A molecular approach to the characterization of the eukaryotic communities of an extreme acidic environment: methods for DNA extraction and denaturing gradient gel electrophoresis analysis

The diversity of the phytobenthonic community present in six acidophilic microbial mats from Río Tinto (Iberian Pyritic Belt, SW Spain) was analysed by optical microscopy and two molecular techniques, denaturing gradient gel electrophoresis (DGGE) and sequence analysis of 18S rDNA cloned gene fragments. Sixteen DNA isolation protocols as well as two commercial DNA extraction kits were tested and their efficiency compared. Purified DNA extracts were amplified by PCR using universal eukaryotic primers and the PCR products analysed by DGGE. Bead-mill homogenization was found to be superior to the other cell lysis methodologies assayed (sonication or freeze-thawing cycles) as it allowed efficiencies of cell disruption of over 95%. The methods combining bead-mill homogenization in the presence of SDS, treatment with chemical extractants (hexadecylmethylammonium bromide or guanidine isothiocyanate) and phenol extraction resulted in DNA preparations that amplified the same number of bands when analysed by DGGE as the two commercial kits assayed. The phylogenetic affiliations of the DGGE bands were determined by a BLAST search, and nine different species related to the Chlorophyta, Ciliophora, Kinetoplastida, Ascomycota, Streptophyta and Colcochaetales taxonomical groups were identified. Similar levels of diversity were found using cloning procedures. Although not all the species observed under the microscope were detected using molecular techniques, e.g. euglenas, heliozoan, or amoebae, DGGE fingerprints showed rather well the level of diversity present in the samples analysed, with limitations similar to cloning techniques.     

Differential subcellular distribution of glucose transporters GLUT1-6 and GLUT9 in human cancer: ultrastructural localization of GLUT1 and GLUT5 in breast tumor tissues

It has been proposed that the enhanced metabolic activity of tumor cells is accompanied by an increased expression of facilitative hexose transporters (GLUTs). However, a previous immunohistochemical analysis of GLUT1 expression in 154 malignant human neoplasms failed to detect the GLUT1 isoform in 87 tumors. We used 146 normal human tissues and 215 tumor samples to reassess GLUT1 expression. A similar number of samples were used to compare the expression of GLUT2-6 and 9. The classical expression of GLUT1-5 in different normal human tissues was confirmed, however, we were unable to detect GLUT2 in human pancreatic islet cells. GLUT6 was principally detected in testis germinal cells and GLUT9 was localized in kidney, liver, heart, and adrenal. In tumor samples, GLUT1, 2, and 5 were the main transporters detected. GLUT1 was the most widely expressed transporter, however, 42% of the samples had very low-to-negative expression levels. GLUT2 was detected in 31% of the samples, being mainly expressed in breast, colon, and liver carcinoma. GLUT5 was detected in 27% of breast and colon adenocarcinoma, liver carcinoma, lymphomas, and testis seminoma samples. In situ RT-PCR and ultrastructural immunohistochemistry confirmed GLUT5 expression in breast cancer. GLUT6 and 9 are not clearly over-expressed in human cancer. The extensive expression of GLUT2 and 5 (glucose/fructose and fructose transporters, respectively) in malignant human tissues indicates that fructose may be a good energy substrate in tumor cells. Our functional data obtained in vitro in different tumor cells support this hypothesis. Additionally, these results suggest that fructose uptake could be used for positron emission tomography imaging and, may possibly represent a novel target for the development of therapeutic agents in different human cancers.     

