Immobilization of lipid vesicles on polymer support via an amphiphilic peptidic anchor: application to a membrane enzyme

To immobilize lipid vesicles on a polymer support, we have used a peptidic anchor with the following sequence: Ala-Ala-Leu-Leu-Leu-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-A la-Ala-Ala-Ala-Ala-Ala-Ala-Trp-Lys-Lys-Lys-Lys-Lys-Lys. This amphiphilic peptide was previously designed in our group to interact spontaneously and strongly with vesicles without perturbing their permeability. At the end of the solid-phase peptide synthesis, the peptide was left on the polymer beads and this novel polymer-peptide system was used for vesicle immobilization. It was shown that this polymer-peptide system could immobilize as much as 200 micromol of lipids per gram of dry resin. The amount of immobilized vesicles was decreased by a reduction of the proportion of the negatively charged lipids in the vesicles, indicating the importance of electrostatic interactions in the immobilization of the vesicles. The integrity of the vesicles was mostly preserved after the immobilization. This new polymer-peptide system was used easily and successfully to immobilize a membrane-bound enzyme, gamma-glutamyl transpeptidase. The activity of the membrane-bound enzyme was studied by monitoring the release of p-nitroaniline. The activity of the enzyme was still retained, even after being re-used eight times, indicating the strong immobilization of the enzyme in its active form. The polymer-peptide support could be regenerated by washing with ethanol and reused.     

A hitchhikers guide to the Galápagos: co-phylogeography of Galápagos mockingbirds and their parasites

Background

Parasites are evolutionary hitchhikers whose phylogenies often track the evolutionary history of their hosts. Incongruence in the evolutionary history of closely associated lineages can be explained through a variety of possible events including host switching and host independent speciation. However, in recently diverged lineages stochastic population processes, such as retention of ancestral polymorphism or secondary contact, can also explain discordant genealogies, even in fully co-speciating taxa. The relatively simple biogeographic arrangement of the Galápagos archipelago, compared with mainland biomes, provides a framework to identify stochastic and evolutionary informative components of genealogic data in these recently diverged organisms.

Results

Mitochondrial DNA sequences were obtained for four species of Galápagos mockingbirds and three sympatric species of ectoparasites - two louse and one mite species. These data were complemented with nuclear EF1α sequences in selected samples of parasites and with information from microsatellite loci in the mockingbirds. Mitochondrial sequence data revealed differences in population genetic diversity between all taxa and varying degrees of topological congruence between host and parasite lineages. A very low level of genetic variability and lack of congruence was found in one of the louse parasites, which was excluded from subsequent joint analysis of mitochondrial data. The reconciled multi-species tree obtained from the analysis is congruent with both the nuclear data and the geological history of the islands.

Conclusions

The gene genealogies of Galápagos mockingbirds and two of their ectoparasites show strong phylogeographic correlations, with instances of incongruence mostly explained by ancestral genetic polymorphism. A third parasite genealogy shows low levels of genetic diversity and little evidence of co-phylogeny with their hosts. These differences can mostly be explained by variation in life-history characteristics, primarily host specificity and dispersal capabilities. We show that pooling genetic data from organisms living in close ecological association reveals a more accurate phylogeographic history for these taxa. Our results have implications for the conservation and taxonomy of Galápagos mockingbirds and their parasites.     

A Multi-Year Record of Methane Flux at the Mer Bleue Bog,Southern Canada

The Mer Bleue peatland is a large ombrotrophic bog with hummock-lawn microtopography, poor fen sections and beaver ponds at the margin. Average growing-season (May–October) fluxes of methane (CH₄) measured in 2002–2003 across the bog ranged from less than 5 mg m⁻² d⁻¹ in hummocks, to greater than 100 mg m⁻² d⁻¹ in lawns and ponds. The average position of the water table explained about half of the variation in the season average CH₄ fluxes, similar to that observed in many other peatlands in Canada and elsewhere. The flux varied most when the water table position ranged between −15 and −40 cm. To better establish the factors that influence this variability, we measured CH₄ flux at approximately weekly intervals from May to November for 5 years (2004–2008) at 12 collars representing the water table and vegetation variations typical of the peatland. Over the snow-free season, peat temperature is the dominant correlate and the difference among the collars’ seasonal average CH₄ flux is partially dependent on water table position. A third important correlate on CH₄ flux is vegetation, particularly the presence of Eriophorum vaginatum, which increases CH₄ flux, as well as differences in the potential of the peat profile to produce and consume CH₄ under anaerobic and aerobic conditions. The combination of peat temperature and water table position with vegetation cover was able to explain approximately 44% of the variation in daily CH₄ flux, based on 1097 individual measurements. There was considerable inter-annual variation in fluxes, associated with varying peat thermal and water table regimes in response to variations in weather, but also by variations in the water level in peripheral ponds, associated with beaver dam activity. Raised water level in the beaver ponds led to higher water tables and increased CH₄ emission in the peatland.     

