Podcasts about Synthase

Gestão humanizada é o tema do Papo Empreendedor dessa semana.Quer aprender sobre carreira, marketing, negócios, inovação e muita motivação?Quarta-feira, às 8h, no Papo Empreendedor da @guaruja929fm você vai conhecer a história do Francisco Deppermann Fortes.Francisco desenvolveu carreira na Gerdau, onde ocupou a posição de Vice-presidente executivo, membro do Comitê Executivo e liderança global das áreas de RH, Gestão, Inovação, TI, Backoffice, SSMA, Engenharia e Industrial. Conduziu o projeto de transformação cultural e digital da Gerdau envolvendo 40 mil colaboradores em 14 países. Par8cipou dos processos de sucessão familiar, desenvolvimento de herdeiros e identificação e mentoring de talentos internos.Atualmente é membro de conselhos e comitês, consultor, mentor e coach de executivos e empreendedores. É membro dos conselhos da Rede Marajó de Serviços, Frota Bank e Rede Frota e ONGs, Instituto C – Criança, Cuidado e Cidadania e nas startups Dêvi, Visto.Bio e Crëam e membro do Comitê de Pessoas, Cultura e Governança da Librelato. Como consultor executa projetos na área de estratégia, governança, cultura, gestão de talentos, inovação, diversidade e digital. É investidor anjo em startups pela WOW, Anjos do Brasil, Bemtevi e Synthase. É professor no curso de Empreendedorismo e Inovação do Hospital Albert Einstein e membro das comissões de Pessoas e de Inovação do IBGC.É formado em Engenharia com especialização na Alemanha e mestrado em Administração pela UFRGS, MBA pelo Insper e com educação execu8va em Stanford, Duke, Insead, HEC e Hong Kong University e formação como Conselheiro no IBGC. É cer8ficado em Coaching pelo Ins8tuto Ecosocial, SixSigma Blackbelt, ScrumMaster, Hogan e LSI Assessment e ferramentas de diagnós8co cultural pela Human Synergis8cs e Barret. Fez imersões em inovação e digital no Vale do Silício e Israel.Não fique de fora dessa!#guarujátáon #papoempreendedor #rádio #marketing #inovação #grandesempreendedores

Celena Kipping, agronomist for SW Missouri and SE Kansas joins me today to talk about the Site of Action (SOA) 2 chemicals. This large chemical family uses a mode of action that inhibits the synthesis of amino acids in the plant. These building blocks of cell membranes are critical to plant development and its life. Please watch this video to understand why it is so important to time applications, use overlapping residuals, and spray weeds when they are small. This video focuses on ALS chemistries in general but is more geared toward fall to winter to spring programs that can get you started with weed control on the right foot. TOP Ag Services is a Beck's Hybrids seed dealer. We provide Hybrid Corn Seed, Soybean Seed, and Wheat Seed. Beck's has access to the best genetics and trait technologies from suppliers worldwide. We are the first to market with the best products & provide the latest, most accurate agronomic information through proven research. If you need agronomic assistance or want to be added to these updates, feel free to reach out via the messaging feature or contact us at topagservices.com/contact

