The evolution of the atpbeta-rbcL intergenic spacer in the epacrids (Ericales) and its systematic and evolutionary implications

Sequence data from the noncoding region separating the plastid genes atpbeta and rbcL were gathered for 27 epacrid taxa, representing all previously recognized infrafamilial groups, and four outgroup taxa (Ericaceae), to address several persistent phylogenetic questions in the group. Parsimony analyses were conducted on these data, as well as on a complementary rbcL sequence dataset assembled from the literature and the combined dataset. The atpbeta-rbcL spacer was notable for the high frequency of insertion-deletion mutations (indels); their distributions were coded as binary characters and included as a adjunct matrix in some of the analyses. The phylogenetic patterns derived from the spacer and rbcL data and the combined analyses, both including and excluding the indel data, concur in resolving seven major lineages corresponding to the tribes of Crayn et al. (1998, Aust. J. Bot. 46, 187-200), viz. Prionoteae, Archerieae, Oligarrheneae, Cosmelieae, Richeeae, Epacrideae, and Styphelieae. The relationships of the tribes and within Styphelieae, however, are not convincingly resolved. Minor conflicts in the positions of some taxa between the spacer and the rbcL trees are poorly supported. Among epacrids, the spacer region provided more cladistically informative characters than rbcL and resulted in trees with lower homoplasy. Further, the spacer data, when analyzed alone and when combined with rbcL, resolved several clades that could not be retrieved on rbcL data alone and provided increased support for many other relationships. The evolution of a putative three-base inversion associated with a hairpin secondary structure in the spacer region is discussed in the light of the inferred phylogeny.     

Sodium-potassium-ATPase electrogenicity in cerebral precapillary arterioles

Electrogenicity of the Na(+)/K(+) pump has the capability to generate a large negative membrane potential independently of ion-channel current. The high background membrane resistance of arterioles may make them susceptible to such an effect. Pump current was detected by patch-clamp recording from smooth muscle cells in fragments of arterioles (diameter 24-58 microm) isolated from pial membrane of rabbit cerebral cortex. The current was 20 pA at -60 mV, and the extrapolated zero current potential was -160 mV. Two methods of estimating the effect of pump electrogenicity on resting potential indicated an average contribution of -35 mV. In 20% of the recordings, block of inward rectifier K(+) channels by 10-100 microM Ba(2+) led to a small depolarization, but hyperpolarization was a more common response. Ba(2+) also inhibited depolarization evoked by 20 mM K(+). In arterioles within intact pial membrane, Ba(2+) failed to evoke constriction but inhibited K(+)-induced constriction. The data suggest that cerebral arterioles are vulnerable to the hyperpolarizing effect of the Na(+)/K(+) pump, excessive effects of which are prevented by depolarizing inward rectifier K(+) current     

Development and application of surface plasmon resonance-based biosensors for the detection of cell-ligand interactions

Surface plasmon resonance (SPR)-based biosensors were investigated with a view to providing a portable, inexpensive alternative to existing technologies for "real-time" biomolecular interaction analysis of whole cell-ligand interactions. A fiber optic SPR-based (FOSPR) biosensor, employing wavelength-dependent SPR, was constructed to enable continuous real-time data acquisition. In addition, a commercially available integrated angle-dependent SPR-based refractometer (ISPR) was modified to facilitate biosensing applications. Solid-phase detection of whole red blood cells (RBCs) using affinity-captured blood group specific antibodies was demonstrated using the BIACORE 1000, BIACORE Probe, FOSPR, and ISPR sensors. Nonspecific binding of RBCs to the hydrogel-based biointerface was negligible. However, the background noise level of the FOSPR-based biosensor was approximately 25-fold higher than that of the widely used BIACORE 1000 system while that of the ISPR-based biosensor was over 100-fold higher. Nevertheless, the FOSPR biosensor was suitable for the analysis of macromolecular analytes contained in crude matrices.     

