Structure of a hexameric RNA packaging motor in a viral polymerase complex

Packaging of the Cystovirus varphi8 genome into the polymerase complex is catalysed by the hexameric P4 packaging motor. The motor is located at the fivefold vertices of the icosahedrally symmetric polymerase complex, and the symmetry mismatch between them may be critical for function. We have developed a novel image-processing approach for the analysis of symmetry-mismatched structures and applied it to cryo-electron microscopy images of P4 bound to the polymerase complex. This approach allowed us to solve the three-dimensional structure of the P4 in situ to 15-A resolution. The C-terminal face of P4 was observed to interact with the polymerase complex, supporting the current view on RNA translocation. We suggest that the symmetry mismatch between the two components may facilitate the ring opening required for RNA loading prior to its translocation.     

The effect of cocoa and polydextrose on bacterial fermentation in gastrointestinal tract simulations

Effects of cocoa mass and supplemented dietary fiber (polydextrose) on microbial fermentation were studied by combining digestion simulations of stomach and small intestine with multi-staged colon simulations. During the four phases of digestion, concentrations of available soluble proteins and reducing sugars reflected in vivo absorption of nutrients in small intestine. In colon simulation vessels, addition of polydextrose to digested cocoa mass significantly increased concentrations of total short-chain fatty acids and butyric acid, from 103 to 468 mM (P<0.01) and from 12 to 22 mM (P<0.01), respectively. Long-chain fatty acid concentrations (decreasing from 1,222 to 240 mM) were mainly affected by the presence of digested cocoa mass. Cocoa mass with or without polydextrose addition significantly decreased production of cadaverine (P<0.02) and branched-chain fatty acids compared to control during colon simulations. Results indicate beneficial effects on metabolism of colonic microbiota after digestion of cocoa mass, and even more so with polydextrose addition.     

Mitogenetic structure of brown bears (Ursus arctos L.) in northeastern Europe and a new time frame for the formation of European brown bear lineages

We estimated the phylogenetic relationships of brown bear maternal haplotypes from countries of northeastern Europe (Estonia, Finland and European Russia), using sequences of mitochondrial DNA (mtDNA) control region of 231 bears. Twenty-five mtDNA haplotypes were identified. The brown bear population in northeastern Europe can be divided into three haplogroups: one with bears from all three countries, one with bears from Finland and Russia, and the third composed almost exclusively of bears from European Russia. Four haplotypes from Finland and European Russia matched exactly with haplotypes from Slovakia, suggesting the significance of the current territory of Slovakia in ancient demographic processes of brown bears. Based on the results of this study and those from the recent literature, we hypothesize that the West Carpathian Mountains have served either as one of the northernmost refuge areas or as an important movement corridor for brown bears of the Eastern lineage towards northern Europe during or after the last ice age. Bayesian analyses were performed to investigate the temporal framework of brown bear lineages in Europe. The molecular clock was calibrated using Beringian brown bear sequences derived from radiocarbon-dated ancient samples, and the estimated mutation rate was 29.8% (13.3%-47.6%) per million years. The whole European population and Western and Eastern lineages formed about 175,000, 70,000 and 25,000 years before present, respectively. Our approach to estimating the time frame of brown bear evolution demonstrates the importance of using an appropriate mutation rate, and this has implications for other studies of Pleistocene populations.     

Electron cryomicroscopy comparison of the architectures of the enveloped bacteriophages phi6 and phi8

The enveloped dsRNA bacteriophages phi6 and phi8 are the two most distantly related members of the Cystoviridae family. Their structure and function are similar to that of the Reoviridae but their assembly can be conveniently studied in vitro. Electron cryomicroscopy and three-dimensional icosahedral reconstruction were used to determine the structures of the phi6 virion (14 A resolution), phi8 virion (18 A resolution), and phi8 core (8.5 A resolution). Spikes protrude 2 nm from the membrane bilayer in phi6 and 7 nm in phi8. In the phi6 nucleocapsid, 600 copies of P8 and 72 copies of P4 interact with the membrane, whereas in phi8 it is only P4 and 60 copies of a minor protein. The major polymerase complex protein P1 forms a dodecahedral shell from 60 asymmetric dimers in both viruses, but the alpha-helical fold has apparently diverged. These structural differences reflect the different host ranges and entry and assembly mechanisms of the two viruses.     