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.07.548115v1?rss=1 Authors: Listian, S. A., Kol, M., Ufelmann, E., Eising, S., Froehlich, F., Walter, S., Holthuis, J. C. M., Barisch, C. Abstract: Dictyostelium discoideum is a professional phagocyte frequently used as experimental model to study cellular processes underlying the recognition, engulfment and infection course of microbial pathogens. Sphingolipids are abundant components of the plasma membrane that bind cholesterol, control vital membrane properties, participate in signal transmission and serve as adhesion molecules in recognition processes relevant to immunity and infection. While the pathway of sphingolipid biosynthesis has been well characterized in plants, animals and fungi, the identity of sphingolipids produced in D. discoideum, an organism at the crossroads between uni- and multicellular life, is not known. Combining lipidomics with a bioinformatics-based cloning strategy for key sphingolipid biosynthetic enzymes, we show here that D. discoideum produces phosphoinositol-containing sphingolipids with predominantly phytoceramide backbones. Cell-free expression of candidate inositol-phosphorylceramide (IPC) synthases from D. discoideum in defined lipid environments enabled identification of an enzyme that selectively catalyses the transfer of phosphoinositol from phosphatidylinositol onto ceramide. The corresponding IPC synthase, DdIPCS1, is non-homologous to but shares multiple sequence motifs with yeast IPC and human sphingomyelin synthases and localizes to the Golgi apparatus as well as the contractile vacuole of D. discoideum. Collectively, these findings open up important opportunities for exploring a role of sphingolipids in phagocytosis and infection across major evolutionary boundaries. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.534899v1?rss=1 Authors: Spaas, J., Van der Stede, T., de Jager, S., van de Waterweg Berends, A., Tiane, A., Baelde, H., Baba, S. P., Eckhardt, M., Wolfs, E., Vanmierlo, T., Hellings, N., Eijnde, B. O., Derave, W. Abstract: Multiple sclerosis (MS) pathology features autoimmune-driven neuroinflammation, demyelination, and failed remyelination. Carnosine is a histidine-containing dipeptide (HCD) with pluripotent homeostatic properties that is able to improve outcomes in an animal MS model (EAE) when supplied exogenously. To uncover if endogenous carnosine is involved in, and protects against, MS-related neuroinflammation, demyelination or remyelination failure, we here studied the HCD-synthesizing enzyme carnosine synthase (CARNS1) in human MS lesions and two preclinical mouse MS models (EAE, cuprizone). We demonstrate that due to its presence in oligodendrocytes, CARNS1 expression is diminished in demyelinated MS lesions and mouse models mimicking demyelination/inflammation, but returns upon remyelination. Carns1-KO mice that are devoid of endogenous HCDs display exaggerated neuroinflammation and clinical symptoms during EAE, which could be partially rescued by exogenous carnosine treatment. Worsening of the disease appears to be driven by a central, not peripheral immune-modulatory, mechanism possibly linked to impaired clearance of the reactive carbonyl acrolein in Carns1-KO mice. In contrast, the presence of CARNS1 and endogenous HCDs does not protect against cuprizone-induced demyelination, and is not required for normal oligodendrocyte precursor cell differentiation and (re)myelin to occur. Exogenously administered carnosine is not effective in blunting demyelination or accelerating remyelination. In conclusion, we show that CARNS1 is diminished in demyelinated MS lesions, which may have detrimental effects on disease progression through weakening the endogenous protection against neuroinflammation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.17.533015v1?rss=1 Authors: Tao, X., Liu, J., Diaz-Perez, Z., Foley, J. R., Stewart, T. M., Casero, R. A., Zhai, G. Abstract: Tauopathy, including Alzheimer Disease (AD), is characterized by Tau protein accumulation and autophagy dysregulation. Emerging evidence connects polyamine metabolism with the autophagy pathway, however the role of polyamines in Tauopathy remains unclear. In the present study we investigated the role of spermine synthase (SMS) in autophagy regulation and tau protein processing in Drosophila and human cellular models of Tauopathy. Our previous study showed that Drosophila spermine synthase (dSms) deficiency impairs lysosomal function and blocks autophagy flux. Interestingly, partial loss-of-function of SMS in heterozygous dSms flies extends lifespan and improves the climbing performance of flies with human Tau (hTau) overexpression. Mechanistic analysis showed that heterozygous loss-of-function mutation of dSms reduces hTau protein accumulation through enhancing autophagic flux. Measurement of polyamine levels detected a mild elevation of spermidine in flies with heterozygous loss of dSms. SMS knock-down in human neuronal or glial cells also upregulates autophagic flux and reduces Tau protein accumulation. Proteomics analysis of postmortem brain tissue from AD patients showed a significant albeit modest elevation of SMS protein level in AD-relevant brain regions compared to that of control brains consistently across several datasets. Taken together, our study uncovers a correlation between SMS protein level and AD pathogenesis and reveals that SMS reduction upregulates autophagy, promotes Tau clearance, and reduces Tau protein accumulation. These findings provide a new potential therapeutic target of Tauopathy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.22.529417v1?rss=1 Authors: Hino, C., Chan, G., Jordaan, G., Chang, S. S., Saunders, J., Bashir, M. T., Hansen, J. E., Gera, J., Weisbart, R. H., Nishimura, R. N. Abstract: Heat shock proteins (HSPs), especially Hsp70 (HSPA1), have been associated with cellular protection from various cellular stresses including heat, hypoxia-ischemia, neurodegeneration, toxins, and trauma. Endogenous HSPs are often synthesized in direct response to these stresses but in many situations are inadequate in protecting cells. The present study addresses the transduction of Hsp70 into cells providing protection from acute oxidative stress by H2O2. The recombinant Fv-Hsp70 protein and two mutant Fv-Hsp70 proteins minus the ATPase domain, and minus the ATPase and terminal lid domains were tested at 0.5 and 1.0 uM concentrations after two different concentrations of H2O2 treatment. All three recombinant proteins protected SH-SY5Y cells from acute H2O2 toxicity. This data indicated that the protein binding domain was responsible for cellular protection. In addition, experiments pretreating cells with inhibitors of antioxidant proteins catalase and gamma-glutamylcysteine synthase (GGCS) before H2O2 resulted in cell death despite treatment with Fv-Hsp70, implying that both enzymes were protected from acute oxidative stress after treatment with Fv-Hsp70. This study demonstrates that Fv-Hsp70 is protective in our experiments primarily by the protein-binding domain. The Hsp70 terminal lid domain was also not necessary for protection. Cellular protection was protective via the antioxidant proteins catalase and GGCS. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

