Outcome-related metabolomic patterns from 1H/31P NMR after mild hypothermia treatments of oxygen–glucose deprivation in a neonatal brain slice model of asphyxia

Human clinical trials using 72 hours of mild hypothermia (32°C–34°C) after neonatal asphyxia have found substantially improved neurologic outcomes. As temperature changes differently modulate numerous metabolite fluxes and concentrations, we hypothesized that 1H/31P nuclear magnetic resonance (NMR) spectroscopy of intracellular metabolites can distinguish different insults, treatments, and recovery stages. Three groups of superfused neonatal rat brain slices underwent 45 minutes oxygen–glucose deprivation (OGD) and then were: treated for 3 hours with mild hypothermia (32°C) that began with OGD, or similarly treated with hypothermia after a 15-minute delay, or not treated (normothermic control group, 37°C). Hypothermia was followed by 3 hours of normothermic recovery. Slices collected at different predetermined times were processed, respectively, for 14.1 Tesla NMR analysis, enzyme-linked immunosorbent assay (ELISA) cell-death quantification, and superoxide production. Forty-nine NMR-observable metabolites underwent a multivariate analysis. Separated clustering in scores plots was found for treatment and outcome groups. Final ATP (adenosine triphosphate) levels, severely decreased at normothermia, were restored equally by immediate and delayed hypothermia. Cell death was decreased by immediate hypothermia, but was equally substantially greater with normothermia and delayed hypothermia. Potentially important biomarkers in the 1H spectra included PCr-1H (phosphocreatine in the 1H spectrum), ATP-1H (adenosine triphosphate in the 1H spectrum), and ADP-1H (adenosine diphosphate in the 1H spectrum). The findings suggest a potential role for metabolomic monitoring during therapeutic hypothermia

Structural analysis of three novel trisaccharides isolated from the fermented beverage of plant extracts

<p>Abstract</p> <p>Background</p> <p>A fermented beverage of plant extracts was prepared from about fifty kinds of vegetables and fruits. Natural fermentation was carried out mainly by lactic acid bacteria (<it>Leuconostoc </it>spp.) and yeast (<it>Zygosaccharomyces </it>spp. and <it>Pichia </it>spp.). We have previously examined the preparation of novel four trisaccharides from the beverage: <it>O</it>-β-D-fructopyranosyl-(2->6)-<it>O</it>-β-D-glucopyranosyl-(1->3)-D-glucopyranose, <it>O</it>-β-D-fructopyranosyl-(2->6)-<it>O</it>-[β-D-glucopyranosyl-(1->3)]-D-glucopyranose, <it>O</it>-β-D-glucopyranosyl-(1->1)-<it>O</it>-β-D-fructofuranosyl-(2<->1)-α-D-glucopyranoside and <it>O</it>-β-D-galactopyranosyl-(1->1)-<it>O</it>-β-D-fructofuranosyl-(2<->1)- α-D-glucopyranoside.</p> <p>Results</p> <p>Three further novel oligosaccharides have been found from this beverage and isolated from the beverage using carbon-Celite column chromatography and preparative high performance liquid chromatography. Structural confirmation of the saccharides was provided by methylation analysis, MALDI-TOF-MS and NMR measurements.</p> <p>Conclusion</p> <p>The following novel trisaccharides were identified: <it>O</it>-β-D-fructofuranosyl-(2->1)-<it>O</it>-[β-D-glucopyranosyl-(1->3)]-β-D-glucopyranoside (named "3^G-β-D-glucopyranosyl β, β-isosucrose"), <it>O</it>-β-D-glucopyranosyl-(1->2)-<it>O</it>-[β-D-glucopyranosyl-(1->4)]-D-glucopyranose (4¹-β-D-glucopyranosyl sophorose) and <it>O</it>-β-D-fructofuranosyl-(2->6)-<it>O</it>-β-D-glucopyranosyl-(1->3)-D-glucopyranose (6²-β-D-fructofuranosyl laminaribiose).</p

Characterisation of Human Embryonic Stem Cells Conditioning Media by 1H-Nuclear Magnetic Resonance Spectroscopy

BACKGROUND: Cell culture media conditioned by human foreskin fibroblasts (HFFs) provide a complex supplement of protein and metabolic factors that support in vitro proliferation of human embryonic stem cells (hESCs). However, the conditioning process is variable with different media batches often exhibiting differing capacities to maintain hESCs in culture. While recent studies have examined the protein complement of conditioned culture media, detailed information regarding the metabolic component of this media is lacking. METHODOLOGY/PRINCIPAL FINDINGS: Using a (1)H-Nuclear Magnetic Resonance ((1)H-NMR) metabonomics approach, 32 metabolites and small compounds were identified and quantified in media conditioned by passage 11 HFFs (CMp11). A number of metabolites were secreted by HFFs with significantly higher concentration of lactate, alanine, and formate detected in CMp11 compared to non-conditioned media. In contrast, levels of tryptophan, folate and niacinamide were depleted in CMp11 indicating the utilisation of these metabolites by HFFs. Multivariate statistical analysis of the (1)H-NMR data revealed marked age-related differences in the metabolic profile of CMp11 collected from HFFs every 24 h over 72 h. Additionally, the metabolic profile of CMp11 was altered following freezing at -20°C for 2 weeks. CM derived from passage 18 HFFs (CMp18) was found to be ineffective at supporting hESCs in an undifferentiated state beyond 5 days culture. Multivariate statistical comparison of CMp11 and CMp18 metabolic profiles enabled rapid and clear discrimination between the two media with CMp18 containing lower concentrations of lactate and alanine as well as higher concentrations of glucose and glutamine. CONCLUSIONS/SIGNIFICANCE: (1)H-NMR-based metabonomics offers a rapid and accurate method of characterising hESC conditioning media and is a valuable tool for monitoring, controlling and optimising hESC culture media preparation

