Native protein sequences are close to optimal for their structures

How large is the volume of sequence space that is compatible with a given protein structure? Starting from random sequences, low free energy sequences were generated for 108 protein backbone structures by using a Monte Carlo optimization procedure and a free energy function based primarily on Lennard–Jones packing interactions and the Lazaridis–Karplus implicit solvation model. Remarkably, in the designed sequences 51% of the core residues and 27% of all residues were identical to the amino acids in the corresponding positions in the native sequences. The lowest free energy sequences obtained for ensembles of native-like backbone structures were also similar to the native sequence. Furthermore, both the individual residue frequencies and the covariances between pairs of positions observed in the very large SH3 domain family were recapitulated in core sequences designed for SH3 domain structures. Taken together, these results suggest that the volume of sequence space optimal for a protein structure is surprisingly restricted to a region around the native sequence.

A molecular analysis of dietary diversity for three archaic Native Americans

DNA was extracted from three fecal samples, more than 2,000 years old, from Hinds Cave, Texas. Amplification of human mtDNA sequences showed their affiliation with contemporary Native Americans, while sequences from pronghorn antelope, bighorn sheep, and cottontail rabbit allowed these animals to be identified as part of the diet of these individuals. Furthermore, amplification of chloroplast DNA sequences identified eight different plants as dietary elements. These archaic humans consumed 2–4 different animal species and 4–8 different plant species during a short time period. The success rate for retrieval of DNA from paleofeces is in strong contrast to that from skeletal remains where the success rate is generally low. Thus, human paleofecal remains represent a source of ancient DNA that significantly complements and may in some cases be superior to that from skeletal tissue.

Total synthesis of cytochrome b562 by native chemical ligation using a removable auxiliary

We have completed the total chemical synthesis of cytochrome b562 and an axial ligand analogue, [SeMet7]cyt b562, by thioester-mediated chemical ligation of unprotected peptide segments. A novel auxiliary-mediated native chemical ligation that enables peptide ligation to be applied to protein sequences lacking cysteine was used. A cleavable thiol-containing auxiliary group, 1-phenyl-2-mercaptoethyl, was added to the α-amino group of one peptide segment to facilitate amide bond-forming ligation. The amine-linked 1-phenyl-2-mercaptoethyl auxiliary was stable to anhydrous hydrogen fluoride used to cleave and deprotect peptides after solid-phase peptide synthesis. Following native chemical ligation with a thioester-containing segment, the auxiliary group was cleanly removed from the newly formed amide bond by treatment with anhydrous hydrogen fluoride, yielding a full-length unmodified polypeptide product. The resulting polypeptide was reconstituted with heme and folded to form the functional protein molecule. Synthetic wild-type cyt b562 exhibited spectroscopic and electrochemical properties identical to the recombinant protein, whereas the engineered [SeMet7]cyt b562 analogue protein was spectroscopically and functionally distinct, with a reduction potential shifted by ≈45 mV. The use of the 1-phenyl-2-mercaptoethyl removable auxiliary reported here will greatly expand the applicability of total protein synthesis by native chemical ligation of unprotected peptide segments.

4-Coumarate:Coenzyme A Ligase in Hybrid Poplar1 : Properties of Native Enzymes, cDNA Cloning, and Analysis of Recombinant Enzymes

The enzyme 4-coumarate:coenzyme A ligase (4CL) is important in providing activated thioester substrates for phenylpropanoid natural product biosynthesis. We tested different hybrid poplar (Populus trichocarpa × Populus deltoides) tissues for the presence of 4CL isoforms by fast-protein liquid chromatography and detected a minimum of three 4CL isoforms. These isoforms shared similar hydroxycinnamic acid substrate-utilization profiles and were all inactive against sinapic acid, but instability of the native forms precluded extensive further analysis. 4CL cDNA clones were isolated and grouped into two major classes, the predicted amino acid sequences of which were 86% identical. Genomic Southern blots showed that the cDNA classes represent two poplar 4CL genes, and northern blots provided evidence for their differential expression. Recombinant enzymes corresponding to the two genes were expressed using a baculovirus system. The two recombinant proteins had substrate utilization profiles similar to each other and to the native poplar 4CL isoforms (4-coumaric acid > ferulic acid > caffeic acid; there was no conversion of sinapic acid), except that both had relatively high activity toward cinnamic acid. These results are discussed with respect to the role of 4CL in the partitioning of carbon in phenylpropanoid metabolism.

