Role of elongation and secondary pathways in S6 amyloid fibril growth

The concerted action of a large number of individual molecular level events in the formation and growth of fibrillar protein structures creates a significant challenge for differentiating between the relative contributions of different self-assembly steps to the overall kinetics of this process. The characterization of the individual steps is, however, an important requirement for achieving a quantitative understanding of this general phenomenon which underlies many crucial functional and pathological pathways in living systems. In this study, we have applied a kinetic modeling approach to interpret experimental data obtained for the aggregation of a selection of site-directed mutants of the protein S6 from Thermus thermophilus. By studying a range of concentrations of both the seed structures, used to initiate the reaction, and of the soluble monomer, which is consumed during the growth reaction, we are able to separate unambiguously secondary pathways from primary nucleation and fibril elongation. In particular, our results show that the characteristic autocatalytic nature of the growth process originates from secondary processes rather than primary nucleation events, and enables us to derive a scaling law which relates the initial seed concentration to the onset of the growth phase.     

Highly specific label-free protein detection from lysed cells using internally referenced microcantilever sensors

We report the investigation of label-free protein detection directly from lysed cells using microcantilever sensors. The integration of an internally referenced microcantilever sensor combined with peptide aptamer technology enables scalable and label-free detection of proteins from a complex biological environment (e.g. cell lysate). The internally referenced microcantilever sensor was found to be effective in minimizing both the effects of thermal drift and non-specific binding interactions with the backside of the cantilever, thereby allowing protein detection in a complex biological background. Highly specific peptide aptamers are used to modify the cantilever surface to specifically detect less than 80nM CDK2 protein from yeast cell lysate. This binding of CDK2 on the microcantilever generates a tensile surface stress of average magnitude equal to 70+/-22mN/m. Similar experiments conducted with quartz crystal microbalance (QCM) technology are consistent with the response observed using microcantilever sensors.     

On the evolution of specialization with a mechanistic underpinning in structured metapopulations

We analyze the evolution of specialization in resource utilization in a discrete-time metapopulation model using the adaptive dynamics approach. The local dynamics in the metapopulation are based on the Beverton-Holt model with mechanistic underpinnings. The consumer faces a trade-off in the abilities to consume two resources that are spatially heterogeneously distributed to patches that are prone to local catastrophes. We explore the factors favoring the spread of generalist or specialist strategies. Increasing fecundity or decreasing catastrophe probability favors the spread of the generalist strategy and increasing environmental heterogeneity enlarges the parameter domain where the evolutionary branching is possible. When there are no catastrophes, increasing emigration diminishes the parameter domain where the evolutionary branching may occur. Otherwise, the effect of emigration on evolutionary dynamics is non-monotonous: both small and large values of emigration probability favor the spread of the specialist strategies whereas the parameter domain where evolutionary branching may occur is largest when the emigration probability has intermediate values. We compare how different forms of spatial heterogeneity and different models of local growth affect the evolutionary dynamics. We show that even small changes in the resource dynamics may have outstanding evolutionary effects to the consumers.     

Prevention of flower formation in dicotyledons

Prevention of flower formation is important, for example for preventing the spread of transgenes from genetically modified plants or the spread of non-native species, for increasing vegetative growth or preventing the formation of allergenic pollen. The aim of this study was to determine whether flowering of dicotyledonous plants can be prevented by genetic manipulation without harmful effects on vegetative growth. Here we describe isolation of the BpMADS1 gene (similar to SEP3, formerly AGL9) from birch and show that it is expressed only in the inflorescences. In tobacco and Arabidopsis, the expression of BpMADS1::GUS was also virtually inflorescence-specific. Transgenic tobacco and Arabidopsis containing a BpMADS1::BARNASE construct grew well. In one tobacco line the formation of the inflorescence was completely prevented; in several other lines the flowers lacked stamens and carpels and therefore were sterile. The final dry weights of the shoots of the sterile tobacco lines were 140–200% of those of controls. In Arabidopsis, some of the transgenic lines containing the BpMADS1::BARNASE construct formed inflorescences. Some of these lines formed never flowers and some others formed occasionally single fertile flowers. Some other lines did not form inflorescences, but formed up to about one hundred leaves, even in long-day conditions. These results suggest that formation of flowers or inflorescences in widely different dicotyledonous plants could be prevented using the BpMADS1::BARNASE construct and that prevention of flowering may lead to increased vegetative mass.     

