An agarose-acrylamide composite native gel system suitable for separating ultra-large protein complexes

An agarose-acrylamide composite native gel (CNG) system has been developed for separating protein complexes of ultra-large molecular sizes (over 500kDa) and for analyzing protein-protein interactions in their native states. Various native gel conditions were explored and techniques were improved to facilitate the formation and performance of the CNG system. We demonstrate here that the CNG technique is capable of resolving a complex of RNA polymerase II and an associated factor from the free components, which had not been previously achieved with other methods. Furthermore, this CNG electrophoresis can be conveniently coupled to second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis for identification of protein components within discrete complexes separated during the CNG run. The CNG technique is particularly suitable for capturing dynamic protein-protein interactions as exemplified here by the formation and demonstration of RNA polymerase II-Fcp1 complex.     

Summer water use by California coastal prairie grasses: fog,drought, and community composition

Plants in the Mediterranean climate region of California typically experience summer drought conditions, but correlations between zones of frequent coastal fog inundation and certain species’ distributions suggest that water inputs from fog may influence species composition in coastal habitats. We sampled the stable H and O isotope ratios of water in non-photosynthetic plant tissue from a variety of perennial grass species and soil in four sites in northern California in order to determine the proportion of water deriving from winter rains and fog during the summer. The relationship between H and O stable isotopes from our sample sites fell to the right of the local meteoric water line (LMWL) during the summer drought, providing evidence that evaporation of water from the soil had taken place prior to the uptake of water by vegetation. We developed a novel method to infer the isotope values of water before it was subjected to evaporation in which we used experimental data to calculate the slope of the δH versus δO line versus the LMWL. After accounting for evaporation, we then used a two-source mixing model to evaluate plant usage of fog water. The model indicated that 28–66% of the water taken up by plants via roots during the summer drought came from fog rather than residual soil water from winter rain. Fog use decreased as distance from the coast increased, and there were significant differences among species in the use of fog. Rather than consistent differences in fog use by species whose distributions are limited to the coast versus those with broader distributions, species responded individualistically to summer fog. We conclude that fogwater inputs can mitigate the summer drought in coastal California for many species, likely giving an advantage to species that can use it over species that cannot.     

亚热带天然次生常绿阔叶林与杉木人工林土壤动物群落特征比较

调查了中亚热带会同林区毗邻的天然次生常绿阔叶林、一代杉木纯林和二代杉木纯林土壤动物群落特征．结果表明,采伐天然林,接着栽植杉木人工林后,土壤动物的多度和多样性均出现明显下降,而土壤动物的生物量和生产力却没有明显差别．杉木连栽对土壤动物的多度、多样性、生物量影响甚少,特别是杉木一、二代土壤动物的生产力接近相等．此项研究结果支持植被是影响土壤动物演替的一个主要原因,也暗示杉木长期连作对土壤动物群落的影响是一个非常缓慢的过程．     

Synthesis of 4-mercapto-l-lysine derivatives: Potential building blocks for sequential native chemical ligation

A general and diastereoselective synthesis of (2S, 4S)-4-mercapto-l-lysine derivative was described. The key features of this synthesis include Zn-mediated diastereoselective Reformatsky reaction and selective reduction of methyl ester with sodium borohydride. Introduction of thiol functional group on lysine side chain proved to be appropriate for dual native chemical ligation. This methodology allows to develop various 4-substituted l-lysine derivatives.     

Human T cell lymphotropic viruses (HTLV-I/II) in South America: Should it be a public health concern?

The presence of the human T cell lymphotropic virus (HTLV-I/II) in South America is well established. Its origin and spectrum in the continent still remain a matter of debate. There are signs now that HTLV-I/II was already present in the Amerindian population coming originally from Asia and that HTLV-I was also introduced with African slave trade and with immigration of individuals from endemic areas of Japan. South America has approximately 350 million inhabitants in its 13 countries. The presence of HTLV-I/II has been reported with impressive numbers in most of them and may be considered endemic in this continent. The distribution of HTLV I/II among native Amerindian populations has shown a geographic clustering of type I in the Andean highlands and Brazilian coast, while type II predominates in lowlands of South America. Although comparability between studies conducted among blood donors in different countries may be difficult, the data indicate that the viruses are also circulating among otherwise healthy individuals. Undoubtedly, HTLV-I/II infection and its related diseases should be considered a public health concern in South America and measures to prevent its spread should be emphasized.     