Regulation of phagocytosis in Dictyostelium by the inositol 5-phosphatase OCRL homolog Dd5P4

Phosphoinositides are involved in endocytosis in both mammalian cells and the amoeba Dictyostelium discoideum. Dd5P4 is the Dictyostelium homolog of human OCRL (oculocerebrorenal syndrome of Lowe); both have a RhoGAP domain and a 5-phosphatase domain that acts on phosphatidylinositol 4,5-bisphosphate/phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). Inactivation of Dd5P4 inhibits growth on liquid medium and on bacteria. Dd5p4-null cells are impaired in phagocytosis of yeast cells. In wild-type cells, PI(3,4,5)P3 is formed and converted to PI(3,4)P2 just before closure of the phagocytic cup. In dd5p4-null cells, a phagocytic cup is formed upon contact with the yeast cell, and PI(3,4,5)P3 is still produced, but the phagocytic cup does not close. We suggest that Dd5P4 regulates the conversion of PI(3,4,5)P3 to PI(3,4)P2 and that this conversion is essential for closure of the phagocytic cup. Phylogenetic analysis of OCRL-like 5-phosphatases with RhoGAP domains reveal that D. discoideum Dd5P4 is a surprisingly close homolog of human OCRL, the protein responsible for Lowe syndrome. We expressed human OCRL in dd5p4-null cells. Growth on bacteria and axenic medium is largely restored, whereas the rate of phagocytosis of yeast cells is partly restored, indicating that human OCRL can functionally replace Dictyostelium Dd5P4.     

Restored cut-away peatland as a sink for atmospheric CO2

In a field study, we examined the relationship between vegetation, abiotic factors and the CO₂ exchange dynamics of a cut-away peatland 20 years after production had ended. The main objective was to determine the effect of rewetting on the CO₂ exchange dynamics, measured separately in Eriophorum vaginatum tussocks and intertussocks (almost non-vegetated surfaces) using closed-chamber techniques, one growing season before and three growing seasons after the rewetting treatment. Rewetting lowered total respiration (R _TOT) and increased gross photosynthesis (P _G), which resulted in a higher incorporation of CO₂ into the system. The seasonal CO₂ balance for the almost continuously submerged section of the rewetted site became positive 2 years after rewetting (9.1 g CO₂-C m⁻²), and it was still higher in the 3rd year (64.5 g CO₂-C m⁻²), i.e. the system accumulated carbon. In the driest section of the rewetted site the seasonal balance increased strongly, but the balance was still negative during the 3 years following rewetting with losses from the system of 44.1, 26.1, 38.3 g CO₂-C m⁻² in 1995, 1996 and 1997 respectively. At the control site seasonal balance during 1995–1997 varied between ecosystem C losses of 41.8 and 95.3 in an area with high Eriophorum cover and between 52.1 and 109.9 g CO₂-C m⁻² with lower cover. Simulation of a cut-away peatland with dense Eriophorum vegetation (Eriophorum cover 70%) showed that if the water level (WT) is low, the seasonal CO₂ balance of the ecosystem can reach the compensation point of no net C change (P _G = R _TOT) only if weather conditions are favourable, but with a high WT the seasonal CO₂ balance would be positive even under varying weather conditions. It can be concluded that with dense Eriophorum vegetation a restored cut-away peatland acts as a functional mire and becomes a sink for atmospheric CO₂. Received: 21 September 1998 / Accepted: 18 May 1999     

Level of Fatty Acid Binding Protein 5 (FABP5) Is Increased in Sputum of Allergic Asthmatics and Links to Airway Remodeling and Inflammation

BackgroundThe inflammatory processes in the upper and lower airways in allergic rhinitis and asthma are similar. Induced sputum and nasal lavage fluid provide a non-invasive way to examine proteins involved in airway inflammation in these conditions.ObjectivesWe conducted proteomic analyses of sputum and nasal lavage fluid samples to reveal differences in protein abundances and compositions between the asthma and rhinitis patients and to investigate potential underlying mechanisms.MethodsInduced sputum and nasal lavage fluid samples were collected from 172 subjects with 1) allergic rhinitis, 2) asthma combined with allergic rhinitis, 3) nonallergic rhinitis and 4) healthy controls. Proteome changes in 21 sputum samples were analysed with two-dimensional difference gel electrophoresis (2D-DIGE), and the found differentially regulated proteins identified with mass spectrometry. Immunological validation of identified proteins in the sputum and nasal lavage fluid samples was performed with Western blot and ELISA.ResultsAltogether 31 different proteins were identified in the sputum proteome analysis, most of these were found also in the nasal lavage fluid. Fatty acid binding protein 5 (FABP5) was up-regulated in the sputum of asthmatics. Immunological validation in the whole study population confirmed the higher abundance levels of FABP5 in asthmatic subjects in both the sputum and nasal lavage fluid samples. In addition, the vascular endothelial growth factor (VEGF) level was increased in the nasal lavage fluid of asthmatics and there were positive correlations between FABP5 and VEGF levels (r=0.660, p<0.001) and concentrations of FABP5 and cysteinyl leukotriene (CysLT) (r=0.535, p<0.001) in the nasal lavage fluid.ConclusionsFABP5 may contribute to the airway remodeling and inflammation in asthma by fine-tuning the levels of CysLTs, which induce VEGF production.