Peroxynitrite mobilizes calcium ions from ryanodine-sensitive stores, a process associated with the mitochondrial accumulation of the cation and the enforced formation of species mediating cleavage of genomic DNA

Peroxynitrite does not directly cause strand scission of genomic DNA. Rather, as we previously reported, the DNA cleavage is largely mediated by H(2)O(2) resulting from the dismutation of superoxide generated in the mitochondria upon peroxynitrite-dependent inhibition of complex III. The present study demonstrates that this process is strictly controlled by the availability of Ca(2+) in the mitochondrial compartment. Experiments using intact as well as permeabilized U937 cells showed that the DNA-damaging response evoked by peroxynitrite is enhanced by treatments causing an increase in mitochondrial Ca(2+) uptake and remarkably reduced under conditions leading to inhibition of mitochondrial Ca(2+) accumulation. An additional, important observation was that the source of the Ca(2+) mobilized by peroxynitrite is the ryanodine receptor; preventing the mobilization of Ca(2+) with ryanodine suppressed the mitochondrial formation of reactive oxygen species and the ensuing DNA strand scission. Identical results were obtained using PC12, C6, and THP-1 cells. These results, along with our previous findings indicating that the DNA damage induced by peroxynitrite is also suppressed by inhibition of the electron flow through complex I, e.g., by rotenone, or by the respiration-deficient phenotype, demonstrate that the mitochondrial formation of DNA-damaging species is critically regulated by the inhibition of complex III and by the availability of Ca(2+).     

Efficacy of <Emphasis Type="Italic">Neoseiulus californicus</Emphasis> and <Emphasis Type="Italic">Phytoseiulus persimilis</Emphasis> in suppression of <Emphasis Type="Italic">Tetranychus urticae</Emphasis> in young clementine plants

Tetranychus urticae is one of the most damaging tetranychid mites affecting clementine orchards in Spain, where natural control is insufficient. Furthermore, in clementine nurseries, tender foliage is highly susceptible to attack and natural enemies are almost always absent. Therefore, acaricides are often used indiscriminately. Alternative control measures are necessary, both in commercial orchards and clementine nurseries. In order to assess the efficacy of inoculative releases of N. californicus and P. persimilis to reduce T. urticae populations in young Spanish clementine plants, a semi-field experiment was conducted and repeated in three seasons (spring, summer and autumn). Phytoseiulus persimilis was highly effective in reducing both T. urticae infestations and the damage level inflicted on plants at both release rates evaluated (40 and 80 phytoseiids/plant) and all three periods considered. By contrast, N. californicus demonstrated low performance under certain conditions. The results of this study could be adapted and transferred to nurseries and young citrus plantations.     

Phylogenetic ecology of widespread uncultured clades of the Kingdom Euryarchaeota

Despite its widespread distribution and high levels of phylogenetic diversity, microbes are poorly understood creatures. We applied a phylogenetic ecology approach in the Kingdom Euryarchaeota (Archaea) to gain insight into the environmental distribution and evolutionary history of one of the most ubiquitous and largely unknown microbial groups. We compiled 16S rRNA gene sequences from our own sequence libraries and public genetic databases for two of the most widespread mesophilic Euryarchaeota clades, Lake Dagow Sediment (LDS) and Rice Cluster-V (RC-V). The inferred population history indicated that both groups have undergone specific nonrandom evolution within environments, with several noteworthy habitat transition events. Remarkably, the LDS and RC-V groups had enormous levels of genetic diversity when compared with other microbial groups, and proliferation of sequences within each single clade was accompanied by significant ecological differentiation. Additionally, the freshwater Euryarchaeota counterparts unexpectedly showed high phylogenetic diversity, possibly promoted by their environmental adaptability and the heterogeneous nature of freshwater ecosystems. The temporal phylogenetic diversification pattern of these freshwater Euryarchaeota was concentrated both in early times and recently, similarly to other much less diverse but deeply sampled archaeal groups, further stressing that their genetic diversity is a function of environment plasticity. For the vast majority of living beings on Earth (i.e. the uncultured microorganisms), how they differ in the genetic or physiological traits used to exploit the environmental resources is largely unknown. Inferring population history from 16S rRNA gene-based molecular phylogenies under an ecological perspective may shed light on the intriguing relationships between lineage, environment, evolution and diversity in the microbial world.     

