Probing the role of packing specificity in protein design

By using a protein-design algorithm that quantitatively considers side-chain packing, the effect of specific steric constraints on protein design was assessed in the core of the streptococcal protein G β1 domain. The strength of packing constraints used in the design was varied, resulting in core sequences that reflected differing amounts of packing specificity. The structural flexibility and stability of several of the designed proteins were experimentally determined and showed a trend from well-ordered to highly mobile structures as the degree of packing specificity in the design decreased. This trend both demonstrates that the inclusion of specific packing interactions is necessary for the design of native-like proteins and defines a useful range of packing specificity for the design algorithm. In addition, an analysis of the modeled protein structures suggested that penalizing for exposed hydrophobic surface area can improve design performance.

The role of Wolbachia bacteria in reproductive incompatibilities and hybrid zones of Diabrotica beetles and Gryllus crickets

A rickettsial bacterium in the genus Wolbachia is the cause of a unidirectional reproductive incompatibility observed between two major beetle pests of maize, the western corn rootworm, Diabrotica virgifera virgifera, and the Mexican corn rootworm, D. v. zeae. These subspecies are allopatric except for two known regions of sympatry in Texas and Mexico. We demonstrate that populations of D. v. virgifera, with the exception of two populations in southern Arizona, are infected with a strain of Wolbachia. Populations of D. v. zeae are not infected. Treatment of D. v. virgifera with tetracycline eliminated the Wolbachia and removed the reproductive incompatibility. Similar patterns of reproductive incompatibility exist among taxa of the cricket genus Gryllus. Gryllus assimilis, G. integer, G. ovisopis, G. pennsylvanicus, and G. rubens are infected with Wolbachia whereas G. firmus is usually not. Populations of G. rubens and G. ovisopis carry the same Wolbachia strain, which is distinct from that of G. integer. G. pennsylvanicus is infected with two Wolbachia strains, that found in G. rubens and one unique to G. pennsylvanicus. Moreover, a proportion of G. pennsylvanicus individuals harbors both strains. Wolbachia may have influenced speciation in some members of the genus Gryllus by affecting the degree of hybridization between species. Given that Wolbachia infections are relatively common in insects, it is likely that other insect hybrid zones may be influenced by infections with Wolbachia.

Genetic instability of cancer cells is proportional to their degree of aneuploidy

Genetic and phenotypic instability are hallmarks of cancer cells, but their cause is not clear. The leading hypothesis suggests that a poorly defined gene mutation generates genetic instability and that some of many subsequent mutations then cause cancer. Here we investigate the hypothesis that genetic instability of cancer cells is caused by aneuploidy, an abnormal balance of chromosomes. Because symmetrical segregation of chromosomes depends on exactly two copies of mitosis genes, aneuploidy involving chromosomes with mitosis genes will destabilize the karyotype. The hypothesis predicts that the degree of genetic instability should be proportional to the degree of aneuploidy. Thus it should be difficult, if not impossible, to maintain the particular karyotype of a highly aneuploid cancer cell on clonal propagation. This prediction was confirmed with clonal cultures of chemically transformed, aneuploid Chinese hamster embryo cells. It was found that the higher the ploidy factor of a clone, the more unstable was its karyotype. The ploidy factor is the quotient of the modal chromosome number divided by the normal number of the species. Transformed Chinese hamster embryo cells with a ploidy factor of 1.7 were estimated to change their karyotype at a rate of about 3% per generation, compared with 1.8% for cells with a ploidy factor of 0.95. Because the background noise of karyotyping is relatively high, the cells with low ploidy factor may be more stable than our method suggests. The karyotype instability of human colon cancer cell lines, recently analyzed by Lengnauer et al. [Lengnauer, C., Kinzler, K. W. & Vogelstein, B. (1997) Nature (London) 386, 623–627], also corresponds exactly to their degree of aneuploidy. We conclude that aneuploidy is sufficient to explain genetic instability and the resulting karyotypic and phenotypic heterogeneity of cancer cells, independent of gene mutation. Because aneuploidy has also been proposed to cause cancer, our hypothesis offers a common, unique mechanism of altering and simultaneously destabilizing normal cellular phenotypes.