Static compression is associated with decreased diffusivity of dextrans in cartilage explants

The chondrocytes of adult articular cartilage rely upon transport phenomena within their avascular extracellular matrix for many biological activities. Therefore, changes in matrix structure which influence cytokine transport parameters may be an important mechanism involved in the chondrocyte response to tissue compression. With this hypothesis in mind, partitioning and diffusion of 3-, 10-, and 40-kDa dextrans conjugated to tetramethylrhodamine, and 430-Da tetramethylrhodamine itself, were measured within statically compressed bovine articular cartilage explants using a novel experimental apparatus and desorption fluorescence method. Partitioning and diffusion were examined as functions of solute molecular weight and matrix proteoglycan density, and diffusion was measured versus static compression up to 35% volumetric strain. In general, partition coefficients and diffusivities were found to decrease with increasing solute molecular weight. In addition, for a given solute, diffusivities decreased significantly with increasing static compression. Results therefore suggest a possible role for transport limitations of relatively large molecular weight solutes within the extracellular matrix in mediating the biological response of chondrocytes to cartilage compression.     

Induced minisatellite germline mutations in herring gulls (Larus argentatus) living near steel mills

Despite widespread industrial release of genotoxic contaminants, little is understood of their role in inducing germline mutations in natural populations. We used multilocus DNA fingerprinting to quantify germline minisatellite mutations in families of herring gulls (Larus argentatus) in three nesting categories: (a) near cities with large steel mills operating coking ovens; (b) near cities without steel mills; and (c) in rural locations removed from point sources of contamination. Gulls nesting near integrated steel mills showed significantly higher mutation rates than gulls from rural locations (Fisher's exact, P=0.0004); urban sites without steel mills fell midway between steel and rural sites (difference from rural; Fisher's exact, P=0.19). Distance of the nesting location of herring gulls from the steel industries' coking ovens was negatively correlated with minisatellite mutation rate demonstrating significant risk for induced germline mutations in cities with steel operations (Kendall Tau; tau=0.119; P<0.0001).     

Sex expression, sex-specific traits, and the effects of salinity on growth and reproduction of Amaranthus cannabinus (Amaranthaceae), a dioecious annual

Amaranthus cannabinus was studied to investigate some of the ecological factors thought to be involved in the evolution of dioecy and to investigate the effects of salinity on sex expression and sex-specific selection. In the field portion of this study, sex ratios, stability of sex expression, spatial distribution, allocation strategies, and phenologies of the sexes were investigated in New Jersey freshwater and salt marsh populations of water hemp. To examine the effects of salinity on vegetative and reproductive development of males and females, plants were grown in the greenhouse at three salinity levels. Adult sex ratios were found to be 1:1. Temporal deviations from a 1:1 sex ratio varied by population and were due to differences in flowering phenology and mortality between the sexes. No plants were observed to change sex expression, and there was no evidence of spatial segregation of the sexes in the field. In both the field and the greenhouse, females allocated more resources to vegetative tissues and had a longer growing period than males. The results of this study suggest that increased reproductive efficiency through sex-specific growth patterns may have been an important selective factor involved in the evolution of dioecy in A. cannabinus.     

Biodegradation of Phosphonomycin by Rhizobium huakuii PMY1

The biodegradation by Rhizobium huakuii PMY1 of up to 10 mM phosphonomycin as a carbon, energy, and phosphorus source with accompanying P_i release is described. This biodegradation represents a further mechanism of resistance to this antibiotic and a novel, phosphate-deregulated route for organophosphonate metabolism by Rhizobium spp.     

Factors affecting the outcome of territorial contests between hatchery and naturally reared coho salmon parr in the laboratory

In aquarium experiments using coho salmon as a model species, prior residents dominated intruders of the same size but intruders with a 6% length advantage were equally matched against prior residents. Prior winning experience (distinct from individual recognition) also strongly influenced competitive success and overcame a prior residence effect. Coho salmon reared in a hatchery dominated size-matched fish from the same parental population reared in a stream. Hatchery-reared salmon also dominated naturally spawned salmon, even when the wild salmon were prior residents. Thus the combined effects of greater size and rearing experience of hatchery-produced salmon were sufficient to overcome a wild salmon's advantage of prior residence. Efforts to rehabilitate salmonid populations must consider such behavioural interactions if displacement of wild fish is to be prevented.     

