Investigating the effect of VEGF glycosylation on glycosaminoglycan binding and protein unfolding

VEGF165 binding to endothelial cells is potentiated by glycosaminoglycans (GAGs). Here, we have investigated the impact of VEGF165 N-glycosylation on GAG binding. Although glycosylated VEGF165 bound to heparin with only slightly higher affinity than non-glycosylated VEGF165, the natural ligand heparan sulfate induced a conformational change only in the glycosylated protein. Unfolding studies of the VEGF proteins indicated a stabilising effect of heparin on the growth factor structure.     

Proteomic analysis of heterotrophy in Synechocystis sp. PCC 6803

To provide an insight into the heterotrophic metabolism of cyanobacteria, a proteomic approach has been employed with the model organism Synechocystis sp. PCC 6803. The soluble proteins from Synechocystis grown under photoautotrophic and light-activated heterotrophic conditions were separated by 2-DE and identified by MALDI-MS or LC-MS/MS analysis. 2-DE gels made using narrow- and micro-range IPG strips allowed quantitative comparison of more than 900 spots. Out of 67 abundant protein spots identified, 13 spots were increased and 9 decreased under heterotrophy, representing all the major fold changes. Proteomic alterations and activity levels of selected enzymes indicate a shift in the central carbon metabolism in response to trophic change. The significant reduction in light-saturated rate of photosynthesis as well as in the expression levels of rubisco and CO(2)-concentrating mechanism proteins under heterotrophy indicates the down-regulation of the photosynthetic machinery. Alterations in the expression level of proteins involved in carbon utilization pathways refer to enhanced glycolysis, oxidative pentose phosphate pathway as well as tricarboxylic acid cycle under heterotrophy. Proteomic evidences also suggest an enhanced biosynthesis of amino acids such as histidine and serine during heterotrophic growth.     

Proteomic characterization of acid stress response in Synechocystis sp. PCC 6803

A comparative proteomic analysis using 2-DE coupled with MALDI-MS and LC-MS/MS was performed in Synechocystis sp. PCC 6803 to identify protein candidates involved in acid stress response in cyanobacteria. Comparison of soluble proteins from the cytoplasmic fraction of cells grown on media set at pH 7.5 and 5.5 using 2-DE identified four proteins, which showed significant changes in the abundance. Surprisingly, several general stress proteins, either the heat shock family proteins or chaperonins, did not show perceptible fold changes in response to acidity. Compared to the cytoplasmic proteome, the periplasmic proteome showed remarkable changes as a function of external pH. Protein expression profiling at different external pH, i.e., 9.0, 7.5, 6.0 and 5.5, allowed classifying the periplasmic proteins depending on their preferential expression patterns towards acidity or alkalinity. Among the acid- and base-induced proteins, oxalate decarboxylase and carbonic anhydrase were already known for their role in pH homeostasis. Several unknown proteins from the periplasm, that showed significant changes in response to pH, provide ideal targets for further studies in understanding pH stress response in cyanobacteria. This study also identified 14 novel proteins, hitherto unknown from the periplasmic space of Synechocystis.     

Prostatic acid phosphatase is an ectonucleotidase and suppresses pain by generating adenosine

Thiamine monophosphatase (TMPase, also known as fluoride-resistant acid phosphatase) is a classic histochemical marker of small-diameter dorsal root ganglia neurons. The molecular identity of TMPase is currently unknown. We found that TMPase is identical to the transmembrane isoform of prostatic acid phosphatase (PAP), an enzyme with unknown molecular and physiological functions. We then found that PAP knockout mice have normal acute pain sensitivity but enhanced sensitivity in chronic inflammatory and neuropathic pain models. In gain-of-function studies, intraspinal injection of PAP protein has potent antinociceptive, antihyperalgesic, and antiallodynic effects that last longer than the opioid analgesic morphine. PAP suppresses pain by functioning as an ecto-5'-nucleotidase. Specifically, PAP dephosphorylates extracellular adenosine monophosphate (AMP) to adenosine and activates A1-adenosine receptors in dorsal spinal cord. Our studies reveal molecular and physiological functions for PAP in purine nucleotide metabolism and nociception and suggest a novel use for PAP in the treatment of chronic pain.     