Pili of Pasteurella multocida of porcine origin

Abstract Using electron microscopy, pili with at least two distinct morphologies were observed on strains of Pasteurella multocida isolated from pigs with atrophic rhinitis. Rigid pili were found on 60–80% of all cells observed. These pili had a strong tendency to lie flat along the side of the outer cell membrane of P. multocida and as a result frequently were difficult to see. After growth in vitro, piliated P. multocida cells produced few pili (approx. 3–5 per cell). Heavily piliated cells were occasionally observed. The second type of pili were curly and also were difficult to visualize. Cells from cultures containing piliated cells failed to attach to red blood cells and to immobilized mucus.     

Use of a Dominant rpsL Allele Conferring Streptomycin Dependence for Positive and Negative Selection in Thermus thermophilus

A spontaneous rpsL mutant of Thermus thermophilus was isolated in a search for new selection markers for this organism. This new allele, named rpsL1, encodes a K47R/K57E double mutant S12 ribosomal protein that confers a streptomycin-dependent (SD) phenotype to T. thermophilus. Models built on the available three-dimensional structures of the 30S ribosomal subunit revealed that the K47R mutation directly affects the streptomycin binding site on S12, whereas the K57E does not apparently affect this binding site. Either of the two mutations conferred the SD phenotype individually. The presence of the rpsL1 allele, either as a single copy inserted into the chromosome as part of suicide plasmids or in multicopy as replicative plasmids, produced a dominant SD phenotype despite the presence of a wild-type rpsL gene in a host strain. This dominant character allowed us to use the rpsL1 allele not only for positive selection of plasmids to complement a kanamycin-resistant mutant strain, but also more specifically for the isolation of deletion mutants through a single step of negative selection on streptomycin-free growth medium.     

G protein-coupled receptor systems and their lipid environment in health disorders during aging

Cells, tissues and organs undergo phenotypic changes and deteriorate as they age. Cell growth arrest and hyporesponsiveness to extrinsic stimuli are all hallmarks of senescent cells. Most such external stimuli received by a cell are processed by two different cell membrane systems: receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). GPCRs form the largest gene family in the human genome and they are involved in most relevant physiological functions. Given the changes observed in the expression and activity of GPCRs during aging, it is possible that these receptors are directly involved in aging and certain age-related pathologies. On the other hand, both GPCRs and G proteins are associated with the plasma membrane and since lipid-protein interactions regulate their activity, they can both be considered to be sensitive to the lipid environment. Changes in membrane lipid composition and structure have been described in aged cells and furthermore, these membrane changes have been associated with alterations in GPCR mediated signaling in some of the main health disorders in elderly subjects. Although senescence could be considered a physiologic process, not all aging humans develop the same health disorders. Here, we review the involvement of GPCRs and their lipid environment in the development of the major human pathologies associated with aging such as cancer, neurodegenerative disorders and cardiovascular pathologies.     

A Natural Variant of Obestatin,Q90L,Inhibits Ghrelin's Action on Food Intake and GH Secretion and Targets NPY and GHRH Neurons in Mice

Background

Ghrelin and obestatin are two gut-derived peptides originating from the same ghrelin/obestatin prepropeptide gene (GHRL). While ghrelin stimulates growth hormone (GH) secretion and food intake and inhibits γ-aminobutyric-acid synaptic transmission onto GHRH (Growth Hormone Releasing Hormone) neurons, obestatin blocks these effects. In Humans, GHRL gene polymorphisms have been associated with pathologies linked to an unbalanced energy homeostasis. We hypothesized that one polymorphism located in the obestatin sequence (Q to L substitution in position 90 of the ghrelin/obestatin prepropeptide, rs4684677) may impact on the function of obestatin. In the present study, we tested the activity of native and Q90L obestatin to modulate ghrelin-induced food intake, GH secretion, cFos activity in GHRH and Neuropeptide Y (NPY) neurons and γ-aminobutyric-acid activity onto GHRH neurons.

Methodology/Principal findings

Food intake, GH secretion and electrophysiological recordings were assessed in C57BL/6 mice. cFos activity was measured in NPY-Renilla-GFP and GHRH-eGFP mice. Mice received saline, ghrelin or ghrelin combined to native or Q90L obestatin (30 nmol each) in the early light phase. Ghrelin stimulation of food intake and GH secretion varied considerably among individual mice with 59–77% eliciting a robust response. In these high-responders, ghrelin-induced food intake and GH secretion were reduced equally by native and Q90L obestatin. In contrast to in vivo observations, Q90L was slightly more efficient than native obestatin in inhibiting ghrelin-induced cFos activation within the hypothalamic arcuate nucleus and the nucleus tractus solitarius of the brainstem. After ghrelin injection, 26% of NPY neurons in the arcuate nucleus expressed cFos protein and this number was significantly reduced by co-administration of Q90L obestatin. Q90L was also more potent that native obestatin in reducing ghrelin-induced inhibition of γ-aminobutyric-acid synaptic transmission onto GHRH neurons.