References Cancer Cell 2020. 40, Issue 4, Pages 365-378.e6 Circulation Research. . 2004 May 28;94(10):1318-24. Mol Cell. 2020 Jun 18;78(6):1192-1206.e10 Cell. 2012 Aug 17;150(4):685-96 Cell Death Differ. 2020 May; 27(5): 1660–1676. --- Send in a voice message: https://anchor.fm/dr-daniel-j-guerra/message Support this podcast: https://anchor.fm/dr-daniel-j-guerra/support

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.03.526715v1?rss=1 Authors: Elezaby, A., Lin, A. J., Vijayan, V., Pokhrel, S., Bechara, L. R., Ostberg, N. P., Queliconi, B. B., Campos, J. C., Ferreira, J. C., Haileselassie, B., Mochly-Rosen, D. Abstract: Cardiac troponin I (cTnI) is a sarcomeric protein critical to myocyte contraction. Unexpectedly, we found that some cTnI localized to the mitochondrial matrix in the heart, inhibited mitochondrial functions when stably expressed in non-cardiac cells and increased opening of the mitochondrial permeability transition pore under oxidative stress. Direct, specific, and saturable binding of cTnI to ATP synthase was demonstrated in vitro, using immune-captured ATP synthase, and in cells using proximity ligation assay. cTnI binding doubled F1F0 ATPase activity, whereas skeletal troponin I and several human mutant cTnI variants associated with familial hypertrophic cardiomyopathy did not. A rationally-designed ten amino acid peptide, P888, inhibited cTnI binding to ATP synthase and cTnI-induced increase in ATPase activity in vitro and reduced cardiac injury following transient ischemia in vivo. We therefore suggest that mitochondria-associated cTnI may inhibit cardiac ATP synthase under basal conditions; pharmacological agents that release this inactivating effect of cTnI and thus preventing ATP hydrolysis during cardiac ischemia may increase the reservoir of functional mitochondria to reduce cardiac injury. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.02.526833v1?rss=1 Authors: Usey, M. M., Huet, D. Abstract: Coiled-coil-helix-coiled-coil-helix (CHCH) domains consist of two pairs of cysteine residues that are oxidized to form disulfide bonds upon mitochondrial import. Proteins containing these domains play important roles in mitochondrial ultrastructure and in the biogenesis, function, and stability of electron transport chain complexes. Interestingly, recent investigations of the Toxoplasma gondii ATP synthase identified subunits containing CHCH domains. As CHCH domain proteins have never been found in any other ATP synthase, their role in T. gondii was unclear. Using conditional gene knockdown systems, we show that two T. gondii ATP synthase subunits containing CHCH domains are essential for the lytic cycle as well as stability and function of the ATP synthase. Further, we illustrated that knockdown disrupts multiple aspects of mitochondrial morphology. Mutation of key residues in the CHCH domains also caused mislocalization of the proteins. This work provides insight into the divergent aspects of the apicomplexan ATP synthase, which could uncover future drug targets. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.02.526754v1?rss=1 Authors: Sharma, R. K., Chafik, A., Bertolin, G. Abstract: Cancer cells often hijack metabolic pathways to obtain the energy required to sustain their proliferation. Understanding the molecular mechanisms underlying cancer cell metabolism is key to fine-tune the metabolic preference of specific tumors, and potentially offer new therapeutic strategies. Here, we show that the pharmacological inhibition of mitochondrial Complex V delays the cell cycle by arresting breast cancer cell models in the G0/G1 phase. Under these conditions, the abundance of the multifunctional protein Aurora kinase A/AURKA is specifically lowered. We then demonstrate that AURKA directly interacts with the mitochondrial Complex V core subunits ATP5F1A and ATP5F1B. Altering the AURKA/ATPF1A/ATPF1B nexus is sufficient to trigger G0/G1 arrest, and this is accompanied by decreased glycolysis and mitochondrial respiration rates. Last, we discover that the roles of the AURKA/ATPF1A/ATPF1B nexus depend on the specific metabolic propensity of triple-negative breast cancer cell lines, where they correlate with cell fate. On one hand, the nexus induces G0/G1 arrest in cells relying on oxidative phosphorylation as the main source of energy. On the other hand, it allows to bypass cell cycle arrest and it triggers cell death in cells with a glycolytic metabolism. Altogether, we provide evidence that AURKA and mitochondrial Complex V subunits cooperate to maintain cell metabolism in breast cancer cells. Our work paves the way to novel anti-cancer therapies targeting the AURKA/ATPF1A/ATPF1B nexus to lower cancer cell metabolism and proliferation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

In this Shortcast, Dr Arthavan Selvanathan of the Queensland Lifespan Metabolic Medicine Service, discusses his team's work: N-acetylglutamate synthase deficiency with associated 3-methylglutaconic aciduria: A case report N-acetylglutamate synthase deficiency with associated 3-methylglutaconic aciduria: A case report Arthavan Selvanathan, et al https://doi.org/10.1002/jmd2.12318

Age Ageing, Volume 48, Issue 6, November 2019, Pages 776–782 Biomedicines. 2020 Aug 6;8(8):277 --- Send in a voice message: https://anchor.fm/dr-daniel-j-guerra/message Support this podcast: https://anchor.fm/dr-daniel-j-guerra/support

This week, we’re welcoming our close friend Etienne Meyer to the show. Together with Joram, he just published a new paper on ATP synthase research and we take the chance to talk about it.

This episode is also available as a blog post: http://biopatrika.com/2021/03/05/interview-ptm-enzyme-tumor-breast-cancer/

On this ID the Future, biochemist Michael Denton delves further into his revelatory new book The Miracle of the Cell. Here he discusses finely tuned chemical bonds. Cellular life would be impossible if strong bonds weren’t just so for some cellular functions, and if weak bonds weren’t just so for others. Each type of bond exists in a Goldilocks zone, neither too strong nor too weak for its purposes. They’re tailored to fit. Denton also explores the miracle enzyme known as ATP synthase and some of the fine-tuning particulars of this life-essential molecular complex. Source