Phosphoenolpyruvate Phosphomutase Activity in an l-Phosphonoalanine-Mineralizing Strain of Burkholderia cepacia

A strain of Burkholderia cepacia isolated by enrichment culture utilized l-2-amino-3-phosphonopropionic acid (phosphonoalanine) at concentrations up to 20 mM as a carbon, nitrogen, and phosphorus source in a phosphate-insensitive manner. Cells contained phosphoenolpyruvate phosphomutase activity, presumed to be responsible for cleavage of the C—P bond of phosphonopyruvate, the transamination product of l-phosphonoalanine; this was inducible in the presence of phosphonoalanine.Organophosphonates are characterized by the presence of a stable, covalent carbon-to-phosphorus (C—P) bond. In the majority of previous studies they have been utilized only under phosphate-limited conditions and only as sole sources of phosphorus for microbial growth (3, 4, 21, 22). The C—P bond may be cleaved by at least three distinct bacterial enzymes: the C—P lyase enzyme complex(es) (17, 24, 25, 27, 28), phosphonoacetaldehyde hydrolase (5, 6, 9, 12), and phosphonoacetate hydrolase (14–16). The latter enzyme is unique in that its expression is independent of the phosphate status of the cell and is inducible solely by phosphonoacetate. It is likely that organophosphonate biodegradation in the environment is mediated largely by a C—P lyase(s), with organisms capable of mineralizing organophosphonates as sources of carbon and energy being rare (2, 13).Phosphonoalanine (2-amino-3-phosphonopropionic acid) is one of the naturally occurring C—P compounds synthesized by lower organisms, such as the sea anemone Zoanthus sociatus (10) and the protozoan Tetrahymena pyriformis (8, 23, 29). In this paper, we report the isolation of a bacterium capable of mineralizing l-phosphonoalanine as a carbon, energy, nitrogen, and phosphorus source independently of the phosphate status of the cell.Enrichment was carried out with a basal mineral salts medium which contained the following (per liter): KCl, 0.2 g; MgSO₄ · 7H₂O, 0.2 g; CaCl₂ · 2H₂O, 0.01 g; ferric ammonium citrate, 1.0 mg; trace element solution (11), 1 ml; and vitamin solution (14), 1 ml. Filter-sterilized (0.22-μm pore size) dl-phosphonoalanine (8 mM) was routinely added as a carbon, energy, nitrogen, and phosphorus source. The pH of the medium was initially adjusted to 7.2, and where required, filter-sterilized solutions of sodium pyruvate as a carbon source (final concentration, 10 g liter⁻¹), NH₄Cl as an inorganic nitrogen source (final concentration, 5 g liter⁻¹), and/or phosphate buffer (final concentration, 1 mM) were added to the medium. Enrichment cultures (25 ml in 250-ml Erlenmeyer flasks) were inoculated with a 0.5% (vol/vol) composite inoculum from an activated sludge plant (Dunmurry, Northern Ireland), a laundry waste disposal lagoon (Summit Lake, Wis.), and a sheep dip disposal site (County Antrim, Northern Ireland). All sites were known to have a history of exposure to organophosphonates. Cultures were incubated at 28°C on an orbital shaker at 100 rpm. Microbial growth was measured by the increase in optical density at 650 nm (OD₆₅₀) using a Pye-Unicam 8265 UV-visible light spectrophotometer (Pye-Unicam Ltd., Cambridge, United Kingdom). Release of inorganic phosphate and ammonium into culture supernatants was monitored by the methods of Fiske and SubbaRow (7) and Weatherburn (30), respectively.Three gram-negative isolates, each capable of growth on 8 mM dl-phosphonoalanine as a carbon, nitrogen, and phosphorus source were obtained following five rounds of serial enrichment. Of these, isolate Pal6 grew most quickly on phosphonoalanine and was chosen for further investigation. It was identified by the National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, Scotland, as a strain of Burkholderia cepacia.When dl-phosphonoalanine (8 mM) was supplied as a carbon, nitrogen, and phosphorus source for growth of B. cepacia Pal6, some 47% of substrate phosphorus and 44% of substrate nitrogen was released concomitantly with growth as P_i and ammonium (results not shown). When the compound was supplied as the sole phosphorus source (Fig. ​(Fig.1),1), transient release of approximately 30% of substrate phosphorus to the medium as P_i was observed; this phenomenon has not previously been reported for the utilization of any organophosphorus compound as a phosphorus source. When B. cepacia Pal6 was grown on dl-phosphonoalanine as a nitrogen and phosphorus (Fig. ​(Fig.2)2) or nitrogen source, removal of 50% of phosphonoalanine from the medium was demonstrated by the method of Moore and Stein (18), along with release of just less than 50% of substrate phosphorus as P_i. A subsequent experiment in which the d- and l-enantiomers were separately supplied as sole sources of phosphorus indicated that only l-phosphonoalanine supported growth of B. cepacia Pal6. It is therefore clear that the catabolism of l-phosphonoalanine by this isolate is independent of the phosphate status of the cell, a marked departure from the many examples of classical pho regulon-controlled biodegradation of organophosphonates reported in the literature (26, 27). Open in a separate windowFIG. 1Growth of B. cepacia Pal6 on phosphonoalanine (1 mM) as the sole phosphorus source, with NH₄Cl as a nitrogen source (5 g liter⁻¹) and pyruvate as a carbon source (10 g liter⁻¹). Symbols: •, OD₆₅₀; ▴, phosphate release.Open in a separate windowFIG. 2Growth of B. cepacia Pal6 on phosphonoalanine (5 mM) as a nitrogen and phosphorus source, with pyruvate as a carbon source (10 g liter⁻¹). Symbols: •, OD₆₅₀; ▴, phosphate release (mM); □, phosphonoalanine remaining in medium (mM).B. cepacia Pal6 was grown on a range of dl-phosphonoalanine concentrations as carbon and nitrogen source in the presence of 1 mM inorganic phosphate. The cell yield was proportional to the concentration of phosphonoalanine supplied up to 20 mM, the highest concentration tested, again with release of less than 50% substrate phosphorus and nitrogen to the medium (results not shown), indicating no toxicity on the part of either the substrate or its breakdown products at these concentrations.In addition to phosphonoalanine, B. cepacia Pal6 was able to utilize 6 of 14 organophosphonate substrates supplied as the sole phosphorus source (Table ​(Table1);1); however, with the exception of 2-aminoethylphosphonic acid (2AEP), no phosphate release was observed during growth on these compounds, suggesting classical pho regulon control of their biodegradation and the involvement of a C—P lyase(s) or similar enzymes. B. cepacia Pal6 was also capable of growing on 2AEP as a carbon, energy, nitrogen, and phosphorus source, with concomitant release of excess phosphorus and nitrogen to the medium as inorganic phosphate and ammonium, respectively. It did not utilize any of the other phosphonates tested as the carbon and/or nitrogen and phosphorus source. The metabolism by B. cepacia Pal6 of 2AEP as a carbon, nitrogen and phosphorus source suggests that a phosphate-deregulated pathway is also responsible for the mineralization of this compound.

