Genetic Variation and Population Structure in Native Americans

We examined genetic diversity and population structure in the American landmass using 678 autosomal microsatellite markers genotyped in 422 individuals representing 24 Native American populations sampled from North, Central, and South America. These data were analyzed jointly with similar data available in 54 other indigenous populations worldwide, including an additional five Native American groups. The Native American populations have lower genetic diversity and greater differentiation than populations from other continental regions. We observe gradients both of decreasing genetic diversity as a function of geographic distance from the Bering Strait and of decreasing genetic similarity to Siberians-signals of the southward dispersal of human populations from the northwestern tip of the Americas. We also observe evidence of: (1) a higher level of diversity and lower level of population structure in western South America compared to eastern South America, (2) a relative lack of differentiation between Mesoamerican and Andean populations, (3) a scenario in which coastal routes were easier for migrating peoples to traverse in comparison with inland routes, and (4) a partial agreement on a local scale between genetic similarity and the linguistic classification of populations. These findings offer new insights into the process of population dispersal and differentiation during the peopling of the Americas.

Structure and function of the glucose PTS transporter from Escherichia coli

The glucose transporter IICB of the Escherichia coli phosphotransferase system (PTS) consists of a polytopic membrane domain (IIC) responsible for substrate transport and a hydrophilic C-terminal domain (IIB) responsible for substrate phosphorylation. We have overexpressed and purified a triple mutant of IIC (mut-IIC), which had recently been shown to be suitable for crystallization purposes. Mut-IIC was homodimeric as determined by blue native-PAGE and gel-filtration, and had an eyeglasses-like structure as shown by negative-stain transmission electron microscopy (TEM) and single particle analysis. Glucose binding and transport by mut-IIC, mut-IICB and wildtype-IICB were compared with scintillation proximity and in vivo transport assays. Binding was reduced and transport was impaired by the triple mutation. The scintillation proximity assay allowed determination of substrate binding, affinity and specificity of wildtype-IICB by a direct method. 2D crystallization of mut-IIC yielded highly-ordered tubular crystals and made possible the calculation of a projection structure at 12Å resolution by negative-stain TEM. Immunogold labeling TEM revealed the sidedness of the tubular crystals, and high-resolution atomic force microscopy the surface structure of mut-IIC. This work presents the structure of a glucose PTS transporter at the highest resolution achieved so far and sets the basis for future structural studies.

Structure determination of channel and transport proteins by high-resolution microscopy techniques

High-resolution microscopy techniques provide a plethora of information on biological structures from the cellular level down to the molecular level. In this review, we present the unique capabilities of transmission electron and atomic force microscopy to assess the structure, oligomeric state, function and dynamics of channel and transport proteins in their native environment, the lipid bilayer. Most importantly, membrane proteins can be visualized in the frozen-hydrated state and in buffer solution by cryo-transmission electron and atomic force microscopy, respectively. We also illustrate the potential of the scintillation proximity assay to study substrate binding of detergent-solubilized transporters prior to crystallization and structural characterization.

Novel sst2-selective somatostatin agonists. Three-dimensional consensus structure by NMR

The 3D NMR structures of six octapeptide agonist analogues of somatostatin (SRIF) in the free form are described. These analogues, with the basic sequence H-DPhe/Phe2-c[Cys3-Xxx7-DTrp8-Lys9-Thr10-Cys14]-Thr-NH2 (the numbering refers to the position in native SRIF), with Xxx7 being Ala/Aph, exhibit potent and highly selective binding to human SRIF type 2 (sst2) receptors. The backbone of these sst2-selective analogues have the usual type-II' beta-turn reported in the literature for sst2/3/5-subtype-selective analogues. Correlating the biological results and NMR studies led to the identification of the side chains of DPhe2, DTrp8, and Lys9 as the necessary components of the sst2 pharmacophore. This is the first study to show that the aromatic ring at position 7 (Phe7) is not critical for sst2 binding and that it plays an important role in sst3 and sst5 binding. This pharmacophore is, therefore, different from that proposed by others for sst2/3/5 analogues.

Three-dimensional consensus structure of sst2-selective somatostatin (SRIF) antagonists by NMR

The three-dimensional NMR structures of seven octapeptide analogs of somatostatin (SRIF), based on octreotide, with the basic sequence H-Cpa/Phe2-c[DCys3-Xxx7-DTrp/DAph(Cbm)8-Lys9-Thr10-Cys14]-Yyy-NH2 (the numbering refers to the position in native SRIF), with Xxx7 being Aph(Cbm)/Tyr/Agl(NMe,benzoyl) and Yyy being Nal/DTyr/Thr, are presented here. Most of these analogs exhibit potent and highly selective binding to sst2 receptors, and all of the analogs are antagonists inhibiting receptor signaling. Based on their consensus 3D structure, the pharmacophore of the sst2-selective antagonist has been defined. The pharmacophore involves the side chains of Cpa2, DTrp/DAph(Cbm)8, and Lys9, with the backbone for most of the sst2-selective antagonists comprised a Type-II' beta-turn. Hence, the sst2-selective antagonist pharmacophore is very similar to the sst2-selective agonist pharmacophore previously described.