Maximum Annual Potential Yields of <Emphasis Type="Italic">Salix miyabeana</Emphasis> SX67 in Southern Quebec and Effects of Coppicing and Stool Age

Aboveground biomass yields of short rotation cultures (SRC) of willow can vary substantially depending on site quality. Among others, aboveground biomass yields depend on climatic conditions, soil properties, age of the SRC, and number of harvesting cycles. In this study, we investigated the effects of coppicing on growth variables (i.e., largest basal stem, height, and aboveground biomass) at ten SRC of Salix miyabeana SX67 established on various soils in southern Quebec. More than 1100 shrubs with stool ages varying between 1 and 15 years were measured. Strain analysis was carried out to calculate past annual aboveground productivities, and maximum annual yield potential was quantified at each site. Annual growth rates were highly variable and depended on site and coppicing history. To achieve optimal stool development and aboveground yields, two to three growing seasons following coppicing were necessary for sandy and clayey sites, respectively. The delays for reaching maximum yields were shortened when soil cation exchange capacity was dramatically low and were prolonged when soil was physically restricting stool development. This lag influenced the total yield of the first rotation and also modulated the magnitude of the increase of aboveground biomass that is generally observed in the second rotation. To increase yields in southern Quebec, our results suggest that it is preferable to extend the length of the first rotation instead of coppicing at the end of the first growing season after establishment.     

Tetraspanin Proteins Regulate Membrane Type-1 Matrix Metalloproteinase-dependent Pericellular Proteolysis

Membrane type-1 matrix metalloproteinase (MT1-MMP) supports tumor cell invasion through extracellular matrix barriers containing fibrin, collagen, fibronectin, and other proteins. Here, we show that simultaneous knockdown of two or three members of the tetraspanin family (CD9, CD81, and TSPAN12) markedly decreases MT1-MMP proteolytic functions in cancer cells. Affected functions include fibronectin proteolysis, invasion and growth in three-dimensional fibrin and collagen gels, and MMP-2 activation. Tetraspanin proteins (CD9, CD81, and TSPAN2) selectively coimmunoprecipitate and colocalize with MT1-MMP. Although tetraspanins do not affect the initial biosynthesis of MT1-MMP, they do protect the newly synthesized protein from lysosomal degradation and support its delivery to the cell surface. Interfering with MT1-MMP-tetraspanin collaboration may be a useful therapeutic approach to limit cancer cell invasion and metastasis.     

Synemin and vimentin are components of intermediate filaments in avian erythrocytes

Synemin, a high-molecular-weight protein associated with intermediate filaments in muscle, and vimentin, an intermediate-filament subunit found in many different cell types, have been identified by immunologic and electrophoretic criteria as components of intermediate filaments in mature avian erythrocytes. Desmin, the predominant subunit of intermediate filaments in muscle, has not been detected in these cells. Two dimensional immunoautoradiography of proteolytic fragments of synemin and vimentin demonstates that the erythrocyte proteins are highly homologous, if not identical, to their muscle counterparts. Double immunoflurorescence reaveals that erythrocyte synemin and vimentin co-localize in a cytoplasmic network of sinuous filaments that extends from the nucleus to the plasma membrane and resists aggregation by colcemid. Erythrocytes that are attached to glass cover slips can be sonicated to remove nuclei and nonadherent regions of the plasma membrane; this leaves elliptical patches of adherent membrane that retain mats of vimentin- and synemin-containing intermediate filaments, as seen by immunofluorescence and rotary shadowing. Similarly, mechanical enucleation of erythrocyte ghosts in suspension allows isolation of plasma membranes that retain a significant fraction of the synemin and vimentin, as assayed by electrophoresis, and intermediate filaments, as seen in thin sections. Both synemin and vimentin remain insoluble along with spectrin and actin, in solutions containing nonionic detergent and high salt. However, brief exposure of isolated membrane to distilled water releases the synemin and vimentin together in nearly pure form, before the release of significant amounts of spectrin and actin. These data suggest that avian erythrocyte intermeditate filaments are somehow anchored to the plasma membrane; erythrocytes may thus provide a simple system for the study of intermediate filaments and their mode of interaction with membranes. In addition, these data, in conjunction with previous data from muscle, indicate that synemin is capable of associating with either desmin or vimentin and may thus perform a special role in the structure or function of intermediate filaments in erythrocytes as well as muscle.     

