Using in-tube extraction and slice selective NMR experiments allow imaging via statistical analysis of metabolic profiles.

Imaging the distribution of metabolites is very powerful in diagnostics but it is also employed in fundamental research. Although NMR spectroscopy is well established for determining metabolic profiles of biological samples, its application is limited to magnetic resonance imaging that can produce images of larger structures, but the number of detectable metabolites is very low. Mass spectrometry imaging on the other hand is well established with pixel sizes in the μm range.

Structural and biophysical characterization of the selenoprotein SelW1 from Chlamydomonas reinhardtii.

Selenoprotein W is widespread among pro- and eukaryotic organisms. It possesses antioxidant activity and plays pivotal roles in mammalian embryonic development and cellular functions. A very simple, prototypical selenoprotein W is SelW1 from Chlamydomonas.

Simple and Rapid (Extraction) Protocol for NMR Metabolomics-Direct Measurement of Hydrophilic and Hydrophobic Metabolites Using Slice Selection.

Investigating the metabolic profiles of solid sample materials with solution nuclear magnetic resonance (NMR) spectroscopy requires the extraction of these metabolites. This is commonly done by using two immiscible solvents such as water and chloroform for extraction. Subsequent solvent removal makes these extraction procedures very time-consuming.

Structural and biophysical characterization of the type VII collagen vWFA2 subdomain leads to identification of two binding sites.

Type VII collagen is an extracellular matrix protein, which is important for skin stability; however, detailed information at the molecular level is scarce. The second vWFA (von Willebrand factor type A) domain of type VII collagen mediates important interactions, and immunization of mice induces skin blistering in certain strains. To understand vWFA2 function and the pathophysiological mechanisms leading to skin blistering, we structurally characterized this domain by X-ray crystallography and NMR spectroscopy.

Maternally Inherited Differences within Mitochondrial Complex I Control Murine Healthspan.

Mitochondrial complex I-the largest enzyme complex of the mitochondrial oxidative phosphorylation machinery-has been proposed to contribute to a variety of age-related pathological alterations as well as longevity. The enzyme complex-consisting proteins are encoded by both nuclear (nDNA) and mitochondrial DNA (mtDNA). While some association studies of mtDNA encoded complex I genes and lifespan in humans have been reported, experimental evidence and the functional consequence of such variants is limited to studies using invertebrate models.

H-NMR spectroscopy shows cellular uptake of HEPES buffer by human cell lines-an effect to be considered in cell culture experiments.

HEPES is commonly used in cell culture media as a buffering substance. Compared to the bicarbonate/CO buffer system, it does not require a CO atmosphere, thereby ensuring stable pH values during handling of cell culture media outside of an incubator. Due to its intrinsic charge, HEPES is considered not to be taken up by cells, which was a prerequisite during buffer development for cell culture by Good and colleagues.

Changes in Neutrophil Metabolism upon Activation and Aging.

Neutrophil activation is an important mechanism of host defense against pathogens. Chronic inflammation and autoimmunity are often associated with abnormalities in phenotype and functions of neutrophils. Since effector functions of immune cells during inflammation are tightly linked to their metabolic state, changes in neutrophil metabolome upon activation have been investigated in this study.

Skin melanocytes and fibroblasts show different changes in choline metabolism during cellular senescence.

Unmodified cells undergo only a limited number of cell divisions until they enter a state termed cellular senescence. Other triggers like cytotoxic compounds can also induce cell senescence. Since cell senescence represents a major mechanism of tumor suppression this cellular state has attracted increasing attention.

A type VII collagen subdomain mutant is thermolabile and shows enhanced proteolytic degradability - Implications for the pathogenesis of recessive dystrophic epidermolysis bullosa?

Type VII collagen is the major constituent of anchoring fibrils. It has a central collagenous domain that is surrounded by a small C-terminal non-collagenous domain (NC2) and a large N-terminal non-collagenous (NC1) domain. Mutations in type VII collagen can lead to hereditary skin blistering disease dystrophic epidermolysis bullosa (DEB).

Metabolic Changes Investigated by Proton NMR Spectroscopy in Cells Undergoing Oncogene-Induced Senescence.

Investigating metabolic changes during different organismal or cellular states is of increasing interest. The combination of a data-rich analytical method like mass spectrometry or NMR spectroscopy with a statistical analysis identifies metabolites that are affected by a certain stimulus. Thus, important information on the underlying molecular pathways can be obtained.

Chemical shift assignments of the fibronectin III like domains 7-8 of type VII collagen.

Type VII collagen (Col7) is important for skin stability. This is underlined by the severe skin blistering phenotype in the Col7 related diseases dystrophic epidermolysis bullosa and epidermolysis bullosa acquisita (EBA). Col7 has a large N-terminal non-collagenous domain (NC1) that is followed by the triple helical collagenous domain.

Radiosensitive Hematopoietic Cells Determine the Extent of Skin Inflammation in Experimental Epidermolysis Bullosa Acquisita.

Animal models have enhanced our understanding of the pathogenesis of autoimmune diseases. For these models, genetically identical, inbred mice have commonly been used. Different inbred mouse strains, however, show a high variability in disease manifestation.

A recombinant fusion protein derived from dog hookworm inhibits autoantibody-induced dermal-epidermal separation ex vivo.

