Protonation status of metal-bound ligands can be determined by quantum refinement

The protonation status of key residues and bound ligands are often important for the function of a protein. Unfortunately, protons are not discerned in normal protein crystal structures, so their positions have to be determined by more indirect methods. We show that the recently developed quantum refinement method can be used to determine the position of protons in crystal structures. By replacing the molecular-mechanics potential, normally used in crystallographic refinement, by more accurate quantum chemical calculations, we get information about the ideal structure of a certain protonation state. By comparing the refined structures of different protonation states, the one that fits the crystallographic raw data best can be decided using four criteria: the R factors, electron density maps, strain energy, and divergence from the unrestrained quantum chemical structure. We test this method on alcohol dehydrogenase, for which the pK(a) of the zinc-bound solvent molecule is experimentally known. We show that we can predict the correct protonation state for both a deprotonated alcohol and a neutral water molecule.     

Targeted gene therapy: frontiers in the development of 'smart drugs'

Chronic diseases, particularly malignancies and immune-mediated inflammatory diseases (IMIDs), are a challenging frontier for clinical diagnosis and treatment, as well as for biomedical research. Current treatment regimens are frequently insufficient and thus new treatment strategies are needed. Novel therapies for disabling such diseases should provide improvements with respect to safety, efficacy and cost. To fulfill these three key criteria, recent research efforts have focused on the development of 'smart drugs'. This review highlights some examples of the rapidly expanding possibilities that current biotechnology has to offer in the development of novel therapeutic strategies for complex diseases such as IMIDs. Special attention is given to advances in, and limitations of, controlled and targeted gene product application in inflammatory diseases.     

An improved method for the solution cyclization of peptides under pseudo-high dilution conditions

Depending on the ring size, the cyclization of peptides often is accompanied by dimerization or cyclodimerization. Hence, these macrocyclizations have to be performed under high dilution conditions. Efficient cyclization of peptides in solution with a minimum amount of solvent succeeds, when a dual syringe pump is used to simultaneously add the linear peptide precursor and a coupling reagent from two separate syringes.     

Crossing the barrier: net flux of 27-hydroxycholesterol into the human brain

Side chain oxidized oxysterols have a unique ability to traverse lipophilic membranes. We tested the hypothesis that there is a net flux of 27-hydroxycholesterol from the circulation into the brain using plasma samples collected from the internal jugular vein and an artery of healthy male volunteers. Two independent studies were performed, one in which total levels of 27-hydroxycholesterol were measured and one in which the free fraction of 27-hydroxycholesterol was measured. In the majority of subjects studied, the level of 27-hydroxycholesterol was higher in the artery than in the vein, and uptake from the circulation was calculated to be about 5 mg/24 h. The distribution of 27-hydroxycholesterol in human brain was found to be consistent with an extracerebral origin, with a concentration gradient from the white to the gray matter--a situation opposite that of 24S-hydroxycholesterol, which os exclusively formed in brain. In view of the fact that the blood-brain barrier is impermeable to cholesterol and that 27-hydroxycholesterol is a potent regulator of several cholesterol-sensitive genes, the flux of 27-hydroxycholesterol into the brain may be and important link between intra- and extracerebral cholesterol homeostasis.     

Structural requirements for activation of latent platelet-derived growth factor CC by tissue plasminogen activator

Platelet-derived growth factor C (PDGF-C) is one of four members in the PDGF family of growth factors, which are known mitogens and survival factors for cells of mesenchymal origin. PDGF-C has a unique two-domain structure consisting of an N-terminal CUB and a conserved C-terminal growth factor domain that are separated by a hinge region. PDGF-C is secreted as a latent dimeric factor (PDGF-CC), which undergoes extracellular removal of the CUB domains to become a PDGF receptor alpha agonist. Recently, the multidomain serine protease tissue plasminogen activator (tPA), a thrombolytic agent used for treatment of acute ischemic stroke, was shown to cleave and activate PDGF-CC. In this study we determine the molecular mechanism of tPA-mediated activation of PDGF-CC. Using various PDGF-CC and tPA mutants, we were able to demonstrate that both the CUB and the growth factor domains of PDGF-C, as well as the kringle-2 domain of tPA, are required for the interaction and cleavage to occur. We also show that Arg231 in PDGF-C is essential for tPA-mediated proteolysis and that the released "free" CUB domain of PDGF-C can act as a competitive inhibitor of the cleavage reaction. Furthermore, we studied how the PDGF-C/tPA axis is regulated in primary fibroblasts and found that PDGF-C expression is down-regulated by hypoxia but induced by transforming growth factor (TGF)-beta1 treatment. Elucidating the regulation and the mechanism of tPA-mediated activation of PDGF-CC will advance our knowledge of the physiological function of PDGF-CC and tPA and may provide new therapeutic opportunities for thrombolytic and cardiovascular therapies.     

