Urocortins in the zebrafish brain and redox regulation of embryonic development through Glutaredoxins

The cellular redox state is a central regulator of embryonic development. Redox homeostasis and thiol redox signaling are modulated through glutaredoxins (Grxs), glutathione dependent thiol-disulfide oxidoreductases. Grxs can reduce protein disulfides and protein-glutathione mixed disulfides (de-glutathionylation) via distinct reaction mechanisms. Although it has been shown that Grxs protect cells against oxidative stress induced apoptosis, known interaction partners are rare and physiological functions of this protein family are poorly understood. This thesis represents the first investigation of dithiol Grxs during vertebrate embryonic development, and we demonstrate that vertebrate specific Grx2 is essential for the formation of a functional brain and cardiovascular system. We characterized a redox circuit, in which Grx2 modulates the activity of a newly identified interaction partner, collapsin response mediator protein 2 (CRMP2), through the reduction of an intra-molecular disulfide. Thereby, Grx2 regulates axonal outgrowth and neuronal survival. Since CRMP2 and Grx2 have already been implicated in various neurological disorders, the redox circuit based on Grx2 might be a promising target for future therapeutic strategies. Additionally, we unraveled that development of functional vessels, heart, and erythrocytes in the zebrafish embryo is dependent on the de-glutathionylation activity of Grx2. These results have high clinical relevance, as defects in the cardiovascular system are a major reason for human embryonic mortality. Moreover, we sought to characterize zebrafish Grx2 (zfGrx2) biochemically and biophysically. Mössbauer spectroscopy as well as size-exclusion chromatography demonstrated the coordination of one [2Fe2S]2+ cluster per zfGrx2 monomer. Further analysis indicated that two out of four additional cysteines, which are conserved across the infraclass of bony fish, are involved in cluster coordination. As this mode of cluster binding is different to all yet described [FeS] Grxs, zfGrx2 might represent a new class of iron-sulfur Grxs. Embryonic development is also regulated by environmental factors, and the stress axis is responsible to mediate the body’s response to those stimuli. Here we investigated the expression pattern of urocortin (UCN) genes, important regulators of the stress axis, in the embryonic zebrafish brain. The specific expression sites indicate that UCNs might modulate locomotor activity through noradrenergic and serotonergic systems

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

Oxidants and Redox Signaling: Perspectives in Cancer Therapy, Inflammation, and Plasma Medicine

[No abstract available

Opposing Effects of Circadian Clock Genes Bmal1 and Period2 in Regulation of VEGF-Dependent Angiogenesis in Developing Zebrafish

SummaryMolecular mechanisms underlying circadian-regulated physiological processes remain largely unknown. Here, we show that disruption of the circadian clock by both constant exposure to light and genetic manipulation of key genes in zebrafish led to impaired developmental angiogenesis. A bmal1-specific morpholino inhibited developmental angiogenesis in zebrafish embryos without causing obvious nonvascular phenotypes. Conversely, a period2 morpholino accelerated angiogenic vessel growth, suggesting that Bmal1 and Period2 display opposing angiogenic effects. Using a promoter-reporter system consisting of various deleted vegf-promoter mutants, we show that Bmal1 directly binds to and activates the vegf promoter via E-boxes. Additionally, we provide evidence that knockdown of Bmal1 leads to impaired Notch-inhibition-induced vascular sprouting. These results shed mechanistic insight on the role of the circadian clock in regulation of developmental angiogenesis, and our findings may be reasonably extended to other types of physiological or pathological angiogenesis

A Systematic Analysis of Metal and Metalloid Concentrations in Eight Zebrafish Recirculating Water Systems

Metals and metalloids are integral to biological processes and play key roles in physiology and metabolism. Nonetheless, overexposure to some metals or lack of others can lead to serious health consequences. In this study, eight zebrafish facilities collaborated to generate a multielement analysis of their centralized recirculating water systems. We report a first set of average concentrations for 46 elements detected in zebrafish facilities. Our results help to establish an initial baseline for trouble-shooting purposes, and in general for safe ranges of metal concentrations in recirculating water systems, supporting reproducible scientific research outcomes with zebrafish

EST-analysis of the thermo-acidophilic red microalga Galdieriasulphuraria reveals potential for lipid A biosynthesis and unveils the pathway of carbon export from rhodoplasts