The N-terminal domain of a K+ channel β subunit increases the rate of C-type inactivation from the cytoplasmic side of the channel

Voltage-gated K+ channels are complexes of membrane-bound, ion-conducting α and cytoplasmic ancillary (β) subunits. The primary physiologic effect of coexpression of α and β subunits is to increase the intrinsic rate of inactivation of the α subunit. For one β subunit, Kvβ1.1, inactivation is enhanced through an N-type mechanism. A second β subunit, Kvβ1.2, has been shown to increase inactivation, but through a distinct mechanism. Here we show that the degree of enhancement of Kvβ1.2 inactivation is dependent on the amino acid composition in the pore mouth of the α subunit and the concentration of extracellular K+. Experimental conditions that promote C-type inactivation also enhance the stimulation of inactivation by Kvβ1.2, showing that this β subunit directly stimulates C-type inactivation. Chimeric constructs containing just the nonconserved N-terminal region of Kvβ1.2 fused with an α subunit behave in a similar fashion to coexpressed Kvβ1.2 and α subunit. This shows that it is the N-terminal domain of Kvβ1.2 that mediates the increase in C-type inactivation from the cytoplasmic side of the pore. We propose a model whereby the N terminus of Kvβ1.2 acts as a weakly binding “ball” domain that associates with the intracellular vestibule of the α subunit to effect a conformational change leading to enhancement of C-type inactivation.

TectoRNA: modular assembly units for the construction of RNA nano-objects

Structural information on complex biological RNA molecules can be exploited to design tectoRNAs or artificial modular RNA units that can self-assemble through tertiary interactions thereby forming nanoscale RNA objects. The selective interactions of hairpin tetraloops with their receptors can be used to mediate tectoRNA assembly. Here we report on the modulation of the specificity and the strength of tectoRNA assembly (in the nanomolar to micromolar range) by variation of the length of the RNA subunits, the nature of their interacting motifs and the degree of flexibility of linker regions incorporated into the molecules. The association is also dependent on the concentration of magnesium. Monitoring of tectoRNA assembly by lead(II) cleavage protection indicates that some degree of structural flexibility is required for optimal binding. With tectoRNAs one can compare the binding affinities of different tertiary motifs and quantify the strength of individual interactions. Furthermore, in analogy to the synthons used in organic chemistry to synthesize more complex organic compounds, tectoRNAs form the basic assembly units for constructing complex RNA structures on the nanometer scale. Thus, tectoRNA provides a means for constructing molecular scaffoldings that organize functional modules in three-dimensional space for a wide range of applications.

The evolution of RNA viruses: A population genetics view

RNA viruses are excellent experimental models for studying evolution under the theoretical framework of population genetics. For a proper justification of this thesis we have introduced some properties of RNA viruses that are relevant for studying evolution. On the other hand, population genetics is a reductionistic theory of evolution. It does not consider or make simplistic assumptions on the transformation laws within and between genotypic and phenotypic spaces. However, such laws are minimized in the case of RNA viruses because the phenotypic space maps onto the genotypic space in a much more linear way than on higher DNA-based organisms. Under experimental conditions, we have tested the role of deleterious and beneficial mutations in the degree of adaptation of vesicular stomatitis virus (VSV), a nonsegmented virus of negative strand. We also have studied how effective population size, initial genetic variability in populations, and environmental heterogeneity shapes the impact of mutations in the evolution of vesicular stomatitis virus. Finally, in an integrative attempt, we discuss pros and cons of the quasispecies theory compared with classic population genetics models for haploid organisms to explain the evolution of RNA viruses.

Sorting and directed transport of membrane proteins during development of hippocampal neurons in culture

Hippocampal neurons in culture develop morphological polarity in a sequential pattern; axons form before dendrites. Molecular differences, particularly those of membrane proteins, underlie the functional polarity of these domains, yet little is known about the temporal relationship between membrane protein polarization and morphological polarization. We took advantage of viral expression systems to determine when during development the polarization of membrane proteins arises. All markers were unpolarized in neurons before axonogenesis. In neurons with a morphologically distinguishable axon, even on the first day in culture, both axonal and dendritic proteins were polarized. The degree of polarization at these early stages was somewhat less than in mature cells and varied from cell to cell. The cellular mechanism responsible for the polarization of the dendritic marker protein transferrin receptor (TfR) in mature cells centers on directed transport to the dendritic domain. To examine the relationship between cell surface polarization and transport, we assessed the selectivity of transport by live cell imaging. TfR-green fluorescent protein-containing vesicles were already preferentially transported into dendrites at 2 days, the earliest time point we could measure. The selectivity of transport also varied somewhat among cells, and the amount of TfR-green fluorescent protein fluorescence on intracellular structures within the axon correlated with the amount of cell surface expression. This observation implies that selective microtubule-based transport is the primary mechanism that underlies the polarization of TfR on the cell surface. By 5 days in culture, the extent of polarization on the cell surface and the selectivity of transport reached mature levels.