Antioxidative treatment of pregnant diabetic rats diminishes embryonic dysmorphogenesis

BACKGROUND: Diabetic pregnancy is still associated with an increased rate of congenital malformations despite extensive clinical efforts to normalize the risk for the offspring. The etiology of diabetic embryopathy is not clear; however, experimental studies have suggested a role for oxidative stress in the teratogenicity of diabetic pregnancy. The antioxidants alpha-tocopherol and ascorbate have improved fetal outcome in diabetic rodent pregnancy when supplemented in moderate to high doses. In the present work we investigated if extremely high doses of either alpha-tocopherol or ascorbate might further improve fetal outcome in offspring of diabetic rats and, in addition, if such treatment may exert any adverse effects of fetal development. METHODS: Nondiabetic and streptozotocin diabetic female rats were fed 2, 5, 10, or 15% alpha-tocopherol or 4, 10, or 15% ascorbate in their diet. RESULTS: Both alpha-tocopherol and ascorbate treatment improved fetal morphology in offspring of diabetic rats. There was a dose-dependent improvement for the alpha-tocopherol supplementation, in which the higher doses diminished fetal dysmorphogenesis more than the 2% diet. The ascorbate supplementation was less dose-dependent; however, the higher doses tended to improve fetal outcome more than the lower doses. No adverse effects of the antioxidants were noted in the offspring with the exception of 1 case of agnathia in a fetus of a nondiabetic rat supplemented with 15% alpha-tocopherol. CONCLUSIONS: These results indicate that very high doses of dietary antioxidants may be needed to normalize the development of the offspring in experimental diabetic pregnancy, but that treatment with such high doses may also have adverse effects in nondiabetic pregnancy.     

Diversity and dominance patterns in Amazon coast dune forest island tree communities

Aiming to better understand richness, dominance, diversity and community changes of coastal vegetation, we studied woody plant communities and soil parameters of equatorial dune forest islands. We investigated four sites isolated by mangroves (Buraco Beach, Bonifácio, Camarauaçu, Apeu-Salvador) and compared data to a paleodune forest with higher floristic and structural complexity (Salinas do Roque) for assessment of the regional species pool. We determined grain size distribution and organic matter content at Buraco Beach, Camarauaçu and Apeu-Salvador. Grain size distribution was similar among sites but upper soil layers at Camarauaçu showed low organic matter contents. We recorded trees and shrubs along several transect plots crossing forested dune ridges and surveyed areas outside of plots for additional species. Richness was lower at Camarauaçu (18 species vs. 25, 26 and 28 at Bonifácio, Apeu-Salvador and Buraco Beach, respectively). Fisher’s α ranged between 3.2 and 4.7, Pielou’s J’ between 0.70 and 0.80. Rényi profiles confirmed lower diversity for Camarauaçu, dominated by a small group of species with high importance values. Within-site beta diversity was lowest at Buraco Beach. Species were mainly wide-spread generalists. We found low richness compared to the Salinas do Roque reference site; null model tests indicated that the species pool of the dune sites was partly shaped by environmental filtering. We attribute differences in species composition and diversity/dominance patterns among the four dune sites to distance to the mainland and stochastic events with effects on non-standard long distance dispersal.     

Warming‐induced upward migration of the alpine treeline in the Changbai Mountains,northeast China

Treeline responses to environmental changes describe an important phenomenon in global change research. Often conflicting results and generally too short observations are, however, still challenging our understanding of climate‐induced treeline dynamics. Here, we use a state‐of‐the‐art dendroecological approach to reconstruct long‐term changes in the position of the alpine treeline in relation to air temperature at two sides in the Changbai Mountains in northeast China. Over the past 160 years, the treeline increased by around 80 m, a process that can be divided into three phases of different rates and drives. The first phase was mainly influenced by vegetation recovery after an eruption of the Tianchi volcano in 1702. The slowly upward shift in the second phase was consistent with the slowly increasing temperature. The last phase coincided with rapid warming since 1985, and shows with 33 m per 1°C, the most intense upward shift. The spatial distribution and age structure of trees beyond the current treeline confirm the latest, warming‐induced upward shift. Our results suggest that the alpine treeline will continue to rise, and that the alpine tundra may disappear if temperatures will increase further. This study not only enhances mechanistic understanding of long‐term treeline dynamics, but also highlights the effects of rising temperatures on high‐elevation vegetation dynamics.     

New barcoded primers for efficient retrieval of cercozoan sequences in high‐throughput environmental diversity surveys,with emphasis on worldwide biological soil crusts

We describe the performance of a new metabarcoding approach to investigate the environmental diversity of a prominent group of widespread unicellular organisms, the Cercozoa. Cercozoa is an immensely large group of protists, and although it may dominate in soil and aquatic ecosystems, its environmental diversity remains undersampled. We designed PCR primers targeting the hypervariable region V4 of the small subunit ribosomal RNA (SSU or 18S) gene, which is the recommended barcode marker for Cercozoa. The length of the amplified fragment (c. 350 bp) is suitable for Illumina MiSeq, the most cost‐effective platform for molecular environmental surveys. We provide barcoded primers, an economical alternative to multiple libraries for multiplex sequencing of over a hundred samples. In silico, our primers matched 68% of the cercozoan sequences of the reference database and performed better than previously proposed new‐generation sequencing primers. In mountain grassland soils and in biological soil crusts from a variety of climatic regions, we were able to detect cercozoan sequences encompassing nearly the whole range of the phylum. We obtained 901 operational taxonomic units (OTUs) at 97% similarity threshold from 26 samples, with c. 50,000 sequences per site, and only 8% of noncercozoan sequences. We could report a further increase in the diversity of Cercozoa, as only 43% of the OTUs were 97%–100% similar to any known sequence. Our study thus provides an advanced tool for cercozoan metabarcoding and to investigate their diversity and distribution in the environment.