The folding mechanism of larger model proteins: role of native structure.

The folding mechanism of a 125-bead heteropolymer model for proteins is investigated with Monte Carlo simulations on a cubic lattice. Sequences that do and do not fold in a reasonable time are compared. The overall folding behavior is found to be more complex than that of models for smaller proteins. Folding begins with a rapid collapse followed by a slow search through the semi-compact globule for a sequence-dependent stable core with about 30 out of 176 native contacts which serves as the transition state for folding to a near-native structure. Efficient search for the core is dependent on structural features of the native state. Sequences that fold have large amounts of stable, cooperative structure that is accessible through short-range initiation sites, such as those in anti-parallel sheets connected by turns. Before folding is completed, the system can encounter a second bottleneck, involving the condensation and rearrangement of surface residues. Overly stable local structure of the surface residues slows this stage of the folding process. The relation of the results from the 125-mer model studies to the folding of real proteins is discussed.

Identification of pentosidine as a native structure for advanced glycation end products in beta-2-microglobulin-containing amyloid fibrils in patients with dialysis-related amyloidosis.

beta-2-Microglobulin (beta-2m) is a major constituent of amyloid fibrils in patients with dialysis-related amyloidosis (DRA). Recently, we found that the pigmented and fluorescent adducts formed nonenzymatically between sugar and protein, known as advanced glycation end products (AGEs), were present in beta-2m-containing amyloid fibrils, suggesting the possible involvement of AGE-modified beta-2m in bone and joint destruction in DRA. As an extension of our search for the native structure of AGEs in beta-2m of patients with DRA, the present study focused on pentosidine, a fluorescent cross-linked glycoxidation product. Determination by both HPLC assay and competitive ELISA demonstrated a significant amount of pentosidine in amyloid-fibril beta-2m from long-term hemodialysis patients with DRA, and the acidic isoform of beta-2m in the serum and urine of hemodialysis patients. A further immunohistochemical study revealed the positive immunostaining for pentosidine and immunoreactive AGEs and beta-2m in macrophage-infiltrated amyloid deposits of long-term hemodialysis patients with DRA. These findings implicate a potential link of glycoxidation products in long-lived beta-2m-containing amyloid fibrils to the pathogenesis of DRA.

