Ca2+-Stimulated Catecholamine Release from alpha-Toxin Permeabilized PC12 Cells

Two possible cellular pathways of catecholamines from the chromaffin vesicles of PC 12 cells to the surrounding medium are explored in this study. The direct one circumventing the cytoplasm can be activated in a-toxin-permeabilized cells with micromolar levels of free Ca2+. Catecholamine metabolites formed in the cytoplasm (i.e., 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxyphenylethanol) are neither formed nor released from the cells under these conditions. However, when vesicular catecholamines were discharged into the cytoplasm by addition of the ionophore nigericin, such metabolites are formed and released into the medium independent of Ca2+. Both types of experiments provide direct evidence for the operation of Ca2+-induced exocytosis of dopamine and noradrenaline in permeabilized PC12 cells. The Ca2+ dependence of dopamine or noradrenaline release, as measured by the determination of the endogenous catecholamines using the high-performance liquid chromatography technique, exhibits two different phases. One is already activated below 1 pM free Ca2+ and plateaus at 1-5 pM free Ca2+, while a second occurs in the presence of larger amounts of free Ca2+ (10-100 pM). Ca2+-induced catecholamine release from the permeabilized cells can be modulated in different ways: It is enhanced by the phorbol ester 12-0-tetradecanoylphorbol 13-acetate and the diacylglycerol 1 -oleyl-2-acetylglycerol provided Mg*+/ATP is present, and it is inhibited by guanosine 5’-0-(3-thiotriphosphate). The latter effect is abolished by pretreatment of the cells with pertussis toxin but not by cholera toxin. Thus, it appears that Ca2+-induced exocytosis can be modulated via the protein kinase C system, as well as via GTP binding proteins

Proteomic Analysis of the Proplastid Envelope Membrane Provides Novel Insights into Small Molecule and Protein Transport across Proplastid Membranes

Bräutigam A, Weber APM. Proteomic Analysis of the Proplastid Envelope Membrane Provides Novel Insights into Small Molecule and Protein Transport across Proplastid Membranes. Molecular Plant. 2009;2(6):1247-1261.Proplastids are undifferentiated plastids of meristematic tissues that synthesize amino acids for protein synthesis, fatty acids for membrane lipid production, and purines and pyrimidines for DNA and RNA synthesis. Unlike chloroplasts, proplastids depend on supply, with reducing power, energy, and precursor metabolites from the remainder of the cell. Comparing proplastid and chloroplast envelope proteomes and the corresponding transcriptomes of leaves and shoot apex revealed a clearly distinct composition of the proplastid envelope. It is geared towards import of metabolic precursors and export of product metabolites for the rapidly dividing cell. The analysis also suggested a new role for the triosephosphate translocator in meristematic tissues, identified the route of organic nitrogen import into proplastids, and detected an adenine nucleotide exporter. The protein import complex contains the import receptors Toc120 and Toc132 and lacks the redox sensing complex subunits of Tic32, Tic55, and Tic62, which mirrors the expression patterns of the corresponding genes in leaves and the shoot apex. We further show that the protein composition of the internal membrane system is similar to etioplasts, as it is dominated by the ATP synthase complex and thus remarkably differs from that of chloroplast thylakoids

The role of membrane transport in metabolic engineering of plant primary metabolism

Weber APM, Bräutigam A. The role of membrane transport in metabolic engineering of plant primary metabolism. Current Opinion in Biotechnology. 2013;24(2):256-262.Plant cells are highly compartmentalized and so is their metabolism. Most metabolic pathways are distributed across several cellular compartments, which requires the activities of membrane transporters to catalyze the flux of precursors, intermediates, and end products between compartments. Metabolites such as sucrose and amino acids have to be transported between cells and tissues to supply, for example, metabolism in developing seeds or fruits with precursors and energy. Thus, rational engineering of plant primary metabolism requires a detailed and molecular understanding of the membrane transporters. This knowledge however still lags behind that of soluble enzymes. Recent advances include the molecular identification of pyruvate transporters at the chloroplast and mitochondrial membranes and of a new class of transporters called SWEET that are involved in the release of sugars to the apoplast

