Motions and negative cooperativity between p97 domains revealed by cryo-electron microscopy and quantised elastic deformational model

p97, a Mg-ATPase belonging to the AAA (ATPase associated with various cellular activities) super family of proteins, has been proposed to function in two distinct cellular pathways, namely homotypic membrane fusion and ubiquitin protein degradation by utilizing differing adaptor complexes. We present the cryo-electron microscopy three-dimensional reconstruction of endogenous p97 in an AMP-PNP bound state at 24 A resolution. It reveals clear nucleotide-dependent differences when compared to our previously published "p97-ADP" reconstruction, including a striking rearrangement of N domains and a positional change of the two ATPase domains, D1 and D2, with respect to each other. The docking of the X-ray structure of N-D1 domains in an ADP bound state indicates that an upward repositioning of N domain is necessary to accommodate the cryo-EM map of "p97-AMP-PNP", suggesting a change in the orientation of N domains upon nucleotide hydrolysis. Furthermore, computational analysis of the deformational motions of p97, performed on the cryo-EM density map and the atomic structure of the N-D1 domains independently, shows the existence of a negative cooperativity between the D1 and D2 rings and the flexibility of the N domains. Together these results allow the identification of functionally important features that offer molecular insights into the dynamics of the proposed p97 chaperone function.     

The supramolecular organization of collagen VI microfibrils

Collagen VI has a ubiquitous distribution throughout connective tissues, and has key roles in linking cells and matrix macromolecules. We have generated three-dimensional reconstructions of collagen VI microfibrils using automated electron tomography (AET) in order to obtain new insights into the organisation of collagen VI in assembled microfibrils. Analysis of the reconstruction data has allowed the resolution of the double-beaded structure into smaller subunits. Volume calculations from the tomography data indicate that ten and six A-domains could be packed into the N and C-terminal regions from each monomer, respectively. A putative location for the globular N-terminal regions of the alpha3 chain, important for microfibril assembly and function, has been identified. Some surfaces of the alpha3 chain N-terminal domains appear to be exposed on the surface of a microfibril, where they may provide an interactive surface for molecules. Analysis of the interbead region provides evidence for complex triple helical supercoiling in microfibrils. Frequently, two strands were visualised emerging from the beaded region and merging into a single interbead region. Measurements taken from the AET data show that there is a decrease in periodicity from dimer/tetramer to microfibrils. Molecular combing reverses this effect by mechanically increasing periodicity to give measurements similar to the component dimers/tetramers. Together, these data have provided important new insights into the organisation and function of these large macromolecular assemblies.     

Evolution of the Vertebrate Cytosolic Malate Dehydrogenase Gene Family: Duplication and Divergence in Actinopterygian Fish

Abstract A general correlation between neural expression and negative charge in isozymes suggests charge represents an adaptation to the neural environment. Interestingly, a notable exception exists in teleost fish. Two cytosolic malate dehydrogenase (MDH) isozymes have different spatial expression patterns in certain fishes: one is expressed in all tissues and the second is expressed primarily in the eye and skeletal muscle. While the neural MDH isozyme is negatively charged, the difference in charge between the two isozymes is not as pronounced as that observed in other gene families (e.g., triosephosphate isomerase and lactate dehydrogenase). Most tetrapods express a single cytosolic MDH isozyme, and it has been demonstrated recently that the pair of isozymes found in teleosts results from a gene duplication sometime after the separation of teleosts and tetrapods, although the exact timing of this duplication has not been inferred. Phylogenetic analyses suggest that the duplication of teleost isozymes occurred during the radiation of actinopterygian fish, consistent with the timing of duplication at other loci. Using inferred amino acid sequences, we examine the pattern of change following the duplication and across the rest of the MDH gene tree. Comparison between the MDH gene family and another gene family that shows a larger charge differential among members (triosephosphate isomerase) indicates that the smaller charge difference between MDH isozymes is best explained by greater constraint on amino acid change directly following the duplication, not greater constraint across the entire gene tree. This difference in constraint might result from the wider pattern of expression of the “neural” MDH isozyme.     

