Optimisation of the surface electrostatics as a strategy for cold adaptation of uracil-DNA N-glycosylase (UNG) from Atlantic cod (Gadus morhua)

Cold-adapted enzymes are characterised by an increased catalytic efficiency and reduced temperature stability compared to their mesophilic counterparts. Lately, it has been suggested that an optimisation of the electrostatic surface potential is a strategy for cold adaptation for some enzymes. A visualisation of the electrostatic surface potential of cold-adapted uracil-DNA N-glycosylase (cUNG) from Atlantic cod indicates a more positively charged surface near the active site compared to human UNG (hUNG). In order to investigate the importance of the altered surface potential for the cold-adapted features of cod UNG, six mutants have been characterised and compared to cUNG and hUNG. The cUNG quadruple mutant (V171E, K185V, H250Q and H275Y) and four corresponding single mutants all comprise substitutions of residues present in the human enzyme. A human UNG mutant, E171V, comprises the equivalent residue found in cod UNG. In addition, crystal structures of the single mutants V171E and E171V have been determined. Results from the study show that a more negative electrostatic surface potential reduces the activity and increases the stability of cod UNG, and suggest an optimisation of the surface potential as a strategy for cold-adaptation of this enzyme. Val171 in cod UNG is especially important in this respect.     

p25alpha Stimulates alpha-synuclein aggregation and is co-localized with aggregated alpha-synuclein in alpha-synucleinopathies

Aggregation of the nerve cell protein alpha-synuclein is a characteristic of the common neurodegenerative alpha-synucleinopathies like Parkinson's disease and Lewy body dementia, and it plays a direct pathogenic role as demonstrated by early onset diseases caused by mis-sense mutations and multiplication of the alpha-synuclein gene. We investigated the existence of alpha-synuclein pro-aggregatory brain proteins whose dysregulation may contribute to disease progression, and we identified the brain-specific p25alpha as a candidate that preferentially binds to alpha-synuclein in its aggregated state. Functionally, purified recombinant human p25alpha strongly stimulates the aggregation of alpha-synuclein in vitro as demonstrated by thioflavin-T fluorescence and quantitative electron microscopy. p25alpha is normally only expressed in oligodendrocytes in contrast to alpha-synuclein, which is normally only expressed in neurons. This expression pattern is changed in alpha-synucleinopathies. In multiple systems atrophy, degenerating oligodendrocytes displayed accumulation of p25alpha and dystopically expressed alpha-synuclein in the glial cytoplasmic inclusions. In Parkinson's disease and Lewy body dementia, p25alpha was detectable in the neuronal Lewy body inclusions along with alpha-synuclein. The localization in alpha-synuclein-containing inclusions was verified biochemically by immunological detection in Lewy body inclusions purified from Lewy body dementia tissue and glial cytoplasmic inclusions purified from tissue from multiple systems atrophy. We suggest that p25alpha plays a pro-aggregatory role in the common neurodegenerative disorders hall-marked by alpha-synuclein aggregates.     

MicroRNA-24 Suppression of N-Deacetylase/N-Sulfotransferase-1 (NDST1) Reduces Endothelial Cell Responsiveness to Vascular Endothelial Growth Factor A (VEGFA)

Heparan sulfate (HS) proteoglycans, present at the plasma membrane of vascular endothelial cells, bind to the angiogenic growth factor VEGFA to modulate its signaling through VEGFR2. The interactions between VEGFA and proteoglycan co-receptors require sulfated domains in the HS chains. To date, it is essentially unknown how the formation of sulfated protein-binding domains in HS can be regulated by microRNAs. In the present study, we show that microRNA-24 (miR-24) targets NDST1 to reduce HS sulfation and thereby the binding affinity of HS for VEGFA. Elevated levels of miR-24 also resulted in reduced levels of VEGFR2 and blunted VEGFA signaling. Similarly, suppression of NDST1 using siRNA led to a reduction in VEGFR2 expression. Consequently, not only VEGFA binding, but also VEGFR2 protein expression is dependent on NDST1 function. Furthermore, overexpression of miR-24, or siRNA-mediated reduction of NDST1, reduced endothelial cell chemotaxis in response to VEGFA. These findings establish NDST1 as a target of miR-24 and demonstrate how such NDST1 suppression in endothelial cells results in reduced responsiveness to VEGFA.     

Topography as a driver of local terrestrial vascular plant diversity patterns

At landscape and regional scales topography is recognized as one of the most important determinants of vascular plant diversity, primarily due to the influence of mountains. As temperature changes markedly over the elevation ranges in mountain areas, topography offers a wide variety of different habitats as well as buffering against climate change. However, for local vegetation, notably in lowland areas, the general importance of topography is less well recognized and the mechanisms by which it exerts influence on local vascular plant diversity are not comprehensively understood. In this review, we provide an overview of the evidence for the different mechanisms involved in topography’s control of local patterns in potential vegetation drivers, namely incident solar energy, wind exposure, hydrology, geochemistry, and biotic conditions. Furthermore, we review the processes through which these factors shape local terrestrial vascular plant diversity patterns and provide directions for future studies on this topic. We find that topography is an important factor for local vascular plant diversity patterns in a broad range of habitats throughout the world, even in relatively flat lowlands. However, the mechanisms involved are varied and complex. Local patterns in soil moisture seem to be affected by topography through more mechanisms than other topographically controlled factors and have a strong and consistent influence on local plant diversity. Hence, local hydrology is probably the main mechanistic factor through which topography influences local terrestrial vascular plant diversity patterns. Future research should focus on employing high‐coverage fine‐resolution topographic data to comprehensively explore the role of topography in controlling local dynamics over large areas. Moreover, we recommend including several different habitats, particularly those in which the role of topography is poorly understood. Finally, we propose to integrate relevant functional topographic variables such as topographic wetness indices instead of simple topographic measures into future investigations.     