Dynamic friction and the origin of the complexity of earthquake sources.

We study a simple antiplane fault of finite length embedded in a homogeneous isotropic elastic solid to understand the origin of seismic source heterogeneity in the presence of nonlinear rate- and state-dependent friction. All the mechanical properties of the medium and friction are assumed homogeneous. Friction includes a characteristic length that is longer than the grid size so that our models have a well-defined continuum limit. Starting from a heterogeneous initial stress distribution, we apply a slowly increasing uniform stress load far from the fault and we simulate the seismicity for a few 1000 events. The style of seismicity produced by this model is determined by a control parameter associated with the degree of rate dependence of friction. For classical friction models with rate-independent friction, no complexity appears and seismicity is perfectly periodic. For weakly rate-dependent friction, large ruptures are still periodic, but small seismicity becomes increasingly nonstationary. When friction is highly rate-dependent, seismicity becomes nonperiodic and ruptures of all sizes occur inside the fault. Highly rate-dependent friction destabilizes the healing process producing premature healing of slip and partial stress drop. Partial stress drop produces large variations in the state of stress that in turn produce earthquakes of different sizes. Similar results have been found by other authors using the Burridge and Knopoff model. We conjecture that all models in which static stress drop is only a fraction of the dynamic stress drop produce stress heterogeneity.

Mechanisms underlying the sensitivity of songbird forebrain neurons to temporal order.

Neurons in the songbird forebrain area HVc (hyperstriatum ventrale pars caudale or high vocal center) are sensitive to the temporal structure of the bird's own song and are capable of integrating auditory information over a period of several hundred milliseconds. Extracellular studies have shown that the responses of some HVc neurons depend on the combination and temporal order of syllables from the bird's own song, but little is known about the mechanisms underlying these response properties. To investigate these mechanisms, we recorded intracellular responses to a set of auditory stimuli designed to assess the degree of dependence of the responses on temporal context. This report provides evidence that HVc neurons encode information about temporal structure by using a variety of mechanisms including syllable-specific inhibition, excitatory postsynaptic potentials with a range of different time courses, and burst-firing nonlinearity. The data suggest that the sensitivity of HVc neurons to temporal combinations of syllables results from the interactions of several cells and does not arise in a single step from afferent inputs alone.

Toward an outline of the topography of a realistic protein-folding funnel.

Experimental information on the structure and dynamics of molten globules gives estimates for the energy landscape's characteristics for folding highly helical proteins, when supplemented by a theory of the helix-coil transition in collapsed heteropolymers. A law of corresponding states relating simulations on small lattice models to real proteins possessing many more degrees of freedom results. This correspondence reveals parallels between "minimalist" lattice results and recent experimental results for the degree of native character of the folding transition state and molten globule and also pinpoints the needs of further experiments.

An evolutive approach for the delineation of local labour markets

This paper presents a new approach to the delineation of local labour markets based on evolutionary computation. The main objective is the regionalisation of a given territory into functional regions based on commuting flows. According to the relevant literature, such regions are defined so that (a) their boundaries are rarely crossed in daily journeys to work, and (b) a high degree of intra-area movement exists. This proposal merges municipalities into functional regions by maximizing a fitness function that measures aggregate intra-region interaction under constraints of inter-region separation and minimum size. Real results are presented based on the latest database from the Census of Population in the Region of Valencia. Comparison between the results obtained through the official method which currently is most widely used (that of British Travel-to-Work Areas) and those from our approach is also presented, showing important improvements in terms of both the number of different market areas identified that meet the statistical criteria and the degree of aggregate intra-market interaction.