Immunohistochemical Characterization and Sensitivity to Human Adenovirus Serotypes 3, 5, and 11p of New Cell Lines Derived from Human Diffuse Grade II to IV Gliomas

BACKGROUND: Oncolytic adenoviruses show promise in targeting gliomas because they do not replicate in normal brain cells. However, clinical responses occur only in a subset of patients. One explanation could be the heterogenic expression level of virus receptors. Another contributing factor could be variable activity of tumor antiviral defenses in different glioma subtypes. METHODS: We established a collection of primary low-passage cell lines from different glioma subtypes (3 glioblastomas, 3 oligoastrocytomas, and 2 oligodendrogliomas) and assessed them for receptor expression and sensitivity to human adenovirus (HAd) serotypes 3, 5, and 11p. To gauge the impact of antiviral defenses, we also compared the infectivity of the oncolytic adenoviruses in interferon (IFN)-pretreated cells with IFN-sensitive Semliki Forest virus (SFV). RESULTS: Immunostaining revealed generally low expression of HAd5 receptor CAR in both primary tumors and derived cell lines. HAd11p receptor CD46 levels were maintained at moderate levels in both primary tumor samples and derived cell lines. HAd3 receptor DSG-2 was reduced in the cell lines compared to the tumors. Yet, at equal multiplicities of infection, the oncolytic potency of HAd5 in vitro in tumor-derived cells was comparable to HAd11p, whereas HAd3 lysed fewer cells than either of the other two HAd serotypes in 72 hours. IFN blocked replication of SFV, while HAds were rather unaffected. CONCLUSIONS: Adenovirus receptor levels on glioma-derived cell lines did not correlate with infection efficacy and may not be a relevant indicator of clinical oncolytic potency. Adenovirus receptor analysis should be preferentially performed on biopsies obtained perioperatively.     

Estimating net primary production of boreal forests in Finland and Sweden from field data and remote sensing

We calculated annual mean stem volume increment (AMSVI) and total litter fall to produce forest net primary production (NPP) maps at 1‐km² and half‐degree resolutions in Finland and Sweden. We used a multi‐scale methodology to link field inventory data reported at plot and forestry district levels through a remotely sensed total plant biomass map derived from 1‐km² AVHRR image. Total litter fall was estimated as function of elevation and latitude. Leaf litter fall, a surrogate for fine root production, was estimated from total litter fall by forest type. The gridded NPP estimates agreed well with previously reported NPP values, based on point measurements. Regional NPP increases from northeast to southwest. It is positively related to annual mean temperature and annual mean total precipitation (strongly correlated with temperature) and is negatively related to elevation at broad scale. Total NPP (TNPP) values for representative cells selected based on three criteria were highly correlated with simulated values from a process‐based model (CEVSA) at 0.5°× 0.5° resolution. At 1‐km² resolution, mean above‐ground NPP in the region was 408 g/m²/yr ranging from 172 to 1091 (standard deviation (SD) = 134). Mean TNPP was 563 (252 to 1426, SD = 176). Ranges and SD were reduced while the mean values of the estimated NPP stayed almost constant as cell size increased from 1‐km² to 0.5°× 0.5°, as expected. Nordic boreal forests seem to have lower productivity among the world boreal forests.     

Air temperature triggers the recovery of evergreen boreal forest photosynthesis in spring

The timing of the commencement of photosynthesis (P^*) in spring is an important determinant of growing‐season length and thus of the productivity of boreal forests. Although controlled experiments have shed light on environmental mechanisms triggering release from photoinhibition after winter, quantitative research for trees growing naturally in the field is scarce. In this study, we investigated the environmental cues initiating the spring recovery of boreal coniferous forest ecosystems under field conditions. We used meteorological data and above‐canopy eddy covariance measurements of the net ecosystem CO₂ exchange (NEE) from five field stations located in northern and southern Finland, northern and southern Sweden, and central Siberia. The within‐ and intersite variability for P^* was large, 30–60 days. Of the different climate variables examined, air temperature emerged as the best predictor for P^* in spring. We also found that ‘soil thaw’, defined as the time when near‐surface soil temperature rapidly increases above 0°C, is not a useful criterion for P^*. In one case, photosynthesis commenced 1.5 months before soil temperatures increased significantly above 0°C. At most sites, we were able to determine a threshold for air‐temperature‐related variables, the exceeding of which was required for P^*. A 5‐day running‐average temperature (T₅) produced the best predictions, but a developmental‐stage model (S) utilizing a modified temperature sum concept also worked well. But for both T₅ and S, the threshold values varied from site to site, perhaps reflecting genetic differences among the stands or climate‐induced differences in the physiological state of trees in late winter/early spring. Only at the warmest site, in southern Sweden, could we obtain no threshold values for T₅ or S that could predict P^* reliably. This suggests that although air temperature appears to be a good predictor for P^* at high latitudes, there may be no unifying ecophysiological relationship applicable across the entire boreal zone.     

Search for the determinants of allergenicity in proteins of the lipocalin family

Three different lines of analysis have been applied to approach the problem of the allergenicity of certain proteins: biological functions, molecular structures and immunological properties. It is immediately obvious that these three are interdependent. The lipocalin family of proteins includes a significant number of allergens. A considerable amount of data is already available of lipocalins and some insights about allergenic determinants can now be presented. However, more information on the molecular structures and immunological parameters of lipocalin allergens is required.