NMR and in silico studies of fucosylated chondroitin sulfate (fCS) and its interactions with selectins

This thesis describes structural studies on the interactions between the fucosylated chondroitin sulfate (fCS) oligosaccharides and human proteins known as selectins. fCS is a carbohydrate obtained from sea cucumbers, that can be classified as a branched glycosaminoglycan (GAG). It has attracted much attention due to its anti-coagulant, anti-inflammatory, antimetastatic and anti-HIV properties and its structure was previously determined by NMR. Selectins constitute a family of proteins involved in cell adhesion processes, such as inflammation, attachment of viral particles and migration of tumour cells. fCS oligosaccharides have been shown to bind to selectins, which is likely a reason behind their biological activity. However, the mechanism of this interaction is currently unknown. The initial part of the thesis describes the experimental work on expression and purification of the recombinant L- and P-selectin constructs in Pichia pastoris, Escherichia coli and HEK 293 cells. The aim of these experiments was to produce two constructs for each selectin, a single domain construct, consisting of the C-type lectin domain only, and a double domain construct, consisting of both the C-type lectin and the EGF-like domains. The intention was that the recombinant proteins would be labelled with 13C and 15N to allow for the in-depth structural NMR studies on the fCS-selectin interaction. Various experimental approaches have been explored, including the use of different cell lines, modifications to construct design, as well as alterations to expression and purification conditions. Although it was not possible to produce soluble selectin constructs in either bacterial or yeast cells, protein expression tests in HEK293 cells, performed in collaboration with the Oxford Protein Production facility (OPPF), led to production of a soluble L-selectin construct, consisting of the L-selectin C-type lectin domain. The produced L-selectin construct, as well as two commercially available constructs of the Land P-selectin extracellular domains, were used in the Saturation Transfer Difference (STD) NMR experiments to provide new information about the nature of the fCS-selectin binding. The STD experiments allowed to identify the regions within the fCS oligosaccharides that are in direct contact with the protein and likely play an important role in this interaction. Experiments on different protein constructs allowed the comparison of fCS binding to P-selectin and to two different recombinant constructs of L-selectin. Results of these studies suggest that the binding occurs via a similar mechanism for both L- and P-selectins and that the fCS oligosaccharides bind to one-domain L-selectin construct with similar affinity as to a larger construct, consisting of the entire extracellular region of the protein. Alongside the experimental work, theoretical in silico studies on the fCS-selectin binding were undertaken as part of this project. The existing X-ray structures of selectin complexes were subjected to Molecular Dynamics (MD) simulations, which allowed to explore the dynamic behaviour of E-selectin upon binding to sialyl Lewis x (sLex). It was found that sLex forms a more favourable interaction with the extended conformation of E-selectin and that the protein in this conformation is characterised by a high degree of interdomain flexibility, with a new type of interdomain movement observed in the MD studies on this complex. In further in silico studies, the fCS oligosaccharides were docked to the existing P-selectin structures. The docking tests were performed on the computationally produced fCS trisaccharides with fucose branches either 2,4 or 3,4-sulfated. Results were evaluated with MD simulations and analysed in the light of current knowledge of selectin-ligand binding and the STD NMR experimental results. The in silico studies allowed to identify a subset of P-selectin residues that are likely involved in the interaction with fCS oligosaccharides in vivo. The conformational behaviour of P-selectin upon binding to fCS was also explored and it was found that the interdomain hinge is flexible during this interaction and allows transition from bent to extended conformational state. Finally, a new NMR method was developed to facilitate the studies of complex carbohydrates, incorporating the concepts of G-matrix Fourier Transform (GFT) NMR into 2D HSQC and 2D HSQC-TOCSY experiments. The method allows to separate peaks in the regions of high spectral overlap, providing information that can simplify the assignment process. The new experiments facilitated the structural evaluation of a sample containing a mixture of oligosaccharides resulting from the depolymerisation of fCS polysaccharide

Adaptation of cellular metabolism to anisosmotic conditions in a glial cell line, as assessed by 13C-NMR spectroscopy

13C-NMR spectroscopy of perchloric acid and lipid extracts of F98 glioma cells showed that volume-regulatory processes under anisosmotic conditions were accompanied by marked alterations in cellular metabolism. Production of alanine, glutamate, and glycine from [U-13C]-glucose is decreased under hypotonic stress and is oppositely increased under hypertonic stress. In contrast, degradation of these molecules is raised under hypotonic conditions and reduced under hypertonic conditions. Furthermore, phospholipid synthesis is decreased under hypertonic stress and increased under hypotonic stress. Obviously, glial metabolism is directed under hypertonic conditions to maintain a high level of small, osmotically active molecules, whereas under hypotonic conditions molecular fragments are increasingly incorporated into the phospholipids and so do not contribute to the osmotic pressure. The latter is evoked by the activation of membrane synthesis process to compensate for stretching and/or damaging of the membranes due to cell swelling