Role of the extracellular loop in the folding of a CFTR transmembrane helical hairpin

The folding of membrane-spanning domains into their native functional forms depends on interactions between transmembrane (TM) helices joined by covalent loops. However, the importance of these covalent linker regions in mediating the strength of helix-helix associations has not been systematically addressed. Here we examine the potential structural impact of cystic fibrosis-phenotypic mutations in the extracellular loop 2 (ECL2) on interactions between the TM3 and TM4 helices of the cystic fibrosis transmembrane conductance regulator (CFTR) in constructs containing CFTR residues 194-241. When the effects of replacements in ECL2 (including the CF-phenotypic mutants E217G and Q220R) were evaluated in a library of wild-type and mutant TM3-ECL2-TM4 hairpin constructs, we found that SDS-PAGE gel migration rates differed over a range of nearly 40% +/- the wild-type position and that decreased migration rates correlate with increasing hairpin alpha-helical content as measured by circular dichroism spectra in sodium dodecyl sulfate micelles. The decreased mobility of TM3/4 constructs by introduction of non-native residues is interpreted in terms of an elongation or "opening" of the helical hairpin and concomitant destabilization of membrane-based helix-helix interactions. Our results support a role for short loop regions in dictating the stability of membrane protein folds and highlight the interplay between membrane-embedded helix-helix interactions and loop conformation in influencing the structure of membrane proteins.     

Distinguishing past from present gene flow along and across a river: the case of the carnivorous marsupial (<Emphasis Type="Italic">Antechinus flavipes</Emphasis>) on southern Australian floodplains

Humans have altered many floodplain ecosystems around the world by clearing vegetation, building towns and regulating river flows. Studies discerning gene flow and population structure of floodplain-dwelling animals are rare yet are necessary for understanding the effects of human actions on native populations. In south-eastern Australia, the yellow-footed antechinus (Antechinus flavipes) is the only carnivorous marsupial on many lowland floodplains, yet our knowledge of impacts of human activities is limited. The control region of mitochondrial DNA and 11 microsatellite DNA markers were used to explore historic and current gene flow in A. flavipes across and along the Murray River. Simulations were carried out to test different migration models. We found evidence for historic gene flow along and across the river but inferred that small towns and farmland or cleared floodplain sections restricted current gene flow along the river. Populations along the river appear to be isolated, and should be maintained at large enough sizes to avoid genetic problems such as inbreeding depression and loss of evolutionary potential. We also investigated whether 50-year-long maintenance of high water levels for irrigation in summer, at the time of juvenile dispersal, has led to restrictions in gene flow across the river. We found no evidence for restrictions to gene flow across the river and suggest that large floods and dropping tree branches may aid dispersal across the river.     

BIODIVERSITY RESEARCH: Diversity and distribution of macroinvertebrates in lentic habitats in massively altered landscapes in south‐eastern Australia

Aim We investigated whether faunas of lentic macroinvertebrates differed among two landscape types: (1) those that are largely covered in forests (presumed to be in a more pre‐human‐impact condition) and (2) those that are completely cleared for agricultural exploitation (massively altered). Location Five pairs of landscapes (each pair referred to as a region) – one of each landscape type – across a 30,000 km² region of north‐central Victoria, Australia. Methods Each individual waterbody was surveyed three times (austral spring 2006, autumn 2007, and spring 2007) for invertebrates. Waterbodies were characterized by measurements of static (e.g. abutting vegetation cover) and labile (e.g. pH) variables. Data were analysed using hierarchical Bayesian models of species richness, α‐ and β‐diversities and functional feeding groups. Assemblage composition was related to landscape and in‐waterbody characteristics. Results Neither measured, nor asymptotic estimates of, species richness differed among landscape types, notwithstanding consistent differences in in‐waterbody habitat characteristics among waterbodies in the two landscape types. There were no discernible differences in patterns of α‐ and β‐diversities at landscape scales relating to landscape type. Habitat diversity of waterbodies at the landscape scale did not affect β‐diversity, although distinct waterbodies within landscapes tended to have more distinct faunas. Main conclusions The lentic macroinvertebrate faunas are relatively homogeneous over the entire region, with little differentiation between wooded and cleared landscapes. The regional fauna may be a homogenized subset of native species, possibly arising from the huge numerical predominance of lentic habitats in agricultural landscapes producing ‘spill‐over’ effects into forested landscapes. Of taxa more frequently found in one or other landscape type, trophic group diversity was greater in forested landscapes.     

A circuit logic for sexually shared and dimorphic aggressive behaviors in Drosophila

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Simultaneous determination of gemcitabine and gemcitabine-squalene by liquid chromatography-tandem mass spectrometry in human plasma

Gemcitabine-squalene is a new prodrug that self-organizes in water forming nanoassemblies. It exhibits better anti-cancer properties in vitro and in vivo than gemcitabine. A liquid chromatography/tandem mass spectrometry assay of gemcitabine-squalene and gemcitabine was developed in human plasma in order to quantitate gemcitabine and its squalene conjugate. After protein precipitation with acetonitrile/methanol (90/10, v/v), the compounds were analyzed by reversed-phase high performance liquid chromatography and detected by tandem mass spectrometry using multiple reaction monitoring. The method was linear over the concentration range of 10-10,000 ng/ml of human plasma for both compounds with an accuracy lower than 10.4% and a precision below 14.8%. The method showed a lower limit of quantitation of 10 ng/ml of human plasma for dFdC and dFdC-SQ. A preliminary in vivo study in mice was shown as application of the method as no significant difference between human and mice plasma for the analysis of dFdC and dFdC-SQ was demonstrated.     

