Functional and structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid nanodiscs

Cellular signaling mediated by the epidermal growth factor receptor (EGFR or ErbB) family of receptor tyrosine kinases plays an important role in regulating normal and oncogenic cellular physiology. While structures of isolated EGFR extracellular domains and intracellular protein tyrosine kinase domains have suggested mechanisms for growth factor-mediated receptor dimerization and allosteric kinase domain activation, understanding how the transmembrane and juxtamembrane domains contribute to transmembrane signaling requires structural studies on intact receptor molecules. In this report, recombinant EGFR constructs containing the extracellular, transmembrane, juxtamembrane, and kinase domains are overexpressed and purified from human embryonic kidney 293 cell cultures. The oligomerization state, overall structure, and functional stability of the purified EGF-bound receptor are characterized in detergent micelles and phospholipid bilayers. In the presence of EGF, catalytically active EGFR dimers can be isolated by gel filtration in dodecyl maltoside. Visualization of the dimeric species by negative stain electron microscopy and single particle averaging reveals an overall structure of the extracellular domain that is similar to previously published crystal structures and is consistent with the C-termini of domain IV being juxtaposed against one another as they enter the transmembrane domain. Although detergent-soluble preparations of EGFR are stable as dimers in the presence of EGF, they exhibit differential functional stability in Triton X-100 versus dodecyl maltoside. Furthermore, the kinase activity can be significantly stabilized by reconstituting purified EGF-bound EGFR dimers in phospholipid nanodiscs or vesicles, suggesting that the environment around the hydrophobic transmembrane and amphipathic juxtamembrane domains is important for stabilizing the tyrosine kinase activity in vitro.     

Honeybee‐assisted wind pollination in bamboo Phyllostachys nidularia (Bambusoideae: Poaceae)?

Floral syndromes and pollination of three species of Phyllostachys bamboos were studied in Central China in 1999 and 2000. All were protogynous. Stigmas were receptive and had pollen deposited on them 2 days before anther dehiscence. The period of anthesis in the three bamboos was 3 days. Individual pollen grains of the three species were similar in size (30–40 μm in diameter) and had features typical of wind-pollinated plants. The ratios of pollen to ovules (p/o ratio) in P . nidularia , P . heteroclada and P . nuda were 6500, 12 700 and 33 000, respectively. Mean pollen loads on each flower (one ovule) of these three species were 7.3, 8.8 and 9.4 grains, respectively. Pollen transfer in P . heteroclada and P . nuda depended on wind, and no flower visitors were seen in the field. However, in P . nidularia , Apis cerana Fab. was a frequent pollen collector observed from 1200 to 1330 h. The visits undertaken by thousands of honeybees resulted in a large number of pollen grains being released from the anthers in a short time (10–15 min) in one day, which accelerated and synchronized the release of pollen from the anthers that seemed to enhance the chance of pollination. Given that honeybees played an indirect role in pollen transfer this could partly explain the low p/o ratio in P . nidularia . Reviewing the literature, we found insect visits to flowers of bamboos were not infrequent phenomena. We suggest pollination efficiency should be considered as one selective factor in attempts to understand mast flowering in bamboo. © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 138 , 1–7.     

Molecular Relationships of the New Guinean Bandicoot Genera Microperoryctes and Echymipera (Marsupialia: Peramelina)

The complete 12S rRNA gene was sequenced for multiple exemplars of the New Guinean bandicoot genera Microperoryctes and Echymipera representing many of the currently recognized subspecies. These two genera are resolved as monophyletic sister taxa but there was no genetic support for the family Peroryctidae proposed by Groves and Flannery (1990). Within Microperoryctes, M. papuensis was sister to M. longicauda. Although there was only weak support for recognition of the subspecies M. l. magnus, our results demonstrate the need for further genetic and morphological studies of variation among populations of Microperoryctes. Haplotype relationships within Echymipera did not reflect current species boundaries. Although E. clara was the most divergent species, no clear separation could be made between E. rufescens and E. kalubu (E. kalubu samples from New Britain were consistently more closely allied with the E. rufescens exemplars collected from north and east of the Central Cordillera than with their congeners). We suggest the need for a major morphological reassessment of New Guinean bandicoot relationships.     

Molecular Evidence for the Major Clades of Placental Mammals

Higher-level relationships among placental mammals, as well as the historical biogeography of this group against the backdrop of continental fragmentation and reassembly, remain poorly understood. Here, we analyze two independent molecular data sets that represent all placental orders. The first data set includes six genes (A2AB, IRBP, vWF, 12S rRNA, tRNA valine, 16S rRNA; total = 5.71 kb) for 26 placental taxa and two marsupials; the second data set includes 2.95 kb of exon 11 of the BRCA1 gene for 51 placental taxa and four marsupials. We also analyzed a concatenation of these data sets (8.66 kb) for 26 placentals and one marsupial. Unrooted and rooted analyses were performed with parsimony, distance methods, maximum likelihood, and a Bayesian approach. Unrooted analyses provide convincing support for a fundamental separation of placental orders into groups with southern and northern hemispheric origins according to the current fossil record. On rooted trees, one or both of these groups are monophyletic depending on the position of the root. Maximum likelihood and Bayesian analyses with the BRCA1 and combined 8.66 kb data sets provide strong support for the monophyly of the northern hemisphere group (Boreoeutheria). Boreoeutheria is divided into Laurasiatheria (Carnivora + Cetartiodactyla + Chiroptera + Eulipotyphla + Perissodactyla + Pholidota) and Euarchonta (Dermoptera + Primates + Scandentia) + Glires (Lagomorpha + Rodentia). The southern hemisphere group is either monophyletic or paraphyletic, depending on the method of analysis used. Within this group, Afrotheria (Proboscidea + Sirenia + Hyracoidea + Tubulidentata + Macroscelidea + Afrosoricida) is monophyletic. A unique nine base-pair deletion in exon 11 of the BRCA1 gene also supports Afrotheria monophyly. Given molecular dates that suggest that the southern hemisphere group and Boreoeutheria diverged in the Early Cretaceous, a single trans-hemispheric dispersal event may have been of fundamental importance in the early history of crown-group Eutheria. Parallel adaptive radiations have subsequently occurred in the four major groups: Laurasiatheria, Euarchonta + Glires, Afrotheria, and Xenarthra.     