Obesity polymorphisms identified in genome-wide association studies interact with n-3 polyunsaturated fatty acid intake and modify the genetic association with adiposity phenotypes in Yup’ik people

n-3 Polyunsaturated fatty acids (n-3 PUFAs) have anti-obesity effects that may modulate risk of obesity, in part, through interactions with genetic factors. Genome-wide association studies (GWAS) have identified genetic variants associated with body mass index (BMI); however, the extent to which these variants influence adiposity through interactions with n-3 PUFAs remains unknown. We evaluated 10 highly replicated obesity GWAS single nucleotide polymorphisms (SNPs) for individual and cumulative associations with adiposity phenotypes in a cross-sectional sample of Yup’ik people (n = 1,073) and evaluated whether genetic associations with obesity were modulated by n-3 PUFA intake. A genetic risk score (GRS) was calculated by adding the BMI-increasing alleles across all 10 SNPs. Dietary intake of n-3 PUFAs was estimated using nitrogen stable isotope ratio (δ¹⁵N) of red blood cells, and genotype–phenotype analyses were tested in linear models accounting for familial correlations. GRS was positively associated with BMI (p = 0.012), PBF (p = 0.022), ThC (p = 0.025), and waist circumference (p = 0.038). The variance in adiposity phenotypes explained by the GRS included BMI (0.7 %), PBF (0.3 %), ThC (0.7 %), and WC (0.5 %). GRS interactions with n-3 PUFAs modified the association with adiposity and accounted for more than twice the phenotypic variation (~1–2 %), relative to GRS associations alone. Obesity GWAS SNPs contribute to adiposity in this study population of Yup’ik people and interactions with n-3 PUFA intake potentiated the risk of fat accumulation among individuals with high obesity GRS. These data suggest the anti-obesity effects of n-3 PUFAs among Yup’ik people may, in part, be dependent upon an individual’s genetic predisposition to obesity.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-013-0340-z) contains supplementary material, which is available to authorized users.     

Compositional and structural characterization of monolayers and bilayers composed of native pulmonary surfactant from wild type mice

This work comprises a structural and dynamical study of monolayers and bilayers composed of native pulmonary surfactant from mice. Spatially resolved information was obtained using fluorescence (confocal, wide field and two photon excitation) and atomic force microscopy methods. Lipid mass spectrometry experiments were also performed in order to obtain relevant information on the lipid composition of this material. Bilayers composed of mice pulmonary surfactant showed coexistence of distinct domains at room temperature, with morphologies and lateral packing resembling the coexistence of liquid ordered (lo)/liquid disordered (ld)-like phases reported previously in porcine lung surfactant. Interestingly, the molar ratio of saturated (mostly DPPC)/non-saturated phospholipid species and cholesterol measured in the innate material corresponds with that of a DOPC/DPPC/cholesterol mixture showing lo/ld phase coexistence at a similar temperature. This suggests that at quasi-equilibrium conditions, key lipid classes in this complex biological material are still able to produce the same scaffold observed in relevant but simpler model lipid mixtures. Also, robust structural and dynamical similarities between mono- and bi-layers composed of mice pulmonary surfactant were observed when the monolayers reach a surface pressure of 30 mN/m. This value is in line with theoretically predicted and recently measured surface pressures, where the monolayer–bilayer equivalence occurs in samples composed of single phospholipids. Finally, squeezed out material attached to pulmonary surfactant monolayers was observed at surface pressures near the beginning of the monolayer reversible exclusion plateau (~ 40 mN/m). Under these conditions this material adopts elongated tubular shapes and displays ordered lateral packing as indicated by spatially resolved LAURDAN GP measurements.     

Total chemical synthesis of dengue 2 virus capsid protein via native chemical ligation: Role of the conserved salt-bridge

The dengue capsid protein C is a highly basic alpha-helical protein of ～100 amino acid residues that forms an emphipathic homodimer to encapsidate the viral genome and to interact with viral membranes. The solution structure of dengue 2 capsid protein C (DEN2C) has been determined by NMR spectroscopy, revealing a large dimer interface formed almost exclusively by hydrophobic residues. The only acidic residue (Glu87) conserved in the capsid proteins of all four serotypes of dengue virus forms a salt bridge with the side chains of Lys45 and Arg55′. To understand the structural and functional significance of this conserved salt bridge, we chemically synthesized an N-terminally truncated form of DEN2C (^WTDEN2C) and its salt bridge-void analog ^E87ADEN2C using the native chemical ligation technique developed by Kent and colleagues. Comparative biochemical and biophysical studies of these two synthetic proteins using circular dichroism spectroscopy, fluorescence polarization, protein thermal denaturation, and proteolytic susceptibility assay demonstrated that the conserved salt bridge contributed to DEN2C dimerization and stability as well as its resistance to proteolytic degradation. Our work provided insight into the role of a fully conserved structural element of the dengue capsid protein C and paved the way for additional functional studies of this important viral protein.     

DNA-instructed acyl transfer reactions for the synthesis of bioactive peptides

We present a method which allows for the translation of nucleic acid information into the output of molecules that interfere with disease-related protein-protein interactions. The method draws upon a nucleic acid-templated reaction, in which adjacent binding of reactive conjugates triggers the transfer of an aminoacyl or peptidyl group from a donating thioester-linked PNA-peptide hybrid to a peptide-PNA acceptor. We evaluated the influence of conjugate structures on reactivity and sequence specificity. The DNA-triggered peptide synthesis proceeded sequence specifically and showed catalytic turnover in template. The affinity of the formed peptide conjugates for the BIR3 domain of the X-linked inhibitor of apoptosis protein (XIAP) is discussed.