Plasmanylethanolamine desaturase (PEDS) (EC 1.14.99.19) introduces the 1-prime double bond into plasmalogens, one of the most abundant phospholipids in the human body. This labile membrane enzyme has ...not been purified and its coding sequence is unknown. Previous assays for this enzyme used radiolabeled substrates followed by multistep processing. We describe here a straight-forward method for the quantification of PEDS in enzyme incubation mixtures using pyrene-labeled substrates and reversed-phase HPLC with fluorescence detection. After stopping the reaction with hydrochloric acid in acetonitrile, the mixture was directly injected into the HPLC system without the need of lipid extraction. The substrate, 1-O-pyrenedecyl-2-acyl-sn-glycero-3-phosphoethanolamine, and the lyso-substrate, 1-O-pyrenedecyl-sn-glycero-3-phosphoethanolamine, were prepared from RAW-12 cells deficient in PEDS activity and were compared for their performance in the assay. Plasmalogen levels in mouse tissues and in cultured cells did not correlate with PEDS levels, indicating that the desaturase might not be the rate limiting step for plasmalogen biosynthesis. Among selected mouse organs, the highest activities were found in kidney and in spleen. Incubation of intact cultivated mammalian cells with 1-O-pyrenedecyl-sn-glycerol, extraction of lipids, and treatment with hydrochloric or acetic acid in acetonitrile allowed sensitive monitoring of PEDS activity in intact cells.
Alzheimer's disease (AD) is a severe and chronic neurodegenerative disorder of the brain. The laboratory diagnosis is limited to the analysis of three biomarkers in cerebrospinal fluid (CSF): ...amyloid-β42 (Aβ42), total tau, and phospho-tau-181 (P-tau-181). However, there is a need to find more biomarkers in CSF that can improve the sensitivity and specificity. The aim of the present study was to analyze endogenous small metabolites (metabolome) in the CSF, which may provide potentially new insights into biochemical processes involved in AD. One hundred CSF samples were dichotomized by normal (n = 50) and pathological decreased Aβ42 and increased tau and P-tau-181 levels (n = 50; correlating to an AD-like pathology). These CSF samples were analyzed using the AbsoluteIDQ® p180 Kit (BIOCRATES Life Sciences), which included 40 acylcarnitines, 21 amino acids, 19 biogenic amines, 15 sphingolipids, and 90 glycerophospholipids. Our data show that two sphingomyelins (SM (d18:1/18:0) and SM (d18:1/18:1)), 5 glycerophospholipids (PC aa C32:0, PC aa C34:1, PC aa C36:1, PC aa C38:4 and PC aa C38:6), and 1 acylcarnitine (C3-DC-M/C5-OH) were significantly altered in the CSF with pathological "AD-like pathology". Sphingomyelin SM (d18:1/18:0) proved to be a specific (76%) and sensitive (66%) biomarker with a defined cut-off of 546 nM. Correct diagnoses for 21 out of 32 unknown samples could be achieved using this SM (d18:1/18:0) cut-off value. In conclusion, the sphingolipid SM (d18:1/18:0) is significantly increased in CSF of patients displaying pathological levels of Aβ42, tau, and P-tau-181.
Afamin, a human plasma glycoprotein and putative transporter of hydrophobic molecules, has been shown to act as extracellular chaperone for poorly soluble, acylated Wnt proteins, forming a stable, ...soluble complex with functioning Wnt proteins. The 2.1-Å crystal structure of glycosylated human afamin reveals an almost exclusively hydrophobic binding cleft capable of harboring large hydrophobic moieties. Lipid analysis confirms the presence of lipids, and density in the primary binding pocket of afamin was modeled as palmitoleic acid, presenting the native O-acylation on serine 209 in human Wnt3a. The modeled complex between the experimental afamin structure and a Wnt3a homology model based on the XWnt8-Fz8-CRD fragment complex crystal structure is compelling, with favorable interactions comparable with the crystal structure complex. Afamin readily accommodates the conserved palmitoylated serine 209 of Wnt3a, providing a structural basis how afamin solubilizes hydrophobic and poorly soluble Wnt proteins.