Interaction of singlet oxygen with DNA and biological consequences.

To study the interaction of singlet oxygen (1O2) with DNA and the biological consequences of 1O2-induced DNA damage, we used the thermodissociable endoperoxide of 3,3'-(1,4 naphthalidene) dipropionate (NDPO2) as a generator of free 1O2 in reactions with (1) 2'-deoxynucleoside 3'-monophosphates (dNps), (2) an oligonucleotide (16-mer) having one deoxyguanine (dG), (3) native and denaturated rat kidney DNA and (4) single-stranded (ss) and double-stranded (ds) bacteriophage M13mp10 DNA. Using both anion exchange and reversed phase HPLC and 32P-postlabeling analyses, it was found that exposure of the various dNps to chemically generated 1O2 led to a detectable reaction with dGp and not with dAp, dCp, d5mCp or Tp. The reaction with dGp led to degradation of this nucleotide and the formation of a large number of reaction products, one of which could be identified as 7-hydro-8-oxo-2'-deoxyguanosine 3'-monophosphate (8-oxo-dGp). A second product could tentatively be identified as a formamido pyrimidine derivative of dGp (Fapy-dGp). When ss DNA, ds DNA or the oligonucleotide were exposed to 1O2, the formation of 8-oxo-dG could also be demonstrated. With the oligonucleotide, we found a so far unidentified reaction product. Under the same reaction conditions the yield of 8-oxo-dG was about 8-fold higher in ss DNA than in ds DNA. In ss DNA 8-oxo-dG seemed to be a more prominent product than in the case of reaction of 1O2 with free dGp. Reaction of 1O2 with ss or ds M13mp10 DNA led to biological inactivation of these DNAs, ss DNA being at least 100-fold more sensitive than ds DNA. It could be concluded that inactivation of the ss DNA must be largely due to 1O2-induced DNA lesions other than 8-oxo-dG. In agreement with the observed preferential reaction of 1O2 with dG most of the so far sequenced mutations, induced by 1O2 in a 144 bp mutation target sequence inserted in the lacZ alpha gene of ss or ds M13mp10 DNA, occurred at a G or G/C base pair respectively. A preference for G(C) to T(A) transversions can be observed for which 8-oxo-dG might have been responsible. In ss DNA a significant number of the mutations are characterized by the fact that a G is deleted.     

Solute size effects on the diffusion in biofilms of Streptococcus mutans

The diffusion of poly(ethylene glycol) (PEG) (MW varying between 200 and 10,000), and of three different types of micelles was examined in Streptococcus mutans biofilms using infrared spectroscopy. PEGs were used because they show limited interactions with biological materials and their weight can be selected in order to cover a wide range of size. The study showed that a considerable fraction at the base of the biofilm was not accessible to the diffusing solute molecules and this inaccessible fraction was very dependent on the size of the diffusing molecules. In parallel, it was found that the diffusion coefficients of these solutes in the biofilms were less than those in water and this reduction was less pronounced for large macromolecules, an effect proposed to be related to their limited penetration. Triton X-100, a neutral detergent, forms micelles that behave like PEG, suggesting that the behaviour observed for neutral macromolecules can be extrapolated to neutral macroassemblies. However, the diffusion, as well as the penetration of sodium dodecylsulphate micelles (a negatively charged surfactant) and cetylpyridinium chloride micelles (positively charged), in the biofilms appeared to be significantly influenced by electrostatic interactions with biofilm components. The present findings provide useful insights associated with the molecular parameters required to efficiently penetrate bacterial biofilms. The study suggests a rationale for the limited bactericidal power of some antibiotics (the large ones). The restricted accessibility of macromolecules and macroassemblies to biofilms must be examined carefully in order to offer guidelines in the development of novel antibacterial treatments.     

A preliminary method for estimating the age of brown kiwi (Apteryx mantelli) embryos

Morphological features of a collection of unknown-age wild kiwi (Apteryx mantelli) embryos from early development to point of hatch are described. Using these features, we assign developmental stages to each embryo and compare the progress of development to similar-staged ostrich (Struthio camelus) and chicken (Gallus gallus) embryos. Two ageing schemes for the kiwi embryos are developed by comparing measurements of their hindlimb segments, bills and crown–rump lengths with those of ostrich and chicken embryos at various stages of development. One of the 20 kiwi embryos was of known age. Both the ostrich model and the chicken model gave identical predictions for the marker and four other embryos. Developmental timing of some features differed between all three species, most markedly in the bill, with growth in the kiwi bill being relatively faster to achieve its larger relative and absolute size at hatch.