Conclusions/Significance

These data support the hypothesis that Q90L obestatin partially blocks ghrelin-induced food intake and GH secretion by acting through NPY and GHRH neurons.     

A simple cell labeling technique by means of lectins linked to fluorochromes for the detection of cells on tissue sections

Summary— We designed a protocol for cell labeling with the lectin wheat germ agglutinin (WGA) linked to the fluorochrome tetramethyl-rhodamine isothiocyanate (TRITC) for effective detection of the B16F10 melanoma and Lewis lung carcinoma (LLc) cells on pulmonary histological sections from C57BL6; mice. We have also determined a suitable concentration of WGA-TRITC (10 μg/ml), which leads to a very intense and homogeneous labeling of the cells, as it avoids cell clumping due to the presence of the lectin WGA. In order to determine to what extent the method affects these tumor cells, we have studied some important aspects related to their metastatic behavior, taking into account three parameters: a) viability and rate of proliferation of the cells cultured in vitro; b) percentage of animals C57BL6 mice) bearing metastasis 15 days after intravenous inoculation with 10⁵ B16F10 or LLc cells; and c) pattern of distribution of tumor foci in lung. There were no significant differences in these three parameters between the WGA-TRITC labeled-cells compared to the cultures of non-labeled cells in either of the cell lines (B16F10, LLc). Thus, we conclude that B16F10 and LLc tumor cells can be labeled following the protocol set-up in our study, as it allows these cells to be neatly identified on tissue sections and it causes no important physiological changes in the cells, with regard to metastatic behavior. These points make this technique very suitable for the detection of B16F10 and LLc cells on histological sections in studying their behavior during the first stages of the metastatic process.     

Soils in warmer and less developed countries have less micronutrients globally

Soil micronutrients are capital for the delivery of ecosystem functioning and food provision worldwide. Yet, despite their importance, the global biogeography and ecological drivers of soil micronutrients remain virtually unknown, limiting our capacity to anticipate abrupt unexpected changes in soil micronutrients in the face of climate change. Here, we analyzed >1300 topsoil samples to examine the global distribution of six metallic micronutrients (Cu, Fe, Mn, Zn, Co and Ni) across all continents, climates and vegetation types. We found that warmer arid and tropical ecosystems, present in the least developed countries, sustain the lowest contents of multiple soil micronutrients. We further provide evidence that temperature increases may potentially result in abrupt and simultaneous reductions in the content of multiple soil micronutrients when a temperature threshold of 12–14°C is crossed, which may be occurring on 3% of the planet over the next century. Altogether, our findings provide fundamental understanding of the global distribution of soil micronutrients, with direct implications for the maintenance of ecosystem functioning, rangeland management and food production in the warmest and poorest regions of the planet.     

Effects of the diatom-Emiliana huxleyi succession on photosynthesis, calcification and carbon metabolism by size-fractioned phytoplankton

Changes in the species composition, photosynthesis, calcification and size-fractionated carbon metabolism by natural phytoplankton assemblages were monitored in three mesocosms under different nutrient conditions during May 1993. In the 3 enclosures, the decline of the diatom-dominated assemblages was followed by the development of a bloom of the coccolithoporid Emiliania huxleyi. Highest growth of E. huxleyi was observed in the mesocosm with a high N : P ratio, suggesting this species is a good competitor at low phosphate concentrations. The transition from diatom- to E. huxleyi-dominated assemblages brought about a sharp reduction of the phytoplankton standing stock and carbon-specific photosynthetic rate. The relative contribution of the smaller size fraction to total photosynthesis increased as the succession progressed. Calcification rate and E. huxleyi cell-specified calcite production were highest during the early stages of development of the E. huxleyi bloom. Distinct changes in the patterns of ¹⁴C allocation into biomolecules were noticed during the diatom-E. huxleyi succession. The diatom-dominated assemblage showed high relative ¹⁴C incorporation into low molecular weight metabolites (LMWM), whereas proteins and, specially, lipids accounted for the largest proportion of carbon incorporation in the E. huxleyi bloom. The patterns of photoassimilated carbon metabolism proved to be strongly dependent on cellular size, as protein relative synthesis was significantly higher in the smaller than in the larger size fraction, irrespective of the nutrient regime and the successional stage. These results are discussed in relation to the ecological and physiological features of small phytoplankton.