Neglected native or undesirable alien? Resolution of a conservation dilemma concerning the pool frog Rana lessonae

Introduced species often pose serious threats to biodiversity, but occasionally confusion arises as to whether a species really is introduced or is in fact an overlooked native. A recent UK conservation dilemma has centred on the status of the pool frog Rana lessonae. This species has been the subject of documented introductions from central and southern Europe since the early 1800s, the accepted position being that all UK R. lessonae populations are descended from these introductions. However, a closer examination of early UK literature sources, and recent discoveries of isolated, native R. lessonae populations in Sweden and Norway, led some herpetologists to question whether the species was in fact present as a native at some locations prior to the introductions. Research was initiated along four major lines of enquiry: genetic, bioacoustic, archaeozoological and archival. A high degree of convergence among the genetic and bioacoustic investigations demonstrated that the potentially native UK pool frogs were closely related to Scandinavian frogs, thus ruling out introductions from further south as a potential origin. Subfossil evidence of pool frogs was found from ca. 1000 years before present, demonstrating that the species occurred in the UK prior to known introductions. Archival sources produced no historical support for introductions from northern Europe. The postglacial history inferred for these northern populations is consistent with the known climatic and geographical conditions. Taken together, the evidence for the native status of the pool frog is compelling, and furthermore the UK population appears to be part of a distinct northern clade.     

Vegetation demographics in Earth System Models: A review of progress and priorities

Numerous current efforts seek to improve the representation of ecosystem ecology and vegetation demographic processes within Earth System Models (ESMs). These developments are widely viewed as an important step in developing greater realism in predictions of future ecosystem states and fluxes. Increased realism, however, leads to increased model complexity, with new features raising a suite of ecological questions that require empirical constraints. Here, we review the developments that permit the representation of plant demographics in ESMs, and identify issues raised by these developments that highlight important gaps in ecological understanding. These issues inevitably translate into uncertainty in model projections but also allow models to be applied to new processes and questions concerning the dynamics of real‐world ecosystems. We argue that stronger and more innovative connections to data, across the range of scales considered, are required to address these gaps in understanding. The development of first‐generation land surface models as a unifying framework for ecophysiological understanding stimulated much research into plant physiological traits and gas exchange. Constraining predictions at ecologically relevant spatial and temporal scales will require a similar investment of effort and intensified inter‐disciplinary communication.     

Design and testing for a nontagged F1-V fusion protein as vaccine antigen against bubonic and pneumonic plague

A two-component recombinant fusion protein antigen was re-engineered and tested as a medical counter measure against the possible biological threat of aerosolized Yersinia pestis. The active component of the proposed subunit vaccine combines the F1 capsular protein and V virulence antigen of Y. pestis and improves upon the design of an earlier histidine-tagged fusion protein. In the current study, different production strains were screened for suitable expression and a purification process was optimized to isolate an F1-V fusion protein absent extraneous coding sequences. Soluble F1-V protein was isolated to 99% purity by sequential liquid chromatography including capture and refolding of urea-denatured protein via anion exchange, followed by hydrophobic interaction, concentration, and then transfer into buffered saline for direct use after frozen storage. Protein identity and primary structure were verified by mass spectrometry and Edman sequencing, confirming a purified product of 477 amino acids and removal of the N-terminal methionine. Purity, quality, and higher-order structure were compared between lots using RP-HPLC, intrinsic fluorescence, CD spectroscopy, and multi-angle light scattering spectroscopy, all of which indicated a consistent and properly folded product. As formulated with aluminum hydroxide adjuvant and administered in a single subcutaneous dose, this new F1-V protein also protected mice from wild-type and non-encapsulated Y. pestis challenge strains, modeling prophylaxis against pneumonic and bubonic plague. These findings confirm that the fusion protein architecture provides superior protection over the former licensed product, establish a foundation from which to create a robust production process, and set forth assays for the development of F1-V as the active pharmaceutical ingredient of the next plague vaccine.     

