The epidermal growth factor receptor juxtamembrane domain has multiple basolateral plasma membrane localization determinants,including a dominant signal with a polyproline core

The epidermal growth factor (EGF) receptor is located predominantly in the basolateral membrane of polarized epithelia, where it plays a pivotal role during organogenesis and tissue homeostasis. We have shown previously that a 22-amino acid sequence in the EGF receptor juxtamembrane domain contains autonomous sorting information necessary for basolateral localization using the Madin-Darby canine kidney epithelial cell model. The goal of this study was to determine the molecular basis of EGF receptor basolateral membrane expression using site-directed mutagenesis to modify specific residues in this region. We now show that this sequence has two different, functionally redundant basolateral sorting signals with distinct amino acid requirements: one dependent on residues (658)LL(659) conforming to well-characterized leucine-based sorting signals, and a second containing a polyproline core comprising residues Pro(667) and Pro(670) ((667)PXXP(670)). Our data also suggest that Arg(662) contributes to the function of the proline-based signal. (667)PXXP(670) was the dominant signal when both motifs were present and was more effective than (658)LL(659) at overriding strong apical sorting signals located in the same molecule. Site-directed mutations at Arg(662), Pro(667), and Pro(670) were also associated with increased apical expression of full-length EGF receptors, demonstrating for the first time that the juxtamembrane region is necessary for accurate polarized expression of the native molecule.     

Structural polarity and functional bile canaliculi in rat hepatocyte spheroids

Primary hepatocytes self-assemble into spheroids that possess tight junctions and microvilli-lined channels. We hypothesized that polarity develops gradually and that the channels structurally and functionally resemble bile canaliculi. Immunofluorescence labeling of apical and basolateral proteins demonstrated reorganization of the membrane proteins into a polarized distribution during spheroid culture. By means of fluorescent dextran diffusion and confocal microscopy, an extensive network of channels was revealed in the interior of the spheroids. These channels connected over several planes and opened to pores on the surface. To examine the content of apical proteins in the channel membranes, the bile canalicular enzyme dipeptidyl peptidase IV (DPPIV) was localized using a fluorogenic substrate, Ala-Pro-cresyl violet. The results show that DPPIV activity is heterogeneously distributed in spheroids and localized in part to channels. Bile acid excretion was then investigated to demonstrate functional polarity. A fluorescent bile acid analogue, fluorescein isothiocyanate-labeled glycocholate, was taken up into the spheroids and excreted into bile canalicular channels. Due to the structural polarity of spheroids and their ability to excrete bile into channels, they are a unique three-dimensional model of in vitro liver tissue self-assembly. (Videoanimations of some results are available at http://hugroup.cems.umn.edu/research_movies).     

Other Perspectives on the Kennewick Man Controversy

Skull Wars: Kennewick Man, Archaeology, and the Battle for Identity. David Hurst Thomas. New York: Basic Books, 2000. 326 pp.
Riddle of the Bones: Politics, Science, Race, and the Story of Kennewick Man. Roger Downey. New York: Copernicus, 2000. 202 pp.     

Functional analysis of the Alternaria brassicicola non-ribosomal peptide synthetase gene AbNPS2 reveals a role in conidial cell wall construction

Alternaria brassicicola is a necrotrophic pathogen causing black spot disease on virtually all cultivated Brassica crops worldwide. In many plant pathosystems fungal secondary metabolites derived from non-ribosomal peptide synthetases (NPSs) are phytotoxic virulence factors or are antibiotics thought to be important for niche competition with other micro-organisms. However, many of the functions of NPS genes and their products are largely unknown. In this study, we investigated the function of one of the A. brassicicola NPS genes, AbNPS2 . The predicted amino acid sequence of AbNPS2 showed high sequence similarity with A. brassicae , AbrePsy1, Cochliobolus heterostrophus , NPS4 and a Stagonospora nodorum NPS. The AbNPS2 open reading frame was predicted to be 22 kb in length and encodes a large protein (7195 amino acids) showing typical NPS modular organization. Gene expression analysis of AbNPS2 in wild-type fungus indicated that it is expressed almost exclusively in conidia and conidiophores, broadly in the reproductive developmental phase. AbNPS2 gene disruption mutants showed abnormal spore cell wall morphology and a decreased hydrophobicity phenotype. Conidia of abnps2 mutants displayed an aberrantly inflated cell wall and an increase in lipid bodies compared with wild-type. Further phenotypic analyses of abnps2 mutants showed decreased spore germination rates both in vitro and in vivo , and a marked reduction in sporulation in vivo compared with wild-type fungus. Moreover, virulence tests on Brassicas with abnps2 mutants revealed a significant reduction in lesion size compared with wild-type but only when aged spores were used in experiments. Collectively, these results indicate that AbNPS2 plays an important role in development and virulence.     