The proteins secreted by parasitic nematodes are evolutionarily optimized molecules with unique capabilities of suppressing the immune response of the host organism. Neutrophil inhibitory factor (NIF), which is secreted by the dog hookworm Ancylostoma caninum, binds to the β2 integrin CD11b/CD18, which is expressed on human neutrophils, eosinophils, monocytes and macrophages and inhibits neutrophil-dependent lung injury and neutrophil invasion of ischaemic brain tissue. Neutrophils are key players in the pathogenesis of subepidermal autoimmune blistering diseases (sAIBDs), and their pathogenic activities are crucially dependent on β2 integrin functionality.

The hinge region of type VII collagen is intrinsically disordered.

Type VII collagen (Col7) is important for skin integrity. As a major component of the anchoring fibrils, Col7 is essential for linking different skin layers together. The central collagenous domain of Col7 contains several interruptions of the collagen triple helix.

The cysteine-rich region of type VII collagen is a cystine knot with a new topology.

Collagens are a group of extracellular matrix proteins with essential functions for skin integrity. Anchoring fibrils are made of type VII collagen (Col7) and link different skin layers together: the basal lamina and the underlying connective tissue. Col7 has a central collagenous domain and two noncollagenous domains located at the N and C terminus (NC1 and NC2), respectively.

B cells, dendritic cells, and macrophages are required to induce an autoreactive CD4 helper T cell response in experimental epidermolysis bullosa acquisita.

In autoimmune bullous dermatoses (AIBD), autoantibodies induce blisters on skin or mucous membranes, or both. Mechanisms of continued autoantibody production and blistering have been well characterized using AIBD animal models. Mechanisms leading to the initial autoantibody production, however, have not been investigated in detail.

Metabolite analysis distinguishes between mice with epidermolysis bullosa acquisita and healthy mice.

Background: Epidermolysis bullosa acquisita (EBA) is a rare skin blistering disease with a prevalence of 0.2/ million people. EBA is characterized by autoantibodies against type VII collagen.

Metabolic changes during cellular senescence investigated by proton NMR-spectroscopy.

Cellular senescence is of growing interest due to its role in tumour suppression and its contribution to organismic ageing. This cellular state can be reached by replicative loss of telomeres or certain stresses in cell culture and is characterized by the termination of cell division; however, the cells remain metabolically active. To identify metabolites that are characteristic for senescent cells, extracts of human embryonic lung fibroblast (WI-38 cell line) have been investigated with NMR spectroscopy.

The vWFA2 domain of type VII collagen is responsible for collagen binding.

Type VII collagen (Col7) is the major component of anchoring fibrils and very important for skin integrity. This is emphasized by the Col7 related skin blistering diseases dystrophic epidermolysis bullosa and epidermolysis bullosa acquisita. Structural data that provides insights into the interaction network of Col7 and thus providing a basis for a better understanding of the pathogenesis of the diseases is missing.

Surprising complexity of the Asf1 histone chaperone-Rad53 kinase interaction.

The histone chaperone Asf1 and the checkpoint kinase Rad53 are found in a complex in budding yeast cells in the absence of genotoxic stress. Our data suggest that this complex involves at least three interaction sites. One site involves the H3-binding surface of Asf11 with an as yet undefined surface of Rad53.

Insight into interactions of the von-Willebrand-factor-A-like domain 2 with the FNIII-like domain 9 of collagen VII by NMR and SPR.

Type VII collagen as component of anchoring fibrils plays an important role in skin architecture, however, no detailed structural information is available. Here, we describe the recombinant expression, isotope labeling, and (1)H, (15)N, (13)C chemical shift assignment of a subdomain of the murine type VII collagen - the von-Willebrand-factor-A-like domain 2 (mvWFA2). vWFA2 interacts with type I collagen and plays a central role in certain skin blistering diseases.

Metabolic changes in autoimmune diseases.

Metabolomics is a recently emerging member of the "omics" technologies. It aims at detecting and quantifying all detectable small molecules, the so-called metabolites, present in a biological sample. Because of the lower number of metabolites compared with the number of genes and proteins identified in genomics and proteomics approaches, data of metabolic studies are less complex and may therefore be more informative.

Saturation transfer difference measurements with SU(VAR)3-9 and S-adenosyl-L-methionine.

Saturation transfer difference NMR measurements were performed to investigate the interaction of S-adenosyl-l-methionine (AdoMet) with SU(VAR)3-9 from Drosophila melanogaster. SU(VAR)3-9 has a SET domain and plays an important role in methylation of lysine-9 of histone H3 which results in gene silencing. We determined the binding epitope of AdoMet and compared it with a crystal structure of another SET protein.

Advanced approaches for the characterization of a de novo designed antiparallel coiled coil peptide.

We report here an advanced approach for the characterization of the folding pattern of a de novo designed antiparallel coiled coil peptide by high-resolution methods. Incorporation of two fluorescence labels at the C- and N-terminus of the peptide chain as well as modification of two hydrophobic core positions by Phe/[15N,13C]Leu enable the study of the folding characteristics and of distinct amino acid side chain interactions by fluorescence resonance energy transfer (FRET) and NMR spectroscopy. Results of both experiments reveal the antiparallel alignment of the helices and thus prove the design concept.

Determination of the binding specificity of the 12S subunit of the transcarboxylase by saturation transfer difference NMR.

In this study we present the characterization of the interaction of biotin and methylmalonyl-CoA (MMCoA) with the carboxyltransferase subunit (12S) from the transcarboxylase (TC) from Propionibacterium shermanii. This biotin dependent multienzyme complex catalyses the transfer of carbon dioxide from methylmalonyl-CoA (MMCoA) to pyruvate. The Saturation Transfer Difference NMR (STD) technique was performed to determine the binding epitope from biotin and MMCoA to the 12S subunit.