Projection structure of DtpD (YbgH), a prokaryotic member of the peptide transporter family

Cellular uptake of di- and tripeptides has been characterized in numerous organisms, and various transporters have been identified. In contrast, structural information on peptide transporters is very sparse. Here, we have cloned, overexpressed, purified, and biochemically characterized DtpD (YbgH) from Escherichia coli, a prokaryotic member of the peptide transporter family. Its homologues in mammals, PEPT1 (SLC15A1) and PEPT2 (SLC15A2), not only transport peptides but also are of relevance for uptake of drugs as they accept a large spectrum of peptidomimetics such as beta-lactam antibiotics, antivirals, peptidase inhibitors, and others as substrates. Uptake experiments indicated that DtpD functions as a canonical peptide transporter and is, therefore, a valid model for structural studies of this family of proteins. Blue native polyacrylamide gel electrophoresis, gel filtration, and transmission electron microscopy of single-DtpD particles suggest that the transporter exists in a monomeric form when solubilized in detergent. Two-dimensional crystallization of DtpD yielded first tubular crystals that allowed the determination of a projection structure at better than 19 A resolution. This structure of DtpD represents the first structural view of a member of the peptide transporter family.

Inversion, Obviation, and Animacy in Native Languages of the Americas: Elements for a Cross-linguistic Survey

Native languages of the Americas whose predicate and clause structure reflect nominal hierarchies show an interesting range of structural diversity not only with respect to morphological makeup of their predicates and arguments but also with respect to the factors governing obviation status. The present article maps part of such diversity. The sample surveyed here includes languages with some sort of nonlocal (third person acting on third person) direction-marking system.

Visualization of cell microtubules in their native state.

BACKGROUND INFORMATION Over the past decades, cryo-electron microscopy of vitrified specimens has yielded a detailed understanding of the tubulin and microtubule structures of samples reassembled in vitro from purified components. However, our knowledge of microtubule structure in vivo remains limited by the chemical treatments commonly used to observe cellular architecture using electron microscopy. RESULTS We used cryo-electron microscopy and cryo-electron tomography of vitreous sections to investigate the ultrastructure of microtubules in their cellular context. Vitreous sections were obtained from organotypic slices of rat hippocampus and from Chinese-hamster ovary cells in culture. Microtubules revealed their protofilament ultrastructure, polarity and, in the most favourable cases, molecular details comparable with those visualized in three-dimensional reconstructions of microtubules reassembled in vitro from purified tubulin. The resolution of the tomograms was estimated to be approx. 4 nm, which enabled the detection of luminal particles of approx. 6 nm in diameter inside microtubules. CONCLUSIONS The present study provides a first step towards a description of microtubules, in addition to other macromolecular assemblies, in an unperturbed cellular context at the molecular level. As the resolution appears to be similar to that obtainable with plunge-frozen samples, it should allow for the in vivo identification of larger macromolecular assemblies in vitreous sections of whole cells and tissues.

Direct visualization of the outer membrane of mycobacteria and corynebacteria in their native state.

The cell envelope of mycobacteria, which include the causative agents of tuberculosis and leprosy, is crucial for their success as pathogens. Despite a continued strong emphasis on identifying the multiple chemical components of this envelope, it has proven difficult to combine its components into a comprehensive structural model, primarily because the available ultrastructural data rely on conventional electron microscopy embedding and sectioning, which are known to induce artifacts. The existence of an outer membrane bilayer has long been postulated but has never been directly observed by electron microscopy of ultrathin sections. Here we have used cryo-electron microscopy of vitreous sections (CEMOVIS) to perform a detailed ultrastructural analysis of three species belonging to the Corynebacterineae suborder, namely, Mycobacterium bovis BCG, Mycobacterium smegmatis, and Corynebacterium glutamicum, in their native state. We provide new information that accurately describes the different layers of the mycobacterial cell envelope and challenges current models of the organization of its components. We show a direct visualization of an outer membrane, analogous to that found in gram-negative bacteria, in the three bacterial species examined. Furthermore, we demonstrate that mycolic acids, the hallmark of mycobacteria and related genera, are essential for the formation of this outer membrane. In addition, a granular layer and a low-density zone typifying the periplasmic space of gram-positive bacteria are apparent in CEMOVIS images of mycobacteria and corynebacteria. Based on our observations, a model of the organization of the lipids in the outer membrane is proposed. The architecture we describe should serve as a reference for future studies to relate the structure of the mycobacterial cell envelope to its function.