Aquatic and terrestrial invertebrate drift in southern Appalachian Mountain streams: implications for trout food resources

1. We characterised aquatic and terrestrial invertebrate drift in six south‐western North Carolina streams and their implications for trout production. Streams of this region typically have low standing stock and production of trout because of low benthic productivity. However, little is known about the contribution of terrestrial invertebrates entering drift, the factors that affect these inputs (including season, diel period and riparian cover type), or the energetic contribution of drift to trout. 2. Eight sites were sampled in streams with four riparian cover types. Drift samples were collected at sunrise, midday and sunset; and in spring, early summer, late summer and autumn. The importance of drift for trout production was assessed using literature estimates of annual benthic production in the southern Appalachians, ecotrophic coefficients and food conversion efficiencies. 3. Abundance and biomass of terrestrial invertebrate inputs and drifting aquatic larvae were typically highest in spring and early summer. Aquatic larval abundances were greater than terrestrial invertebrates during these seasons and terrestrial invertebrate biomass was greater than aquatic larval biomass in the autumn. Drift rates of aquatic larval abundance and biomass were greatest at sunset. Inputs of terrestrial invertebrate biomass were greater than aquatic larvae at midday. Terrestrial invertebrate abundances were highest in streams with open canopies and streams adjacent to pasture with limited forest canopy. 4. We estimate the combination of benthic invertebrate production and terrestrial invertebrate inputs can support 3.3–18.2 g (wet weight) m⁻² year⁻¹ of trout, which is generally lower than values considered productive [10.0–30.0 g (wet weight) m⁻² year⁻¹]. 5. Our results indicate terrestrial invertebrates can be an important energy source for trout in these streams, but trout production is still low. Any management activities that attempt to increase trout production should assess trout food resources and ensure their availability.     

Congeneric phylogeography: hypothesizing species limits and evolutionary processes in Patagonian lizards of the Liolaemus boulengeri group (Squamata: Liolaemini)

In poorly known groups for which data are insufficient to develop biologically plausible model‐based approaches to phylogeographical analyses, a ‘first hypotheses’ protocol is suggested as offering the best way to generate hypotheses for subsequent model‐based tests. Preliminary hypotheses are formulated about species boundaries and population processes in three species complexes of the Liolaemus boulengeri group in the context of mtDNA ‘congeneric phylogeography’. The temperate South American Liolaemus provides a model with ancient and recent allopatric divergence across ecologically and geologically complex landscapes, incipient speciation, secondary contact, and discordance between molecular and morphological patterns of variation. Moderately dense sampling of widely distributed ‘inertial’ species in the Patagonian Steppe has revealed hidden genetic and probably species diversity, and also hinted at demographic and historical processes that may have shaped the histories of these taxa. Five of the seven focal species of the present study were paraphyletic for mtDNA genealogies, suggesting that they represent complexes of species, and nested clade phylogeographical analysis (NCPA) analyses suggest that different historical and demographic processes have shaped the observed patterns. Introgression and incomplete lineage sorting are hypothesized as being the cause of some of the observed paraphyly. Provisional delimitations of species are proposed and NCPA is used to generate hypotheses of population history, all of which are subject to further testing. Multi‐faceted studies, involving phylogenetic assessments of independent molecular markers and morphological variation across codistributed taxa with estimates of niche breadths in a landscape context, will likely yield the most promising returns for cross‐validation of hypotheses of population and speciation histories. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89 , 241–275.     

Phylogeographic diversification of antelope squirrels (Ammospermophilus) across North American deserts

We investigated the biogeographic history of antelope squirrels, genus Ammospermophilus, which are widely distributed across the deserts and other arid lands of western North America. We combined range‐wide sampling of all currently recognized species of Ammospermophilus with a multilocus data set to infer phylogenetic relationships. We then estimated divergence times within identified clades of Ammospermophilus using fossil‐calibrated and rate‐calibrated molecular clocks. Lastly, we explored generalized distributional changes of Ammospermophilus since the last glacial maximum using species distribution models, and assessed responses to Quaternary climate change by generating demographic parameter estimates for the three wide‐ranging clades of A. leucurus. From our phylogenetic estimates we inferred strong phylogeographic structure within Ammospermophilus and the presence of three well‐supported major clades. Initial patterns of historical divergence were coincident with dynamic alterations in the landscape of western North America, and the formation of regional deserts during the Late Miocene and Pliocene. Species distribution models and demographic parameter estimates revealed patterns of recent population expansion in response to glacial retreat. When combined with evidence from co‐distributed taxa, the historical biogeography of Ammospermophilus provides additional insight into the mechanisms that impacted diversification of arid‐adapted taxa across the arid lands of western North America. We propose species recognition of populations of the southern Baja California peninsula to best represent our current understanding of evolutionary relationships among genetic units of Ammospermophilus. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 949–967.