Effect of a gap on gene flow between otherwise adjacent transgenic <Emphasis Type="Italic">Brassica napus</Emphasis> crops

The chloroplast DNA diversity of 33 accessions belonging to 16 species of five sections in Allium subgenus Rhizirideum was studied by analysing the sequence of three fragments: the trnL-F intergenic spacer, the rps 16 intron and rbcL (rubisco large subunit). The three sections Cepa, Schoenoprasum and Rhizirideum, representing the majority of the included species, each possess a separate clade after phylogenetic analysis. Exceptions to this general rule are the placement of Allium pskemense (section Cepa) connected to Allium senescens (section Rhizirideum) and Alium roylei, taking an intermediate position between sections Cepa and Schoenoprasum. Both species were located in their own section after nuclear DNA analysis. A range of crossing experiments has been carried out. The different position of A. roylei when comparing cpDNA and nDNA diversity was not confirmed with the production of hybrid seeds after crossing A. roylei with species other than those of section Cepa. The different position of A. pskemense in the cpDNA and the nDNA tree can not be compared to its crossability, since only a few crossing experiments are reported for this species. The hypothesis that a shorter distance between two species in a cpDNA tree compared to their distance in a nDNA tree will indicate interfertility at a certain level, is neither confirmed nor rejected by the currently available results.Communicated by R. Hagemann     

Infectious exacerbations of chronic obstructive pulmonary disease associated with respiratory viruses and non-typeable Haemophilus influenzae

Infectious exacerbations of chronic obstructive pulmonary disease (COPD) have been reported to occur with both viral and bacterial pathogens. In this study, 35 exacerbations associated with the isolation of non-typeable Haemophilus influenzae from sputum were identified as part of a prospective longitudinal study. Samples from these patients were subjected to immunoassays to identify a new immune response to the homologous isolate of non-typeable H. influenzae to more accurately assess a bacterial etiology. These patients also were studied carefully for evidence of viral infection using viral culture, serology and polymerase chain reaction-based assays. Sixteen of 35 exacerbations (45.7%) were associated with evidence of acute viral infection and 11 of the 35 exacerbations (31.4%) were associated with the development of new serum IgG to homologous non-typeable H. influenzae. Overall, evidence of infection with a respiratory virus or non-typeable H. influenzae was seen in 24 of 35 exacerbations (68.6%). No association between viral infection and immune response to non-typeable H. influenzae was observed, although a trend toward an immune response to non-typeable H. influenzae and absence of viral infection was seen. The results show that exacerbations in adults with COPD were associated with infection caused by virus alone, non-typeable H. influenzae alone, or virus and non-typeable H. influenzae simultaneously.     

Quantitative time series reconstruction of Eemian temperature at three European sites using pollen data

Botanical data are widely used as terrestrial proxy data for climate reconstructions. Using a newly established method based on probability density functions (pdf-method), the temperature development throughout the last interglacial, the Eemian, is reconstructed for the two German sites Bispingen and Gröbern and the French site La Grande Pile. The results are compared with previous reconstructions using other methods. After a steep increase in January as well as July temperatures in the early phase of the interglacial, the reconstructed most probable climate appears to be slightly warmer than today. While the temperature is reconstructed as relatively stable throughout the Eemian, a certain tendency towards cooler January temperatures is evident. January temperatures decreased from approx. 2–3 °C in the early part to approx. –3 °C in the later part at Bispingen, and from approx. 2 °C to approx. –1 °C at Gröbern and La Grande Pile. A major drop to about –8 °C marks the very end of the interglacial at all three sites. While these results agree well with other proxy data and former reconstructions based on the indicator species method, the results differ significantly from reconstructions based on the modern pollen analogue technique (pollen transfer functions). The lack of modern analogues is assumed to be the main reason for the discrepancies. It is concluded that any reconstruction method needs to be evaluated carefully in this respect if used for periods lacking modern analogous plant communities.     

Update on animal models developed for analyses of estrogen receptor biological activity

Targeted disruption of the different ER genes has generated experimental animal models that are very useful in evaluating the distinct and cooperative roles of the two estrogen receptors, ER and ERβ, in reproductive but also non-reproductive tissues of both sexes. Phenotypic analysis has provided definitive experimental findings for estrogen receptor mediated physiological actions, involving ER in uterine, mammary gland and neuroendocrine sites. ERβ is involved most dramatically in the ovary as is ER. More detailed studies in combination with tissue specific or inducible ER knock outs will be important for future research.