Lakes in the era of global change: moving beyond single-lake thinking in maintaining biodiversity and ecosystem services

The Anthropocene presents formidable threats to freshwater ecosystems. Lakes are especially vulnerable and important at the same time. They cover only a small area worldwide but harbour high levels of biodiversity and contribute disproportionately to ecosystem services. Lakes differ with respect to their general type (e.g. land-locked, drainage, floodplain and large lakes) and position in the landscape (e.g. highland versus lowland lakes), which contribute to the dynamics of these systems. Lakes should be generally viewed as ‘meta-systems’, whereby biodiversity is strongly affected by species dispersal, and ecosystem dynamics are contributed by the flow of matter and substances among locations in a broader waterscape context. Lake connectivity in the waterscape and position in the landscape determine the degree to which a lake is prone to invasion by non-native species and accumulation of harmful substances. Highly connected lakes low in the landscape accumulate nutrients and pollutants originating from ecosystems higher in the landscape. The monitoring and restoration of lake biodiversity and ecosystem services should consider the fact that a high degree of dynamism is present at local, regional and global scales. However, local and regional monitoring may be plagued by the unpredictability of ecological phenomena, hindering adaptive management of lakes. Although monitoring data are increasingly becoming available to study responses of lakes to global change, we still lack suitable integration of models for entire waterscapes. Research across disciplinary boundaries is needed to address the challenges that lakes face in the Anthropocene because they may play an increasingly important role in harbouring unique aquatic biota as well as providing ecosystem goods and services in the future.     

Shifting stoichiometry: Long-term trends in stream-dissolved organic matter reveal altered C:N ratios due to history of atmospheric acid deposition

Dissolved organic carbon (DOC) and nitrogen (DON) are important energy and nutrient sources for aquatic ecosystems. In many northern temperate, freshwater systems DOC has increased in the past 50 years. Less is known about how changes in DOC may vary across latitudes, and whether changes in DON track those of DOC. Here, we present long-term DOC and DON data from 74 streams distributed across seven sites in biomes ranging from the tropics to northern boreal forests with varying histories of atmospheric acid deposition. For each stream, we examined the temporal trends of DOC and DON concentrations and DOC:DON molar ratios. While some sites displayed consistent positive or negative trends in stream DOC and DON concentrations, changes in direction or magnitude were inconsistent at regional or local scales. DON trends did not always track those of DOC, though DOC:DON ratios increased over time for ~30% of streams. Our results indicate that the dissolved organic matter (DOM) pool is experiencing fundamental changes due to the recovery from atmospheric acid deposition. Changes in DOC:DON stoichiometry point to a shifting energy-nutrient balance in many aquatic ecosystems. Sustained changes in the character of DOM can have major implications for stream metabolism, biogeochemical processes, food webs, and drinking water quality (including disinfection by-products). Understanding regional and global variation in DOC and DON concentrations is important for developing realistic models and watershed management protocols to effectively target mitigation efforts aimed at bringing DOM flux and nutrient enrichment under control.     

Age and source of different forms of carbon released from boreal peatland streams during spring snowmelt in E. Finland

Isotopic data are increasingly being used to quantify and understand the processes that control the release of carbon (C) from northern peatlands. We used δ¹³C and ¹⁴C measurements to investigate the source and age of different forms of aquatic C (DOC, POC and evasion CO₂) released from 2 contrasting (undrained v drained) forested peatland catchments at the end of the winter snowmelt period in boreal E Finland. The δ¹³C_VPDB values of DOC (range ?28.3 to ?28.8 ‰) were generally more ¹³C depleted than evasion CO₂ (range ?22.7 to ?31.5 ‰) and showed no clear differences between the pre-flood, flood and post-flood periods. Both forms of C had evidence of bomb-¹⁴C (i.e. >100%modern), indicating that they contained substantial quantities of C fixed since the mid AD 1950s. However, DOC was ¹⁴C enriched compared to evasion CO₂, with ¹⁴C concentrations suggesting that, on average, DOC-C was ~5–6 years younger than evasion CO₂–C, with the most recently fixed C being released when temperatures were highest. POC was significantly depleted in ¹⁴C with conventional (uncalibrated) radiocarbon ages of 805–1135 BP. In contrast to other studies, the isotopic compositions of DOC and evasion CO₂ were very similar, suggesting a predominantly single and consistent C source (decomposition of soil organic matter; SOM) during the snowmelt period. Whilst we found no evidence to suggest that old (pre-bomb) C was being released at the end of the winter period, the drained site was associated with more ¹⁴C depleted and ¹³C enriched evasion CO₂, suggesting a closer link to the atmospheric CO₂ pool. Our isotopic data suggest that the various forms of C released to the aquatic system from these forested Finnish peatlands are closely related, largely unaffected by drainage and (at least in the case of evasion CO₂ and DOC) indicate strong connectivity between C cycling in the soil–plant–water system.