Needs-based family support – Perception, structures and challenges in practical implementation

such as low-threshold access to services. Beside structural framework conditions in context of family support, the question arises as to what extent the subjective perception of preventive family support is one more aspect of utilisation and to what extent preventive family support actually matches the needs of families. This Study asks as well what connotations and attitudes do families have regarding family support services? How important do families consider these offers and what are their needs? Based on a mixed-methods design, the Citizens’ Survey on Family Support was conducted by means of a standardised questionnaire in combination with interviews of family support users and non-users in order to elaborate deeper meaning structures through the qualitative analysis method of grounded theory. Summary survey results point out that family support in Germany includes a wide range of offers, which can promote a broad array of familial interests and competences, but not all families, diverse as they are, feel consciously addressed – or else they see obstacles to using family services. Our qualitative results point out that ‘family support’ as a term is neither clearly identified nor properly understood by many citizens – or else it has different connotations. However, those families who do use the services – within the framework of transitioning to parenthood – feel supported in their psychosocial adaptations and regulatory processes. In relation to results and as compared to other EU countries, practical implications for further developments in family support approaches are discussed

High-throughput colorimetric method for the parallel assay of glyoxylic acid and ammonium in a single extract

Bräutigam A, Gagneul D, Weber APM. High-throughput colorimetric method for the parallel assay of glyoxylic acid and ammonium in a single extract. Analytical Biochemistry. 2007;362(1):151-153

Comparative Proteomics of Chloroplast Envelopes from C-3 and C-4 Plants Reveals Specific Adaptations of the Plastid Envelope to C-4 Photosynthesis and Candidate Proteins Required for Maintaining C-4 Metabolite Fluxes (vol 148, pg 568, 2008)

Bräutigam A, Hoffmann-Benning S, Weber APM. Comparative Proteomics of Chloroplast Envelopes from C-3 and C-4 Plants Reveals Specific Adaptations of the Plastid Envelope to C-4 Photosynthesis and Candidate Proteins Required for Maintaining C-4 Metabolite Fluxes (vol 148, pg 568, 2008). Plant Physiology. 2008;148(3):1734

The Plastid Outer Envelope – A Highly Dynamic Interface between Plastid and Cytoplasm

Plastids are the defining organelles of all photosynthetic eukaryotes. They are the site of photosynthesis and of a large number of other essential metabolic pathways, such as fatty acid and amino acid biosyntheses, sulfur and nitrogen assimilation, and aromatic and terpenoid compound production, to mention only a few examples. The metabolism of plastids is heavily intertwined and connected with that of the surrounding cytosol, thus causing massive traffic of metabolic precursors, intermediates, and products. Two layers of biological membranes that are called the inner (IE) and the outer (OE) plastid envelope membranes bound the plastids of Archaeplastida. While the IE is generally accepted as the osmo-regulatory barrier between cytosol and stroma, the OE was considered to represent an unspecific molecular sieve, permeable for molecules of up to 10 kDa. However, after the discovery of small substrate specific pores in the OE, this view has come under scrutiny. In addition to controlling metabolic fluxes between plastid and cytosol, the OE is also crucial for protein import into the chloroplast. It contains the receptors and translocation channel of the TOC complex that is required for the canonical post-translational import of nuclear-encoded, plastid-targeted proteins. Further, the OE is a metabolically active compartment of the chloroplast, being involved in, e.g., fatty acid metabolism and membrane lipid production. Also, recent findings hint on the OE as a defense platform against several biotic and abiotic stress conditions, such as cold acclimation, freezing tolerance, and phosphate deprivation. Moreover, dynamic non-covalent interactions between the OE and the endomembrane system are thought to play important roles in lipid and non-canonical protein trafficking between plastid and endoplasmic reticulum. While proteomics and bioinformatics has provided us with comprehensive but still incomplete information on proteins localized in the plastid IE, the stroma, and the thylakoids, our knowledge of the protein composition of the plastid OE is far from complete. In this article, we report on the recent progress in discovering novel OE proteins to draw a conclusive picture of the OE. A “parts list” of the plastid OE will be presented, using data generated by proteomics of plastids isolated from various plant sources

MGST1, a GSH transferase/peroxidase essential for development and hematopoietic stem cell differentiation.