A Modified,High Yield Procedure for the Synthesis of Unlabeled and 14C-Labeled 4-Methylene-DL-glutamic Acid

This paper describes the complete chemical synthesis of 4-methylene-DL-glutamic acid from diethylmalonate, formaldehyde and diethyl acetamidomalonate. The amino acid was obtained pure following ion-exchange chromatography and/or crystallization from hot water in an overall yield of 30% based on the amount of diethylmalonate used. Several physico-chemical characteristics of the synthetic compound were determined, including ir and pmr spectra, chromatography on paper, retention time on an amino acid analyzer, pK values and melting point; all properties cf the synthetic material were found to be identical to those seen with the naturally occurring L-isomer. The procedure for obtaining gram quantities of the unlabeled compound has also been modified for the synthesis of high specific activity (10.6 mCi/nmol) 4-methylene-[2-¹⁴C]-DL-glutamic acid.     

Cytochrome P450scc-dependent metabolism of 7-dehydrocholesterol in placenta and epidermal keratinocytes

The discovery that 7-dehydrocholesterol (7DHC) is an excellent substrate for cytochrome P450scc (CYP11A1) opens up new possibilities in biochemistry. To elucidate its biological significance we tested ex vivo P450scc-dependent metabolism of 7DHC by tissues expressing high and low levels of P450scc activity, placenta and epidermal keratinocytes, respectively. Incubation of human placenta fragments with 7DHC led to its conversion to 7-dehydropregnenolone (7DHP), which was inhibited by dl-aminoglutethimide, and stimulated by forskolin. Final proof for P450scc involvement was provided in isolated placental mitochondria where production of 7DHP was almost completely inhibited by 22R-hydroxycholesterol. 7DHC was metabolized by placental mitochondria at a faster rate than exogenous cholesterol, under both limiting and saturating conditions of substrate transport, consistent with higher catalytic efficiency (k(cat)/K(m)) with 7DHC as substrate than with cholesterol. Ex vivo experiments showed five 5,7-dienal intermediates with MS spectra of dihydroxy and mono-hydroxy-7DHC and retention time corresponding to 20,22(OH)(2)7DHC and 22(OH)7DHC. The chemical structure of 20,22(OH)(2)7DHC was defined by NMR. 7DHP was further metabolized by either placental fragments or placental microsomes to 7-dehydroprogesterone as defined by UV, MS and NMR, and to an additional product with a 5,7-dienal structure and MS corresponding to hydroxy-7DHP. Furthermore, epidermal keratinocytes transformed either exogenous or endogenous 7DHC to 7DHP. 7DHP inhibited keratinocytes proliferation, while the product of its pholytic transformation, pregcalciferol, lost this capability. In conclusion, tissues expressing P450scc can metabolize 7DHC to biologically active 7DHP with 22(OH)7DHC and 20,22(OH)(2)7DHC serving as intermediates, and with further metabolism to 7-dehydroprogesterone and (OH)7DHP.     

Maintenance of Y receptor dimers in epithelial cells depends on interaction with G-protein heterotrimers

Treatment of CHO cells expressing human Y receptors (Y₁, Y₂ or Y4 subtype) with pertussis toxin results in a large decrease in functional receptors, with a preferential loss of heteropentameric assemblies of receptor dimers and G-protein trimers. This occurs in parallel to inactivation of the nucleotide site of Gi α subunits, with a half period of about 4 h. The loss could be mainly due to proteolysis at the level of recycling/perinuclear endosomes, and of receptor completion in the ER, since it is reduced by co-treatment with ammonium chloride, an inhibitor of particulate proteinases. Antagonists do not strongly decrease the heteropentameric fraction. These findings indicate that the upkeep of Y receptor dimers in epithelial cell lines depends on the association of receptor oligomers with functional Gi α subunits. This interaction could use the juxtamembrane helix 8 in the fourth intracellular domain, and could also be supported by the C-terminal helix of the third intracellular loop, as outlined in the companion review (Parker et al., Amino Acids, doi:, 2010).