Weber APM, Oesterhelt C, Gross W, et al. EST-analysis of the thermo-acidophilic red microalga Galdieriasulphuraria reveals potential for lipid A biosynthesis and unveils the pathway of carbon export from rhodoplasts. Plant Molecular Biology. 2004;55(1):17-32.When we think of extremophiles, organisms adapted to extreme environments, prokaryotes come to mind first. However, the unicellular red micro-alga Galdieria sulphuraria (Cyanidiales) is a eukaryote that can represent up to 90% of the biomass in extreme habitats such as hot sulfur springs with pH values of 0-4 and temperatures of up to 56 degreesC. This red alga thrives autotrophically as well as heterotrophically on more than 50 different carbon sources, including a number of rare sugars and sugar alcohols. This biochemical versatility suggests a large repertoire of metabolic enzymes, rivaled by few organisms and a potentially rich source of thermo-stable enzymes for biotechnology. The temperatures under which this organism carries out photosynthesis are at the high end of the range for this process, making G. sulphuraria a valuable model for physical studies on the photosynthetic apparatus. In addition, the gene sequences of this living fossil reveal much about the evolution of modern eukaryotes. Finally, the alga tolerates high concentrations of toxic metal ions such as cadmium, mercury, aluminum, and nickel, suggesting potential application in bioremediation. To begin to explore the unique biology of G. sulphuraria, 5270 expressed sequence tags from two different cDNA libraries have been sequenced and annotated. Particular emphasis has been placed on the reconstruction of metabolic pathways present in this organism. For example, we provide evidence for (i) a complete pathway for lipid A biosynthesis; (ii) export of triose-phosphates from rhodoplasts; (iii) and absence of eukaryotic hexokinases. Sequence data and additional information are available at http://genomics.msu.edu/galdieria

Zebrafish heart development is regulated via glutaredoxin 2 dependent migration and survival of neural crest cells

Glutaredoxin 2 is a vertebrate specific oxidoreductase of the thioredoxin family of proteins modulating the intracellular thiol pool. Thereby, glutaredoxin 2 is important for specific redox signaling and regulates embryonic development of brain and vasculature via reversible oxidative posttranslational thiol modifications. Here, we describe that glutaredoxin 2 is also required for successful heart formation. Knock-down of glutaredoxin 2 in zebrafish embryos inhibits the invasion of cardiac neural crest cells into the primary heart field. This leads to impaired heart looping and subsequent obstructed blood flow. Glutaredoxin 2 specificity of the observed phenotype was confirmed by rescue experiments. Active site variants of glutaredoxin 2 revealed that the (de)-glutathionylation activity is required for proper heart formation. Our data suggest that actin might be one target during glutaredoxin 2 regulated cardiac neural crest cell migration and embryonic heart development. In summary, this work represents further evidence for the general importance of redox signaling in embryonic development and highlights additionally the importance of glutaredoxin 2 during embryogenesis

Inhibitors of PD-1/PD-L1 and ERK1/2 impede the proliferation of receptor positive and triple-negative breast cancer cell lines

Purpose!#!Triple-negative breast cancer (TNBC) is characterized by an unfavorable prognosis and missing systemic therapeutic approaches beside chemotherapy. Targeting the immune checkpoint PD-1/PD-L1 showed promising results in breast cancer and especially in TNBC. The extracellular signal-regulated kinase 1/2 (ERK1/2) is an important driver of carcinogenesis. Here, the effect of combined PD-1/PD-L1 and ERK1/2 inhibitor treatment is investigated of cell growth and intracellular impact of breast cancer cell lines.!##!Methods!#!The IC!##!Results!#!Both inhibitors PD-1/PD-L1 and ERK1/2 impeded the proliferation of TNBC to a higher extent than of non-TNBC. By combined treatment, cell lines were inhibited either synergistically or additively. ERK1/2 and S6 phosphorylation were reduced and expressions of c-Fos and FosL were diminished after ERK1/2 inhibitor as single and combined treatment. Between genes involved in immune modulation, IL-8 was upregulated in TNBC cells after combined treatment.!##!Conclusion!#!In conclusion, combination of PD-1/PD-L1 and ERK1/2 inhibitors showed favorable effects for a new therapy strategy, with better results in TNBC cell lines than in non-TNBC cells. The effects have to be validated in models that can reflect the interaction between immune and tumor cells like the situation in the tumor micro-environment