Soil quality response to land-use change for sugarcane expansion in Brazil

Globally, increasing demands for biofuels have intensified the rate of land-use change (LUC) for expansion of bioenergy crops. In Brazil, the world\'s largest sugarcane-ethanol producer, sugarcane area has expanded by 35% (3.2 Mha) in the last decade. Sugarcane expansion has resulted in extensive pastures being subjected to intensive mechanization and large inputs of agrochemicals, which have direct implications on soil quality (SQ). We hypothesized that LUC to support sugarcane expansion leads to overall SQ degradation. To test this hypothesis we conducted a field-study at three sites in the central-southern region, to assess the SQ response to the primary LUC sequence (i.e., native vegetation to pasture to sugarcane) associated to sugarcane expansion in Brazil. At each land use site undisturbed and disturbed soil samples were collected from the 0-10, 10-20 and 20-30 cm depths. Soil chemical and physical attributes were measured through on-farm and laboratory analyses. A dataset of soil biological attributes was also included in this study. Initially, the LUC effects on each individual soil indicator were quantified. Afterward, the LUC effects on overall SQ were assessed using the Soil Management Assessment Framework (SMAF). Furthermore, six SQ indexes (SQI) were developed using approaches with increasing complexity. Our results showed that long-term conversion from native vegetation to extensive pasture led to soil acidification, significant depletion of soil organic carbon (SOC) and macronutrients [especially phosphorus (P)] and severe soil compaction, which creates an unbalanced ratio between water- and air-filled pore space within the soil and increases mechanical resistance to root growth. Conversion from pasture to sugarcane improved soil chemical quality by correcting for acidity and increasing macronutrient levels. Despite those improvements, most of the P added by fertilizer accumulated in less plant-available P forms, confirming the key role of organic P has in providing available P to plants in Brazilian soils. Long-term sugarcane production subsequently led to further SOC depletions. Sugarcane production had slight negative impacts on soil physical attributes compared to pasture land. Although tillage performed for sugarcane planting and replanting alleviates soil compaction, our data suggested that the effects are short-term with persistent, reoccurring soil consolidation that increases erosion risk over time. These soil physical changes, induced by LUC, were detected by quantitative soil physical properties as well as by visual evaluation of soil structure (VESS), an on-farm and user-friendly method for evaluating SQ. The SMAF efficiently detected overall SQ response to LUC and it could be reliably used under Brazilian soil conditions. Furthermore, since all of the SQI values developed in this study were able to rank SQ among land uses. We recommend that simpler and more cost-effective SQI strategies using a small number of carefully chosen soil indicators, such as: pH, P, K, VESS and SOC, and proportional weighting within of each soil sectors (chemical, physical and biological) be used as a protocol for SQ assessments in Brazilian sugarcane areas. The SMAF and SQI scores suggested that long-term conversion from native vegetation to extensive pasture depleted overall SQ, driven by decreases in chemical, physical and biological indicators. In contrast, conversion from pasture to sugarcane had no negative impacts on overall SQ, mainly because chemical improvements offset negative impacts on biological and physical indicators. Therefore, our findings can be used as scientific base by farmers, extension agents and public policy makers to adopt and develop management strategies that sustain and/or improving SQ and the sustainability of sugarcane production in Brazil.

Identifying sociodemographic differences in Intimate Partner Violence among immigrant and native women in Spain: a cross-sectional study

Objectives: To analyze whether sociodemographics and social support have a different or similar effect on the likelihood of Intimate Partner Violence in immigrants and natives, and to estimate prevalences and associations between different types of IPV depending on women's birthplace. Methods: Cross-sectional study of 10,048 women (18–70 years) attending primary healthcare in Spain (2006–2007). Outcome: Current Intimate Partner Violence (psychological, physical and both). Sociodemographics and social support were considered first as explicative and later as control variables. Results: Similar Intimate Partner Violence sociodemographic and social support factors were observed among immigrants and natives. However, these associations were stronger among immigrants, except in the case of poor social support (adjusted odds ratio natives 4.36 and adjusted odds ratio immigrants 4.09). When these two groups were compared, immigrants showed a higher likelihood of IPV than natives (adjusted odds ratios 1.58). Conclusion: Immigrant women are in a disadvantaged Intimate Partner Violence situation. It is necessary that interventions take these inequalities into account.

Changes in Access to Health Services of the Immigrant and Native-Born Population in Spain in the Context of Economic Crisis

Aim: To analyze changes in access to health care and its determinants in the immigrant and native-born populations in Spain, before and during the economic crisis. Methods: Comparative analysis of two iterations of the Spanish National Health Survey (2006 and 2012). Outcome variables were: unmet need and use of different healthcare levels; explanatory variables: need, predisposing and enabling factors. Multivariate models were performed (1) to compare outcome variables in each group between years, (2) to compare outcome variables between both groups within each year, and (3) to determine the factors associated with health service use for each group and year. Results: unmet healthcare needs decreased in 2012 compared to 2006; the use of health services remained constant, with some changes worth highlighting, such as the decline in general practitioner visits among autochthons and a narrowed gap in specialist visits between the two populations. The factors associated with health service use in 2006 remained constant in 2012. Conclusion: Access to healthcare did not worsen, possibly due to the fact that, until 2012, the national health system may have cushioned the deterioration of social determinants as a consequence of the financial crisis. Further studies are necessary to evaluate the effects of health policy responses to the crisis after 2012.

Paugvik : a nineteenth-century native village on Bristol Bay, Alaska / Don E. Dumond, James W. VanStone.

n.s. no.24(1995)