Knockdown of Nav1.6a Na+ channels affects zebrafish motoneuron development

In addition to rapid signaling, electrical activity provides important cues to developing neurons. Electrical activity relies on the function of several different types of voltage-gated ion channels. Whereas voltage-gated Ca2+ channel activity regulates several aspects of neuronal differentiation, much less is known about developmental roles of voltage-gated Na+ channels, essential mediators of electrical signaling. Here, we focus on the zebrafish Na+ channel isotype, Nav1.6a, which is encoded by the scn8a gene. A restricted set of spinal neurons, including dorsal sensory Rohon-Beard cells, two motoneuron subtypes with different axonal trajectories, express scn8a during embryonic development. CaP, an early born primary motoneuron subtype with ventrally projecting axons expresses scn8a, as does a class of secondary motoneurons with axons that project dorsally. To test for developmental roles of scn8a, we knocked down Nav1.6a protein using antisense morpholinos. Na+ channel protein and current amplitudes were reduced in neurons that express scn8a. Furthermore, Nav1.6a knockdown altered axonal morphologies of some but not all motoneurons. Dorsally projecting secondary motoneurons express scn8a and displayed delayed axonal outgrowth. By contrast, CaP axons developed normally, despite expression of the gene. Surprisingly, ventrally projecting secondary motoneurons, a population in which scn8a was not detected, displayed aberrant axonal morphologies. Mosaic analysis indicated that effects on ventrally projecting secondary motoneurons were non cell-autonomous. Thus, voltage-gated Na+ channels play cell-autonomous and non cell-autonomous roles during neuronal development.     

Role of NPR-C natriuretic receptor in nitric oxide system activation induced by atrial natriuretic peptide

Atrial natriuretic peptide (ANP) exerts its hypotensive, natriuretic and diuretic effects, almost in part, through the activation of nitric oxide synthase (NOS). The aim was to investigate the natriuretic receptor type and the signaling cascade involved in NOS activation induced by ANP. Male Wistar rats were sacrificed and NOS activity was determined in kidney, aorta and heart with L-[U14C]-arginine, as substrate. ANP and cANP (4-23), a selective NPR-C ligand, increased NOS activity in all tissues. ANP induced a more marked activation in aorta and kidney than cANP (4-23), but no difference in atria NOS activation was observed. NOS activity induced by both peptides was blunted by nifedipine (L-type channel blocker) and calmidazolium (calmodulin antagonist) in heart and aorta. In kidney, nifedipine and calmidazolium abolished NOS activity stimulated by cANP (4-23) but only partially inhibited NOS activity elicited by ANP. Gi inhibition with pertussis toxin abolished NOS activity stimulated by ANP and cANP in atria but only partially inhibited the increased NOS activity induced by ANP and cANP in kidney, aorta and ventricle. Our results show that NPR-C receptor would mediate the activation of NOS by ANP in atria. In kidney, aorta and ventricle, NOS activation would also involve NPR-A and/or B. ANP would interact with NPR-C coupled via Gi to activation Ca2+ -dependent NOS.     

Activation of the 12-lipoxygenase and signal transducer and activator of transcription pathway during neointima formation in a model of the metabolic syndrome

Insulin resistance (IR) is associated with an increased risk of cardiovascular diseases. The obese Zucker rat (ZR) is a model of IR that shows markedly increased insulin and triglyceride concentrations without major changes in glucose. In this study, we evaluated the response of obese and lean ZR to carotid balloon injury and determined potential mechanisms and treatments. The neointima-to-media ratio of obese ZR was greater than that of lean ZR, starting at 14 days after injury, and persisted until at least day 30. An enhanced inflammatory response to balloon injury in the obese ZR was reflected by significantly higher ED1-positive macrophage cells in the injured vessel wall compared with that in lean ZR at 3, 7, and 14 days after balloon injury. Inflammatory mediators 12-lipoxygenase (12-LO) and STAT4 were studied in neointimal lesions. Expression of 12-LO RNA was increased beginning at day 7 and showed increases of 4.3-fold on day 14 and 7-fold on day 30 in obese ZR compared with lean animals. Staining of phosphorylated STAT4 (PSTAT4), the activated form of STAT4, in lesions from obese ZR was also increased compared with that in leans. We tested the effects of a novel anti-inflammatory agent, lisofylline (LSF), in the obese ZR. LSF markedly reduced neointimal formation in the obese ZR. LSF also reduced monocyte/macrophage infiltration into the vessel wall and the activation of PSTAT4. These studies suggest both the presence of an exaggerated injury response in the insulin-resistant obese ZR model and that inflammation plays a major role in mediating neointimal growth.     