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.30.320408v1?rss=1 Authors: Sobti, M., Walshe, J. L., Zeng, Y. C., Ishmukhametov, R., Stewart, A. G. Abstract: F1Fo ATP synthase functions as a biological rotary generator that makes a major contribution to cellular energy production. Proton flow through the Fo motor generates rotation of the central stalk, inducing conformational changes in the F11 motor that catalyzes ATP production via flexible coupling. Here we present a range of cryo-EM structures of E. coli ATP synthase in different rotational and inhibited states observed following a 45 second incubation with 10 mM MgATP. The structures generated describe multiple changes that occur following addition of MgATP, with the inhibitory C-terminal domain of subunit {epsilon} ({epsilon}CTD) disassociating from the central stalk to adopt a condensed ''down'' conformation. The transition to the {epsilon}CTD down state increases the torsional flexibility of the central stalk allowing its foot to rotate by ~50{degrees}, with further flexing in the peripheral stalk enabling the c-ring to rotate by two sub-steps in the Fo motor. Truncation mutants lacking the second helix of the {epsilon}CTD suggest that central stalk rotational flexibility is important for F1Fo synthase function. Overall this study identifies the potential role played by torsional flexing within the rotor and how this could be influenced by the {epsilon} subunit. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.18.303636v1?rss=1 Authors: Spikes, T. E., Montgomery, M. G., Walker, J. E. Abstract: The ATP synthase complexes in mitochondria make the ATP required to sustain life by a rotary mechanism. Their membrane domains are embedded in the inner membranes of the organelle and they dimerize via interactions between their membrane domains. The dimers form extensive chains along the tips of the cristae with the two rows of monomeric catalytic domains extending into the mitochondrial matrix at an angle to each other. When the interaction between membrane domains is disrupted in living cells, the morphology of the cristae is affected severely. By analysis of particles of purified dimeric bovine ATP synthase by cryo-electron microscopy, we have shown that the angle between the central rotatory axes of the monomeric complexes varies between ca. 76o and ca. 95o. Some variations in this angle arise directly from the catalytic mechanism of the enzyme, and others are independent of catalysis. The monomer-monomer interaction is mediated mainly by j-subunits attached to the surface of wedge shaped protein-lipid structures in the membrane domain of the complex, and the angular variation arises from rotational and translational changes in this interaction, and combinations of both. The structures also suggest how the dimeric ATP synthases might be interacting with each other to form the characteristic rows along the tips of the cristae via other inter-wedge contacts, moulding themselves to the range of oligomeric arrangements observed by tomography of mitochondrial membranes, and at the same time allowing the ATP synthase to operate under the range of physiological conditions that influence the structure of the cristae. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.06.225375v1?rss=1 Authors: Guo, H., Courbon, G. M., Bueler, S. A., Mai, J., Liu, J., Rubinstein, J. L. Abstract: Tuberculosis (TB), the leading cause of death by infectious disease worldwide, is increasingly resistant to first line antibiotics. Developed from a screen against Mycobacterium smegmatis, bedaquiline can sterilize even latent M. tuberculosis infections that may otherwise persist for decades and has become a cornerstone of treatment for multidrug resistant and extensively-drug resistant TB. Bedaquiline targets mycobacterial ATP synthase, an essential enzyme in the obligate aerobic Mycobacterium genus. However, how the drug binds the intact enzyme is unknown. We determined the structure of M. smegmatis ATP synthase with and without bedaquiline. The drug-free structure reveals hook-like extensions from the enzyme's subunits that inhibit ATP hydrolysis in low-energy conditions, such as during latent infections. Bedaquiline binding induces global conformational changes in ATP synthase, creating tight binding pockets at the interface of subunits a and c. These binding sites explain the drug's structure-activity relationship and its potency as an antibiotic for TB. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.21.212928v1?rss=1 Authors: Minen, R. I., Martinez, M. P., Iglesias, A. A., Figueroa, C. M. Abstract: Raffinose (Raf) protects plant cells during seed desiccation and under different abiotic stress conditions. The biosynthesis of Raf starts with the production of UDP-galactose by UDP-sugar pyrophosphorylase (USPPase) and continues with the synthesis of galactinol by galactinol synthase (GolSase). Galactinol is then used by Raf synthase to produce Raf. In this work, we report the biochemical characterization of USPPase (BdiUSPPase) and GolSase 1 (BdiGolSase1) from Brachypodium distachyon. The catalytic efficiency of BdiUSPPase was similar with galactose 1-phosphate and glucose 1-phosphate, but 5-to 17-fold lower with other sugar 1-phosphates. The catalytic efficiency of BdiGolSase1 with UDP-galactose was three orders of magnitude higher than with UDP-glucose. A structural model of BdiGolSase1 allowed us to determine the residues putatively involved in the binding of substrates. Among these, we found that Cys261 lies within the putative catalytic pocket. BdiGolSase1 was inactivated by oxidation with diamide and H2O2. The activity of the diamide-oxidized enzyme was recovered by reduction with dithiothreitol or E. coli thioredoxin, suggesting that BdiGolSase1 is redox-regulated. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.18.159665v1?rss=1 Authors: Saundh, S., Patnaik, D., Gagne, S., Bishop, J., Lipsit, S., Amat, S., Pujari, N., Krishnan, A. K., Bigsby, R., Murphy, M., Tsai, L.-H., Haggarty, S., Leung, A. K.-W. Abstract: Glycogen Synthase Kinase 3-beta (GSK3{beta}) is a critical regulator of several cellular pathways involved in neuroplasticity and is a potential target for neurotherapeutic development in the treatment of neuropsychiatric and neurodegenerative diseases. The majority of efforts to develop inhibitors of GSK3{beta} have been focused on developing small molecule inhibitors that compete with ATP through direct interaction with the ATP binding site. This strategy has presented selectivity challenges due to the evolutionary conservation of this domain within the kinome. The Disrupted in Schizophrenia (DISC1) protein, has previously been shown to bind and inhibit GSK3{beta} activity. Here, we report the characterization of a 44-mer peptide derived from human DISC1 (hDISCtide) that is sufficient to both bind and inhibit GSK3{beta} in a non-competitive mode that is distinct from classical ATP competitive inhibitors. Based on multiple independent biochemical and biophysical assays, we propose that hDISCtide interacts at two distinct regions of GSK3{beta}: an inhibitory region that partially overlaps with the binding site of FRATide, a well-known GSK3{beta} binding peptide, and a specific binding region that is unique to hDISCtide. Taken together, our findings present a novel avenue for developing a peptide-based selective inhibitor of GSK3{beta}. Copy rights belong to original authors. Visit the link for more info

