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.

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.

La evaluación continua en la docencia del urbanismo: experiencia aplicada a través de Google Apps

El sistema tradicional de evaluación continua implica asumir, en muchas ocasiones, algunas dificultades en cuanto al seguimiento y valoración del trabajo del alumnado. La existencia de grupos numerosos y la necesidad de agilizar la comunicación profesor-alumno y entre alumnos, nos lleva a investigar las posibilidades que ofrecen las nuevas tecnologías con el fin de actualizar los métodos de aprendizaje y facilitar la valoración del trabajo de los alumnos dentro y fuera del aula. La presente experiencia docente se centra en estudiar las posibilidades que ofrece Google, a través de distintas aplicaciones web o Apps -Groups, Forms, Drive, Mail y Calendar- e incorporarlas como herramientas integradas en el trabajo del aula. Concretamente, se aplica simultáneamente esta metodología a dos asignaturas diferentes del área de Urbanística y Ordenación del Territorio -Urbanismo 2 y Urbanismo 4- en las que se integra al alumnado en un grupo virtual a través del cual, se realizan distintas actividades -resolución de dudas, intercambio de información, exámenes, etc.- que permiten al profesor gestionar y evaluar, de forma eficiente, cuestiones como la asistencia y la participación en la asignatura conociendo de forma inmediata la respuesta de los estudiantes ante los conceptos trabajados.

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

n.s. no.24(1995)

The native flowers and ferns of the United States in their botanical, horticultural and popular aspects / by Thomas Meehan ; illustrated by chromolithographs.

2