Optimisation of the Factor VIII yield in mammalian cell cultures by reducing the membrane bound fraction

In vivo, clotting Factor VIII (FVIII) circulates in plasma bound to von Willebrand factor (vWF), and the vWF:FVIII complex prevents binding of FVIII to phosphatidylserine (PS). Activation of FVIII by thrombin releases FVIII from vWF, and subsequently FVIII binds to PS exposed on activated platelets and forms the tenase complex together with clotting Factor IX. In vitro, during serum free production of recombinant FVIII (rFVIII), production cells also expose PS, and since vWF is not present to hinder interaction of secreted rFVIII with PS, rFVIII is partly associated with the cell membrane of the production cells. Recently, we showed that as much as 90% of secreted rFVIII is bound to transiently transfected production cells during serum free conditions. In this study, we investigated the effect of including vWF in the serum free medium, and demonstrate that addition of vWF results in release of active membrane bound rFVIII to the culture medium. Moreover, the attachment of rFVIII to cell membranes of un-transfected HEK293 cells was studied in the presence of compounds that competes for interactions between rFVIII and PS. Competitive assays between iodinated rFVIII (¹²⁵I-rFVIII) and annexin V or ortho-phospho-l-serine (OPLS) demonstrated that annexin V and OPLS were able to reduce the membrane bound fraction of rFVIII by 70% and 30%, respectively. Finally, adding OPLS to CHO cells stably expressing FVIII increased the yield by 50%. Using this new knowledge, the recovery of rFVIII could be increased considerably during serum free production of this therapeutic protein.     

Oxidative folding of nerve growth factor can be mediated by the pro-peptide of neurotrophin-3

We have previously shown that the pro-peptide of human nerve growth factor (NGF) facilitates oxidative folding of the mature part. For the analysis of functional specificities of the pro-peptides of NGF and the related neurotrophin-3 (NT-3) with respect to structure formation, chimeric proteins with swapped pro-peptides were generated. Neither the structure nor the stability of the mature domains was influenced by the heterologous pro-peptides. For the pro-peptide of NT-3 fused to the mature part of NGF, stabilization of the pro-peptide moiety by the NGF part was observed. Folding kinetics and renaturation yields of this chimeric protein were comparable to those of proNGF. Our results demonstrate functional interchangeability between the pro-peptides of NGF and NT-3 with respect to their role in assisting oxidative folding of the mature part.     

Population fluctuations and regulation in great snipe: a time-series analysis

1. During the last centuries, the breeding range of the great snipe Gallinago media has declined dramatically in the western part of its distribution. To examine present population dynamics in the Scandinavian mountains, we collected and analysed a 19-year time series of counts of great snipe males at leks in central Norway, 1987-2005. 2. The population showed large annual fluctuations in the number of males displaying at lek sites (range 45-90 males at the peak of the mating season), but no overall trend. 3. We detected presence of direct density-dependent mechanisms regulating this population. Inclusion of the density-dependent term in a Ricker-type model significantly improved the fit with observed data (evaluated with Parametric Bootstrap Likelihood Ratio tests and Akaike's Information Criterion for small sample size). 4. An analysis of (a number of a priori likely) environmental covariates suggests that the population dynamics were affected by conditions influencing reproduction and survival of offspring during the summer, but not by conditions influencing survival at the wintering grounds in Africa. This is in contrast to many altricial birds breeding in the northern hemisphere, and supports the idea that population dynamics of migratory nidifugous birds are more influenced by conditions during reproduction. 5. Inclusion of these external factors into our model improved the detectability of density dependence. This illustrates that allowing for external effects may increase statistical power of density dependence tests and thus be of particular importance in relatively short time series. 6. In our best model of the population dynamics, two likely density-independent offspring survival covariates explained 47.3% of the variance in great snipe numbers (predation pressure estimated by willow grouse reproductive success and food availability estimated by the amount of precipitation in June), whereas density dependence explained 35.5%. Demographic stochasticity and unidentified environmental stochasticity may account for the remaining 17.2%.     

A sexually neutral discrete Markov model for given sum males + females

An inhomogeneous discrete Markov model is formulated for sexual random mating with diploid male and female individuals. The generations are nonoverlapping and of given sizes. The genetic variation is in a sexually neutral allele with two varieties, giving three different genotypes. Taking sex as a marker, the Markov model works with six genotypes. The sex of each offspring is random. This implies a probability of extinction, giving the model an algorithmic nature. We compute expected genotype frequencies, their standard deviations and fixation probabilities.     

Characterizing morphological (co)variation using structural equation models: Body size,allometric relationships and evolvability in a house sparrow metapopulation

Body size plays a key role in the ecology and evolution of all organisms. Therefore, quantifying the sources of morphological (co)variation, dependent and independent of body size, is of key importance when trying to understand and predict responses to selection. We combine structural equation modeling with quantitative genetics analyses to study morphological (co)variation in a meta‐population of house sparrows (Passer domesticus). As expected, we found evidence of a latent variable “body size,” causing genetic and environmental covariation between morphological traits. Estimates of conditional evolvability show that allometric relationships constrain the independent evolution of house sparrow morphology. We also found spatial differences in general body size and its allometric relationships. On islands where birds are more dispersive and mobile, individuals were smaller and had proportionally longer wings for their body size. Although on islands where sparrows are more sedentary and nest in dense colonies, individuals were larger and had proportionally longer tarsi for their body size. We corroborated these results using simulations and show that our analyses produce unbiased allometric slope estimates. This study highlights that in the short term allometric relationships may constrain phenotypic evolution, but that in the long term selection pressures can also shape allometric relationships.