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•The molecular structure of human glycoprotein afamin is reported•Afamin is a lipid transporter and marker for metabolic syndrome and various cancers•Afamin solubilizes hydrophobic, acylated Wnt proteins in active form•The afamin-Wnt3a complex model suggests a structural basis for Wnt solubilization
Naschberger et al. report the structure of afamin, a human plasma glycoprotein and transporter of hydrophobic molecules, thought to solubilize the hydrophobic Wnt signaling proteins. The authors' model of the Wnt3a-afamin complex, based on the crystal structure of afamin and a homology model of Wnt3a, has implications for embryonic intercellular signaling.
The spatial and temporal characterization of geochemical tracers over Alpine glacierized catchments is particularly difficult, but fundamental to quantify groundwater, glacier melt, and rain water ...contribution to stream runoff. In this study, we analysed the spatial and temporal variability of δ2H and electrical conductivity (EC) in various water sources during three ablation seasons in an 8.4‐km2 glacierized catchment in the Italian Alps, in relation to snow cover and hydro‐meteorological conditions. Variations in the daily streamflow range due to melt‐induced runoff events were controlled by maximum daily air temperature and snow covered area in the catchment. Maximum daily streamflow decreased with increasing snow cover, and a threshold relation was found between maximum daily temperature and daily streamflow range. During melt‐induced runoff events, stream water EC decreased due to the contribution of glacier melt water to stream runoff. In this catchment, EC could be used to distinguish the contribution of subglacial flow (identified as an end member, enriched in EC) from glacier melt water to stream runoff, whereas spring water in the study area could not be considered as an end member. The isotopic composition of snow, glacier ice, and melt water was not significantly correlated with the sampling point elevation, and the spatial variability was more likely affected by postdepositional processes. The high spatial and temporal variability in the tracer signature of the end members (subglacial flow, rain water, glacier melt water, and residual winter snow), together with small daily variability in stream water δ2H dynamics, are problematic for the quantification of the contribution of the identified end members to stream runoff, and call for further research, possibly integrated with other natural or artificial tracers.
Primary successions of glacier forelands are unique model systems to investigate community dynamics and assembly processes. However, successional changes of plant and insect communities have been ...mainly analysed separately. Therefore, changes in plant–insect interactions along successional gradients on glacier forelands remain unknown, despite their relevance to ecosystem functioning. This study assessed how successional changes of the vegetation influenced the composition of the flower-visiting insect assemblages of two plant species,
Leucanthemopsis alpina
(L.) Heyw. and
Saxifraga bryoides
L., selected as the only two insect-pollinated species occurring along the whole succession. In addition, we investigated the links between reproductive output of these plants and pollinator abundance through experimental exclusion of pollinators. Plant community structure changed along the succession, affecting the distribution and the abundance of insects via idiosyncratic responses of different insect functional groups.
L. alpina
interacted with ubiquitously distributed pollinators, while
S. bryoides
pollinators were positively associated with insect-pollinated plant species density and
S. bryoides
abundance. With succession proceeding, insect assemblages became more functionally diverse, with the abundance of parasitoids, predators and opportunists positively related to an increase in plant cover and diversity. The reproductive output of both plant species varied among successional stages. Contrary to our expectation, the obligate insect-pollinated
L. alpina
showed a reproductive output rather independent from pollinator abundance, while the reproductive output of the self-fertile
S. bryoides
seemed linked to pollinator abundance. Observing ecological interactions and using functional traits, we provided a mechanistic understanding of community assembly processes along a successional gradient. Plant community diversity and cover likely influenced insect community assembly through bottom-up effects. In turn, pollinators regulate plant reproductive output through top-down control. We emphasise that dynamics of alpine plant and insect communities may be structured by biotic interactions and feedback processes, rather than only be influenced by harsh abiotic conditions and stochastic events.
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