We show for the first time that, in contrast to other glutathione transferases and peroxidases, deletion of microsomal glutathione transferase 1 (MGST1) in mice is embryonic lethal. To elucidate why, we used zebrafish development as a model system and found that knockdown of MGST1 produced impaired hematopoiesis. We show that MGST1 is expressed early during zebrafish development and plays an important role in hematopoiesis. High expression of MGST1 was detected in regions of active hematopoiesis and co-expressed with markers for hematopoietic stem cells. Further, morpholino-mediated knock-down of MGST1 led to a significant reduction of differentiated hematopoietic cells both from the myeloid and the lymphoid lineages. In fact, hemoglobin was virtually absent in the knock-down fish as revealed by diaminofluorene staining. The impact of MGST1 on hematopoiesis was also shown in hematopoietic stem/progenitor cells (HSPC) isolated from mice, where it was expressed at high levels. Upon promoting HSPC differentiation, lentiviral shRNA MGST1 knockdown significantly reduced differentiated, dedicated cells of the hematopoietic system. Further, MGST1 knockdown resulted in a significant lowering of mitochondrial metabolism and an induction of glycolytic enzymes, energetic states closely coupled to HSPC dynamics. Thus, the non-selenium, glutathione dependent redox regulatory enzyme MGST1 is crucial for embryonic development and for hematopoiesis in vertebrates

The dicotyledonous NAD malic enzyme C4 plant Cleome gynandra displays age-dependent plasticity of C4 decarboxylation biochemistry

Sommer M, Bräutigam A, Weber APM. The dicotyledonous NAD malic enzyme C4 plant Cleome gynandra displays age-dependent plasticity of C4 decarboxylation biochemistry. Plant Biology. 2012;14(4):621-629.The C4 photosynthetic pathway enriches carbon dioxide in the vicinity of Rubisco, thereby enabling plants to assimilate carbon more efficiently. Three canonical subtypes of C4 exist, named after their main decarboxylating enzymes: NAD-dependent malic enzyme type, NADP-dependent malic enzyme type and phosphoenolpyruvate carboxykinase type. Cleome gynandra is known to perform NAD-ME type C4 photosynthesis. To further assess the mode of C4 in C. gynandra and its manifestation in leaves of different age, total enzyme activities of eight C4-related enzymes and the relative abundance of 31 metabolites were measured. C. spinosa was used as a C3 control. C. gynandra was confirmed as an NAD-ME type C4 plant in mid-aged leaves, whereas a mixed NAD-ME and PEPCK type was observed in older leaves. Young leaves showed a C3-C4 intermediate state with respect to enzyme activities and metabolite abundances. Comparative transcriptome analysis of mid-aged leaves of C. gynandra and C. spinosa showed that the transcript of only one aspartate aminotransferase (AspAT) isoform is highly abundant in C. gynandra. However, the canonical model of the NAD-ME pathway requires two AspATs, a mitochondrial and a cytosolic isoform. Surprisingly, our results indicate the existence of only one highly abundant AspAT isoform. Using GFP-fusion, this isozyme was localised exclusively to mitochondria. We propose a revised model of NAD-ME type C4 photosynthesis in C. gynandra, in which both AspAT catalysed reactions take place in mitochondria and PEPCK catalyses an alternative decarboxylating pathway

Multiomics surface receptor profiling of the NCI-60 tumor cell panel uncovers novel theranostics for cancer immunotherapy.

BACKGROUND Immunotherapy with immune checkpoint inhibitors (ICI) has revolutionized cancer therapy. However, therapeutic targeting of inhibitory T cell receptors such as PD-1 not only initiates a broad immune response against tumors, but also causes severe adverse effects. An ideal future stratified immunotherapy would interfere with cancer-specific cell surface receptors only. METHODS To identify such candidates, we profiled the surface receptors of the NCI-60 tumor cell panel via flow cytometry. The resulting surface receptor expression data were integrated into proteomic and transcriptomic NCI-60 datasets applying a sophisticated multiomics multiple co-inertia analysis (MCIA). This allowed us to identify surface profiles for skin, brain, colon, kidney, and bone marrow derived cell lines and cancer entity-specific cell surface receptor biomarkers for colon and renal cancer. RESULTS For colon cancer, identified biomarkers are CD15, CD104, CD324, CD326, CD49f, and for renal cancer, CD24, CD26, CD106 (VCAM1), EGFR, SSEA-3 (B3GALT5), SSEA-4 (TMCC1), TIM1 (HAVCR1), and TRA-1-60R (PODXL). Further data mining revealed that CD106 (VCAM1) in particular is a promising novel immunotherapeutic target for the treatment of renal cancer. CONCLUSION Altogether, our innovative multiomics analysis of the NCI-60 panel represents a highly valuable resource for uncovering surface receptors that could be further exploited for diagnostic and therapeutic purposes in the context of cancer immunotherapy