TABLE 1

Range of organophosphonate substrates utilized by B. cepacia Pal6 as the sole phosphorus source

In vivo pharmaco-proteomic analysis of hydroxyurea induced changes in the sickle red blood cell membrane proteome

Hydroxyurea (HU) is an effective drug for the treatment of sickle cell disease (SCD). The main clinical benefit of HU is thought to derive from its capacity to increase fetal hemoglobin (HbF) production. However, other effects leading to clinical benefit, such as improved blood rheology, have been suggested. In order to understand HU-induced changes at the proteomic level, we profiled sickle RBC membranes from of HU-treated and untreated patients. Our previous in vitro profiling studies on sickle RBC membranes identified a significant increase in predominantly anti-oxidant enzymes, protein repair and degradation components and a few RBC cytoskeletal proteins. In the present study, using 2D-DIGE (Two-Dimensional Difference In-Gel Electrophoresis) and tandem mass spectrometry, we detected 32 different proteins that significantly changed in abundance in the HU treatment group. The proteins that significantly increased in abundance were mostly membrane skeletal components involved in the regulation of RBC shape and flexibility, and those showing a significant decrease were components of the protein repair and degradation machinery. RBC palmitoylated membrane protein 55 (p55) is significantly increased in abundance at low (in vitro) and high (in vivo) concentrations of HU. Palmitoylated p55 may be an important target of HU-dependent regulation of the sickle RBC membrane, consistent with our earlier in vitro studies.