Field determination of denitrification in water-logged forest soils

Abstract Denitrification rates were measured as N₂O production in two water-logged forest soils at monthly intervals. The effect of acetylene inhibition and the addition of nitrate, glucose, acetate and celloboise in field incubations was examined.
N₂O release from the two soils was very low, 26 mg N₂m⁻²y⁻¹ in ash and 178 mg N₂ in alder. In acetylene inhibited incubations N₂O production was higher, 296 and 486 mg N₂m⁻² y⁻¹ in ash and alder respectively. After addition of nitrate and C-sources to a 10 mM concentration, denitrification rates increased to 5–15 times higher values.
The denitrification rates below 4°C were low and most N₂O was produced in late spring and summer.
The highest rate of denitrification during a 50 h incubation experiment occurred between 3 and 23 h.     

Interactions of the monomeric and dimeric flavones apigenin and amentoflavone with the plasma membrane of L929 cells; a fluorescence study

Flavonoids are ubiquitous polyphenolic compounds, found in vascular plants, which are endowed with a large variety of biological effects. Some of these effects have been assumed to result from interactions with the cell plasma membrane. In order to investigate the nature of these interactions a fluorescence study was performed with two flavonoids, currently used in one of the laboratories: apigenin and its homologous dimer amentoflavone. After preliminary assays with DPH in several types of phospholipid liposomes, the effects of these flavonoids on the membrane of mouse L929 fibroblasts were compared, using the non-permeant probe TMA-DPH. Amentoflavone, unlike apigenin, induced a static quenching effect, which denoted an important, but reversible, association of the molecule with the plasma membrane. In addition, amentoflavone treatment induced a dose-dependent increase in TMA-DPH fluorescence anisotropy, which could be interpreted as an increase in membrane lipidic order. For apigenin, the effect was much less important. Moreover, exploiting the capacity of TMA-DPH to label endocytic compartments, it was shown that, after association with the membrane, amentoflavone is not internalized into the cell. Possible correlations of these membrane effects with other biological properties are discussed.     

Biomimetic N-terminal alkylation of peptoid analogues of surfactant protein C

Surfactant protein C (SP-C) is a hydrophobic lipopeptide that is critical for lung function, in part because it physically catalyzes the formation of surface-associated surfactant reservoirs. Many of SP-C's key biophysical properties derive from its highly stable and hydrophobic α-helix. However, SP-C's posttranslational modification with N-terminal palmitoyl chains also seems to be quite important. We created a new (to our knowledge) class of variants of a synthetic, biomimetic family of peptide mimics (peptoids) that allow us to study the functional effects of biomimetic N-terminal alkylation in vitro. Mimics were designed to emulate the amphipathic patterning, helicity, and hydrophobicity of SP-C, and to include no, one, or two vicinal amide-linked, N-terminal octadecyl chains (providing a reach equivalent to that of natural palmitoyl chains). Pulsating bubble surfactometry and Langmuir-Wilhelmy surface balance studies showed that alkylation improved biomimetic surface activities, yielding lower film compressibility and lower maximum dynamic surface tensions. Atomic force microscopy studies indicated that alkyl chains bind to and retain segregated interfacial surfactant phases at low surface tensions by inducing 3D structural transitions in the monolayer's fluid-like phase, forming surfactant-associated reservoirs. Peptoid-based SP-C mimics are easily produced and purified, and offer much higher chemical and secondary structure stability than polypeptide-based mimics. In surfactant replacements intended for medical use, synthetic SP mimics reduce the odds of pathogen contamination, which may facilitate the wider use of surfactant treatment of respiratory disorders and diseases.     

Metabolic status rather than cell cycle signals control quiescence entry and exit

Quiescence is defined as a temporary arrest of proliferation, yet it likely encompasses various cellular situations. Our knowledge about this widespread cellular state remains limited. In particular, little is known about the molecular determinants that orchestrate quiescence establishment and exit. Here we show that upon carbon source exhaustion, budding yeast can enter quiescence from all cell cycle phases. Moreover, using cellular structures that are candidate markers for quiescence, we found that the first steps of quiescence exit can be triggered independently of cell growth and proliferation by the sole addition of glucose in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. Importantly, glucose needs to be internalized and catabolized all the way down to glycolysis to mobilize quiescent cell specific structures, but, strikingly, ATP replenishment is apparently not the key signal. Altogether, these findings strongly suggest that quiescence entry and exit primarily rely on cellular metabolic status and can be uncoupled from the cell cycle.     