Distribution of anuran amphibians in massively altered landscapes in south-eastern Australia: effects of climate change in an aridifying region

Aim We explored whether the anuran amphibian faunas differed among landscapes that are relatively intact (largely covered in forests and woodlands) and others that are completely converted to agriculture. We also used historical data sets to assess the current condition of the anuran fauna in a region predicted to experience, and experiencing, severe drying and warming. Location Five pairs of landscapes (each of c. 20 km²) – one in each pair being almost completely wooded and the other cleared for agriculture – across a 30,000 km² region of northern Victoria, Australia. Methods Sites were repeatedly surveyed in the austral winter–spring breeding seasons of 2006 and 2007, with records collected of numbers of calling males and the presence of egg masses and tadpoles. We characterized the sites using static (e.g. dimensions, surrounding physiognomic characteristics such as tree cover) and labile (e.g. pH, dissolved oxygen) variables. Data were analysed using hierarchical Bayesian models. Results For calling males, landscape type did not affect densities or species richness measures. The availability of a grassy verge around water bodies was an important predictor for most species, but other labile and static variables seemed not to be important. Fewer than half of the species historically known from the region were recorded. There were no important predictors of occurrence of egg masses or tadpoles. Reproduction effectively may have failed over the period, with fewer than one in four sites showing evidence of egg masses or tadpoles. Main conclusions The proportion of sites at which some well‐studied species (e.g. Crinia signifera, Limnodynastes dumerilii) were recorded has dropped substantially since the 1970s, as have average densities of calling males of Crinia spp. The remnant anuran fauna appears to be dominated by resilient and hardy species with low current diversity. The on‐going (12+ years) drought in these landscapes suggests a bleak long‐term prognosis for the few remaining species of anuran amphibians.     

Decline and potential recovery of Yellow-footed Antechinus in parts of south-eastern Australia: A perspective with implications for management

Summary   The extent and intensity of European-induced changes to ecosystems in south-eastern Australia mean that remaining habitats, despite being degraded, are of high conservation value. Given the extinction of several species of native mammals in the last 160 years in the area, and conservation concerns regarding others, it is important to provide conservation managers with sufficient information to prevent further extinctions and maintain evolutionary potential of the species. A native, carnivorous marsupial, the Yellow-footed Antechinus ( Antechinus flavipes ) exists within these massively altered landscapes. We present conceptual models, derived from the literature, of persistence of populations of Yellow-footed Antechinus both before European colonization and in the 21st century. We conclude that preservation of large trees, restoration of fallen-timber volumes, spring flooding of floodplains and presence of vegetation corridors between forests should be undertaken to prevent local extinctions of Yellow-footed Antechinus. From historic and current gene flow, we identify remnant woodland and forest groupings that we consider should be managed as coherent units.     

Positional dependence of non-native polar mutations on folding of CFTR helical hairpins

Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) cause CF disease by altering the biosynthesis, maturation, folding and ion conductance of this protein. Our laboratory has focused on expression and structural analysis of the CFTR transmembrane (TM) domains using two-TM segments (i.e., helix-loop-helix constructs) which we term ‘helical hairpins’; these represent the minimal model of tertiary contacts between two helices in a membrane. Previous studies on a library of TM3/4 hairpins of the first CFTR TM domain suggested that introduction of non-native polar residues into TM4 can compromise CFTR function through side chain-side chain H-bonding interactions with native Q207 in TM3 [Choi, M. Y., Cardarelli, L., Therien, A. G., and Deber, C. M. Non-native interhelical hydrogen bonds in the cystic fibrosis transmembrane conductance regulator domain modulated by polar mutations, Biochemistry 43 (2004) 8077-8083]. In the present work, we combine gel shift assays with a series of NMR experiments for comparative structural characterization of the wild type TM3/4 hairpin and its mutants V232D, I231D, Q207N/V232E. Over 95% of the backbone resonances of a ¹⁵N,¹³C-labelled V232D-TM3/4 construct in the membrane-mimetic environment of perfluorooctanoate (PFO) micelles were successfully assigned, and the presence and boundaries of helical segments within TM3 and TM4 were defined under these conditions. Comparative analysis of ¹⁵N and ¹H chemical shift variations among HSQC spectra of WT-, V232D-, I231D- and Q207N/V232E-TM3/4 indicated that hairpin conformations vary with the position of a polar mutation (i.e., V232D and I231D vs. WT), but remain similar when hairpins with identically-positioned polar partners are compared (i.e., V232D vs. Q207N-V232E). The overall findings suggest that a polar mutation in a TM helix can potentially distort native interfacial packing determinants in membrane proteins such as CFTR, with consequences that may lead to disease.