Intelligent Design, Echolocation, Hummingbird tongue, ATP Synthase, Dr David Berlinski, Evolution Vs. God Movie Excerpt. 

The TWiM team reveals how ribosome modification resuscitates bacterial persister cells, and explain how a phage tail fiber protein exploits rotation of flagella to move towards the cell membrane. Become a patron of TWiM. Links for this episode: Ribosome modification resuscitates persister cells (Environ Micro) Flagellotrophic phage targets host with tail fiber (Mol Micro) Hydrodynamics of phage migration along flagella (Phys Rev Fluids) Music used on TWiM is composed and performed by Ronald Jenkees and used with permission. Send your microbiology questions and comments to twim@microbe.tv  

In this episode Dr Wagner talks to us about his recent paper 'Role of neuronal nitric oxide synthase in the estrogenic attenuation of cannabinoid-induced changes in energy homeostasis' and highlights the need for gender specific therapies for metabolic disorders.

The TWiPwalas solve the case of the Woman with a Worm in Her Eye, and discuss the role of nitric oxide in the resistance of rats to Schistosoma japonicum. Hosts: Vincent Racaniello, Dickson Despommier, and Daniel Griffin Become a patron of TWiP. Links for this episode: Nitric oxide blocks schistosome development (PNAS) Photo credit: Dave the Sheep Shearer Letters read on TWiP 139 This episode is brought to you by Blue Apron. Blue Apron is the #1 fresh ingredient and recipe delivery service in the country. See what’s on the menu this week and get 3 meals free with your first purchase – WITH FREE SHIPPING – by going to blueapron.com/twip. Case Study for TWiP 139 Seen at Columbia Medical Center, a crossover. Woman in 30s returns to US after 2 years in Peace Corp, Cameroon and Gabon. On medical exam 2 years earlier: eosiniphilia noted, no diagnosis reached. Now comes to NYC 2 years later to attend grad school, again eosinophilia noted. Asymptomatic. Send your case diagnosis, questions and comments to twip@microbe.tv Music by Ronald Jenkees

