha-R) enzyme, which is expressed in the prostate as two isozymes, 5alpha-R1 and 5alpha-R2. In this study, sulpiride, a prolactin-secretion inductor, was administered to male rats. mRNA levels of 5alpha-R1 and 5alpha-R2 were measured in prostate of controls and sulpiride-treated rats, using one-step quantitative RT-PCR coupled with laser-induced fluorescence capillary electrophoresis (LIF-CE). Results demonstrated that sulpiride-induced hyperprolactinemia is associated with an increase in mRNA levels of both 5alpha-R1 and 5alpha-R2 in prostate of adult rats. Although a direct effect of sulpiride on prostate gland cannot be ruled out, hyperprolactinemia may be a factor to be considered in aging males, in whom prostatic diseases such as BPH and PCa are more frequent.     

Modulation of the proliferation and differentiation of human mesenchymal stem cells by copper

Copper plays important functional roles in bone metabolism and turnover. It is known that it is essential for normal growth and development of the skeleton in humans and in animals. Although at present the exact role that copper plays in bone metabolism is unknown, bone abnormalities are a feature of severe copper deficiency. Osteoblasts are derived from mesenchymal stem cells (MSCs) present in bone marrow stroma, which are able to differentiate into bone, adipocytes, and other cell phenotypes. Excess adipogenesis in postmenopausal women may occur at the expense of osteogenesis and, therefore, may be an important factor in the fragility of postmenopausal bone. The purpose of this study was to evaluate whether an increase of the extracellular concentration of copper affects the ability of MSCs to differentiate into osteoblasts or adipocytes. The results showed that copper modified both the differentiation and the proliferative activity of MSCs obtained from postmenopausal women. Copper (50 microM) diminished the proliferation rate of MSCs, increasing their ability to differentiate into the osteogenic and the adipogenic lineages. Copper induced a 2-fold increase in osteogenic differentiation of MSCs, measured as a increase in calcium deposition. Copper (5 and 50 microM) diminished the expression of alkaline phosphatase (50 and 80%, respectively), but induced a shift in the expression of this enzyme to earlier times during culture. Copper also induced a 1.3-fold increase in the adipogenic differentiation of MSCs. It is concluded that copper stimulates MSC differentiation, and that this is preferentially towards the osteogenic lineage.     

An antioxidant redox system in the nucleus of wheat seed cells suffering oxidative stress

Cereal seed cells contain different mechanisms for protection against the oxidative stress that occurs during maturation and germination. One such mechanism is based on the antioxidant activity of a 1-Cys peroxiredoxin (1-Cys Prx) localized in the nuclei of aleurone and scutellum cells. However, nothing is known about the mechanism of activation of this enzyme. Here, we describe the pattern of localization of NADPH thioredoxin reductase (NTR) in developing and germinating wheat seeds using an immunocytochemical analysis. The presence of NTR in transfer cells, vascular tissue, developing embryo and root meristematic cells, agrees with the localization of thioredoxin h (Trx h ), and supports the important function of the NTR/Trx system in cell proliferation and communication. Interestingly, NTR is found in the nuclei of seed cells suffering oxidative stress, thus showing co-localization with Trx h and 1-Cys Prx. To test whether the NTR/Trx system serves as a reductant of the 1-Cys Prx, we cloned a full-length cDNA encoding 1-Cys Prx from wheat, and expressed the recombinant protein in Escherichia coli . Using the purified components, we show NTR-dependent activity of the 1-Cys Prx. Mutants of the 1-Cys Prx allowed us to demonstrate that the peroxidatic residue of the wheat enzyme is Cys46, which is overoxidized in vitro under oxidant conditions. Analysis of extracts from developing and germinating seeds confirmed 1-Cys Prx overoxidation in vivo . Based on these results, we propose that NADPH is the source of the reducing power to regenerate 1-Cys Prx in the nuclei of seed cells suffering oxidative stress, in a process that is catalyzed by NTR.     

The Hoopoe's Uropygial Gland Hosts a Bacterial Community Influenced by the Living Conditions of the Bird

Molecular methods have revealed that symbiotic systems involving bacteria are mostly based on whole bacterial communities. Bacterial diversity in hoopoe uropygial gland secretion is known to be mainly composed of certain strains of enterococci, but this conclusion is based solely on culture-dependent techniques. This study, by using culture-independent techniques (based on the 16S rDNA and the ribosomal intergenic spacer region) shows that the bacterial community in the uropygial gland secretion is more complex than previously thought and its composition is affected by the living conditions of the bird. Besides the known enterococci, the uropygial gland hosts other facultative anaerobic species and several obligated anaerobic species (mostly clostridia). The bacterial assemblage of this community was largely invariable among study individuals, although differences were detected between captive and wild female hoopoes, with some strains showing significantly higher prevalence in wild birds. These results alter previous views on the hoopoe-bacteria symbiosis and open a new window to further explore this system, delving into the possible sources of symbiotic bacteria (e.g. nest environments, digestive tract, winter quarters) or the possible functions of different bacterial groups in different contexts of parasitism or predation of their hoopoe host.