Preparative laser capture microdissection and single-pot cell wall material preparation: a novel method for tissue-specific analysis

In adaptation to their function the walls of plant cell display tissue-specific variations of composition according to their developmental stage, cell type and stress of various origin. It is therefore important to obtain a precise analytical data describing the cell wall composition with respect to these different factors. In the present work, laser capture microdissection (LCM) was used for isolating different tissues from the stem of Urtica dioica L. at a semi-preparative scale. The technique was associated for the first time to a one-pot sequential cell wall preparation and hydrolysis for the carbohydrate analysis of each cell type. The results demonstrate that the combination of LCM and micro-analytical methods can provide individual cell type composition and should improve our knowledge of the biochemical diversity of cell walls in plants. This approach will be of potential interest for the understanding of the effects of stress or genetic engineering on the composition of the cell walls.     

Eukaryotic community distribution and its relationship to water physicochemical parameters in an extreme acidic environment, Rio Tinto (southwestern Spain)

The correlation between water physicochemical parameters and eukaryotic benthic composition was examined in Río Tinto. Principal component analysis showed a high inverse relationship between pH and most of the heavy metals analyzed as well as Dunaliella sp., while Chlamydomonas sp. abundance was positively related. Zn, Cu, and Ni clustered together and showed a strong inverse correlation with the diversity coefficient and most of the species analyzed. These eukaryotic communities seem to be more influenced by the presence of heavy metals than by the pH.     

Assessment of genetic and epigenetic variation in hop plants regenerated from sequential subcultures of organogenic calli

Organogenic calli induced from internodal segments were subcultured three times. Regenerated plants obtained from each subculture were analysed by molecular methods. No major genetic rearrangements were detected in the callus-derived plants since none of the amplified fragment-length polymorphism (AFLP) loci were found to be polymorphic. However, epigenetic changes due to a demethylation process were detected by methylation-sensitive amplified polymorphism (MSAP) technique. The results allowed inference of the possible relationship among the plants derived from different calli subcultures and the in vitro control. The plants recovered from the first and second callus subcultures clustered with the in vitro control pools in the phenogram while the regenerants from the third callus subculture showed the highest genetic distance with the controls. This is the first study reporting data about the genetic stability of callus-derived Humulus lupulus L. plants.     

Modulation of the physical properties of dielaidoylphosphatidylethanolamine membranes by a dirhamnolipid biosurfactant produced by Pseudomonas aeruginosa

Rhamnolipids are bacterial biosurfactants produced by Pseudomonas spp. These compounds have been shown to present several interesting biological activities, restricting the growth of Bacillus subtilis and showing zoosporicidal activity on zoosporic phytopathogens. It has been suggested that the interaction with the membrane could be the ultimate responsible for these actions. Therefore, it is of great interest to get insight into the molecular mechanism of the interaction of purified rhamnolipids with the various phospholipid components of biological membranes. In this paper we report on the phase behaviour of mixtures of dielaidoylphosphatidylethanolamine (DEPE) with a purified dirhamnolipid (DiRL) fraction from Pseudomonas aeruginosa, as studied by a number of physical techniques such as differential scanning calorimetry, FTIR, small angle X-ray (SAX) diffraction and dynamic light scattering. Our data indicate that the presence of DiRL counteracts the tendency of DEPE to form vesicular aggregates of large size, forming vesicles of smaller diameter which most probably have a lower lamellarity index. The partial phase diagram obtained from calorimetric data shows a complex behaviour with a solid-phase immiscibility. X-ray diffraction shows that DiRL has a bilayer stabilizing effect, impeding formation of the inverted hexagonal-HII phase of DEPE. The presented data are discussed focussing into how DiRL/DEPE interactions could help to explain the membrane perturbing activities of this biosurfactant.