A fluorescence polarization assay using an engineered human respiratory syncytial virus F protein as a direct screening platform

Human respiratory syncytial virus (hRSV) typically affects newborns and young children. Even though it can cause severe and, in some cases, lifelong respiratory infections, there are currently no Food and Drug Administration (FDA)-approved therapeutics that control this virus. The hRSV F protein facilitates viral fusion, a critical extracellular event that can be targeted for therapeutic intervention by disrupting the assembly of a postfusion 6-helix bundle (6HB) within the hRSV F protein. Here we report the development of a fluorescence polarization (FP) assay using an engineered hRSV F protein 5-helix bundle (5HB). We generated the 5HB and validated its ability to form a 6HB in an FP assay. To test the potential of 5HB as a screening tool, we then investigated a series of truncated peptides derived from the “missing” sixth helix. Using this FP-based 5HB system, we have successfully demonstrated that short peptides can prevent 6HB formation and serve as potential hRSV fusion inhibitors. We anticipate that this new 5HB system will provide an effective tool to identify and study potential antivirals to control hRSV infection.     

NMEGylation: a novel modification to enhance the bioavailability of therapeutic peptides

We have evaluated "NMEGylation"--the covalent attachment of an oligo-N-methoxyethylglycine (NMEG) chain--as a new form of peptide/protein modification to enhance the bioavailability of short peptides. OligoNMEGs are hydrophilic polyethylene glycol-like molecules made by solid-phase synthesis, typically up to 40 monomers in length. They have been studied as nonfouling surface coatings and as monodisperse mobility modifiers for free-solution conjugate capillary electrophoresis. However, polyNMEGs have not been demonstrated before this work as modifiers of therapeutic proteins. In prior published work, we identified a short peptide, "C20," as a potential extracellular inhibitor of the fusion of human respiratory syncytial virus with mammalian cells. The present study was aimed at improving the C20 peptide's stability and solubility. To this end, we synthesized and studied a series of NMEGylated C20 peptide-peptoid bioconjugates comprising different numbers of NMEGs at either the N- or C-terminus of C20. NMEGylation was found to greatly improve this peptide's solubility and serum stability; however, longer polyNMEGs (n > 3) deleteriously affected peptide binding to the target protein. By incorporating just one NMEG monomer, along with a glycine monomer as a flexible spacer, at C20's N-terminus (NMEG-Gly-C20), we increased both solubility and serum stability greatly, while recovering a binding affinity comparable to that of unmodified C20 peptide. Our results suggest that NMEGylation with an optimized number of NMEG monomers and a proper linker could be useful, more broadly, as a novel modification to enhance bioavailability and efficacy of therapeutic peptides.     

High Prevalence of Anemia in Children and Adult Women in an Urban Population in Southern Brazil

This population-based study was designed to detect the prevalence of anemia in a healthy population of children (18 months to 7 years) and women (14 to 30 years) tested in 2006–2007 in the state of Rio Grande do Sul, Brazil as part of an effort to tackle this massive problem that still affects so many people in the XXI century. Anemia was defined according to the WHO. Capillary blood was measured and socioeconomic status was determined according to the Brazilian Association of Market Research Agencies. The median prevalence of anemia in 2198 children was 45.4% and in 1999 women 36.4%. Anemia decreased with age during childhood; although significantly more prevalent in lower classes individuals, it was also high in the upper classes. There are indirect evidences that the lack of iron supplementation and/or iron fortified food may play a role in it. Professionals and society wise measures of education have to be implemented in order to address possible biologic factors involved in childhood psychosocial development in southern Brazil.     

Topical Gene Electrotransfer to the Epidermis of Hairless Guinea Pig by Non-Invasive Multielectrode Array

Topical gene delivery to the epidermis has the potential to be an effective therapy for skin disorders, cutaneous cancers, vaccinations and systemic metabolic diseases. Previously, we reported on a non-invasive multielectrode array (MEA) that efficiently delivered plasmid DNA and enhanced expression to the skin of several animal models by in vivo gene electrotransfer. Here, we characterized plasmid DNA delivery with the MEA in a hairless guinea pig model, which has a similar histology and structure to human skin. Significant elevation of gene expression up to 4 logs was achieved with intradermal DNA administration followed by topical non-invasive skin gene electrotransfer. This delivery produced gene expression in the skin of hairless guinea pig up to 12 to 15 days. Gene expression was observed exclusively in the epidermis. Skin gene electrotransfer with the MEA resulted in only minimal and mild skin changes. A low level of human Factor IX was detected in the plasma of hairless guinea pig after gene electrotransfer with the MEA, although a significant increase of Factor IX was obtained in the skin of animals. These results suggest gene electrotransfer with the MEA can be a safe, efficient, non-invasive skin delivery method for skin disorders, vaccinations and potential systemic diseases where low levels of gene products are sufficient.