Tourism potential analysis of the castles of Vinalopó, Spain: an opportunity for social and tourist development of cultural heritage

Within the overall framework of the renewal process of coastal tourist destinations, cultural heritage has frequently been used as a key argument for the introduction and development of strategies for the diversification and differentiation of the traditional tourist product based on sun and sand. This is the situation of the province of Alicante, identified with the Costa Blanca geotourism brand, where there are important economic issues that could contribute to the renewal of this coastal tourist destination. One of the most significant heritage values of this space consists of a series of medieval fortresses located along the axis of the Vinalopó River, which has acted since prehistoric times as a natural route from within the provincial mainland to the coast. It is precisely the argument of this historical, territorial and landscape content that has been used repeatedly in recent years to develop initiatives aimed at the creation of a tourist product, currently inexistent, based on the route of the castles of Vinalopó. This communication aims to analyse the degree of tourism potential of the fortresses located in the towns of Biar, Banyeres de Mariola, Sax, Villena, Novelda, Elda, Petrer and Elche, which constitute the core of municipalities where these medieval fortresses are located, finally pointing out some proposals for the creation of a heritage tourism product.

The Composition of Holocene Aeolianites; Western Eyre Peninsula, Great Australian Bight, South Australia

The Australian southern continental margin is the world’s largest site of cool-water carbonate deposition, and the Great Australian Bight is its largest sector. The Eyre Peninsula is fringed by coastal beaches with aeolianites and marks the eastern edge of the Great Australian Bight. Five shoreline transects of varying lengths spanned a 150km longitudinal distance and at each the intertidal, beach, dune and secondary dune environments were sampled, for a total of 18 samples. Sediments are a mixture of modern, relict, and Cenozoic carbonates, and quartz grains. Carbonate aeolianites on the western Eyre Peninsula are mostly composed of modern carbonate grains: predominantly molluscs (23-33%) and benthic foraminifera (10-26%), locally abundant coralline algae (3-28%), echinoids (2-22%), and bryozoans (2-14%). Cenozoic grain abundance ranges from 1-6% whereas relict grain abundance ranges from 0-17%. A southward increase in bryozoan particles correlates with a nutrient element abundance and decrease in temperature due to a large seasonal coastal upwelling system that drives 2-3 major upwelling events per year, bringing cold, nutrient rich, Sub-Antarctic Surface Water (<12°C) onto the shelf. In southern, mostly wind protected locations, the beach and dune sediment compositions are similar, indicating that wind energy has successfully carried all sediment components of the beach into the adjacent dunes. In northern, exposed locations, the composition is not the same everywhere, and trends indicate that relative wind energy has the ability to impact grain composition through preferential wind transport. Aeolianite composition is therefore a function of both upwelling and the degree of coastal exposure.

Aristotelian predicables, universality and realism: the logic of comparison in topics as denying the view that Aristotle was a realist

Aristotle is reportedly held to have been a Moderate Realist in that he would maintain that a concept derives from an act of grasping a mind-independent universal object that exists somehow inside of the many different things which the concept is predicated of. As far as a universal is independent of mind, it would stand for the proper object of a concept that subsumes a given number of things as its own instantiations. But we claim that Aristotle rejected such a view and instead did perceive and comprehend universality as a feature of thought rather than as a feature of reality in its own right. As showed in the chapters of Topics regarding the so-called logic of comparison (with the support of Albert the Great’s commentary), each predicate can be more or less consistent with the attribute of the subject of which it may be predicated. Both essential and accidental attributes assume a definite degree of being related to the degree of belonging to substance. Unlike particular things, the universality of a concept is to be understood always in comparison with another concept according to a hierarchy of predicates in terms of universality degree arranged by comparative terms such as ‘more’, ‘less’, and ‘likewise’. What is really mind-independent are the truth conditions which make a universal true when exclusively referring to a set of things identically meant by the same predicate whose universality is given by the place occupied in the hierarchy of predicates.

The assessment of the level of knowledge about dietary fibre among the portuguese population

The aim of this work was to assess the degree of knowledge about dietary fibre (DF), as influenced by factors such as age, gender, level of education and living environment. For this, a descriptive cross- sectional study was undertaken by means of a questionnaire and the total number of respondents was 382. The results revealed that the participants had a not satisfying global level of knowledge (3.59 ± 0.64, on a scale from 1 to 5). The overall knowledge was found lower for young adults aged 19 to 30 years, when compared to adults aged over 31 and up to 65 years, being this difference statistically significant (p=0.030). However, no statistical significant differences were found between genders, levels of education or living environments. The low level of knowledge demonstrated by the enquired alerts for the need to plan some additional actions to further inform the Portuguese population about dietary fibre.