Design, synthesis, and SAR of heterocycle-containing antagonists of the human CCR5 receptor for the treatment of HIV-1 infection

Replacement of the large hydantoin-indole moiety from our previous work with a variety of smaller heterocyclic analogues gave rise to potent CCR5 antagonists having binding affinity comparable to the hydantoin analogues. The synthesis, SAR, and biological profiles of this class of antagonists are described.     

Spectral shifts of mammalian ultraviolet-sensitive pigments (short wavelength-sensitive opsin 1) are associated with eye length and photic niche evolution

Retinal opsin photopigments initiate mammalian vision when stimulated by light. Most mammals possess a short wavelength-sensitive opsin 1 (SWS1) pigment that is primarily sensitive to either ultraviolet or violet light, leading to variation in colour perception across species. Despite knowledge of both ultraviolet- and violet-sensitive SWS1 classes in mammals for 25 years, the adaptive significance of this variation has not been subjected to hypothesis testing, resulting in minimal understanding of the basis for mammalian SWS1 spectral tuning evolution. Here, we gathered data on SWS1 for 403 mammal species, including novel SWS1 sequences for 97 species. Ancestral sequence reconstructions suggest that the most recent common ancestor of Theria possessed an ultraviolet SWS1 pigment, and that violet-sensitive pigments evolved at least 12 times in mammalian history. We also observed that ultraviolet pigments, previously considered to be a rarity, are common in mammals. We then used phylogenetic comparative methods to test the hypotheses that the evolution of violet-sensitive SWS1 is associated with increased light exposure, extended longevity and longer eye length. We discovered that diurnal mammals and species with longer eyes are more likely to have violet-sensitive pigments and less likely to possess UV-sensitive pigments. We hypothesize that (i) as mammals evolved larger body sizes, they evolved longer eyes, which limited transmittance of ultraviolet light to the retina due to an increase in Rayleigh scattering, and (ii) as mammals began to invade diurnal temporal niches, they evolved lenses with low UV transmittance to reduce chromatic aberration and/or photo-oxidative damage.     

Evidence for cooperative effects in the binding of polyvalent metal ions to pure phosphatidylcholine bilayer vesicle surfaces

An internal NMR monitor for the study of lanthanide ion (Ln³⁺) binding to phospholipid bilayer membranes has been developed. The dimethylphosphate anion, DMP^?, forms labile complexes with Ln³⁺ in aqueous solution and in solutions also containing bilayer dispersions. The hyperfine shift in the DMP^? resonance induced by Pr³⁺ ions has been used to determine the overall thermodynamic formation constants for the Pr(DMP)²⁺ and Pr(DMP)₂⁺ complexes: 81 (M^?1) and 349 (M^?2) at 52°C; the limiting hyperfine shift (³¹P) at 52°C is 91.5 ppm downfield. These parameters, applied to the observed DMP^? hyperfine shift in the presence of the membrane, establish both the free Pr³⁺ concentration and the amount of Pr³⁺ bound to the phospholipid surface. Extensive data for the binding of Pr³⁺ to the outer surfaces of sonicated vesicles yield a limiting hyperfine shift per Pr³⁺ of 181.6 ppm downfield for the dipalmitoylphosphatidylcholine ³¹P resonance at 52°C, clearly demonstrating that the binding stoichiometry is two DPPCs per Pr³⁺. A Hill analysis indicates that the binding data are more anti-cooperative than a realistic Langmuir isotherm, yet more cooperative than a Stern isotherm incorporating electrostatic considerations at the Debye-Hückel level. Fittings to specific models lead to a cooperative model in which tense (T) sites, with low affinity for Pr³⁺, present in the absence of metal ions, quickly give way to relaxed (R) sites (two DPPCs per site), with much higher affinity for Pr³⁺, as the amount of Pr³⁺ bound to the surface increases. The intrinsic equilibrium constants for the binding of Pr³⁺ to DPPC vesicles are 2 M^?1 and 3 000 M^?1 for the T and R sites, respectively, at 52°C. The distribution coefficient between these sites ([R]/[T]) in the absence of Ln³⁺ is 0.14 at 52°C. We picture the binding site conversion as a head-group conformational change involving mostly the choline moiety. Sketchy results for binding on the inside vesicle surface indicate that the overall affinity for Pr³⁺ is significantly greater and suggest that the site stoichiometry may be different.