The TWiP Titans solve the case of the Man from Queens with a Blister Burster, and explain the role of inflammatory monocytes during Leishmania infection of the skin. Hosts: Vincent Racaniello, Dickson Despommier, and Daniel Griffin Become a patron of TWiP. Links for this episode: Inflammatory monocytes and skin Leishmania (PLoS Path) Dracunculiasis (TWiP 37) Photo: Daniel using a LifeStraw Letters read on TWiP 136 This episode is brought to you by Blue Apron. Blue Apron is the #1 fresh ingredient and recipe delivery service in the country. See what’s on the menu this week and get your first 3 meals free with your first purchase – WITH FREE SHIPPING – by going to blueapron.com/twip. Case Study for TWiP 136 Patient seen by Daniel in India, 18 yo Islamic college student, left home, living in dorms in south, Hindu couple prepare meals, called dorm parents. He is being seen because developed lump in left side of neck, 1-2 cm mass. Previously completely healthy, no med/surg, no allergies. Prays multiple times a day, observes dietary restrictions. Afebrile, normal, but has 2 cm firm nontender lump inside interior portion of sternocleido mastoid muscle. Not tender. End of November, rainy season. No screens on dorm windows. No animal contact. Ultrasound done, and was helpful. Noticed in his neck over several weeks. Send your case diagnosis, questions and comments to twip@microbe.tv Music by Ronald Jenkees

Dr. Nathan Pennell, Cleveland Clinic, evaluates chemotherapy sensitivity assays, describing the difficulties inherent in predicting response to chemotherapy agents.

Dr. Nathan Pennell, Cleveland Clinic, evaluates chemotherapy sensitivity assays, describing the difficulties inherent in predicting response to chemotherapy agents.

Dr. Nathan Pennell, Cleveland Clinic, evaluates chemotherapy sensitivity assays, describing the difficulties inherent in predicting response to chemotherapy agents.

Macrophage-derived foam cells play a critical role in all stages of atherosclerosis, from the earliest discernable lesions to complex plaques. oxLDL is thought to be a main trigger for endothelial release of pro-inflammatory cytokines, subsequently causing transmigration of the monocytes into the vessel wall. Moreover, formation of macrophage-derived foam cells is mainly induced by oxLDL. Deposition of macrophage-derived foam cells in the lesions is induced by oxLDL uptake, as this uptake causes migratory arrest of the cells. Therefore, reversion of migratory arrest of macrophage-derived foam cells might enable these cells to leave the plaques resulting in reduction of plaque sizes. Our results show that iNOS participates in the mechanisms of oxLDL induced inhibition of macrophage-derived foam cell migration. Inhibition of iNOS expression completely reversed oxLDL mediated migratory arrest of macrophage-derived foam cells. Inhibition of iNOS was associated with enhanced phosphorylation of focal adhesion kinase (FAK) and subseqent actin polymerization. Furthermore, the p-FAK triggered increase in actin polymerization is dependent on iNOS mediated increased oxidative stress. Our results suggest that iNOS may be an interesting target gene to reverse the process of atherosclerosis.

Thu, 5 Sep 2013 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18674/ https://edoc.ub.uni-muenchen.de/18674/1/Angouri_Razeghi_Jafar.pdf Angouri Razeghi, Jafar ddc:570, ddc:5

Hosts: Vincent Racaniello, Rich Condit, Dickson Despommier, Alan Dove, and Kathy Spindler The complete TWiV team discusses the amazing finding that cyclic GMP-AMP synthase is a cytosolic innate immune DNA sensor. Links for this episode: Zhijian J. Chen (Wikipedia) Sensing the dark side of DNA (Science) cGAMP is an endogenous second messenger (Science) cGAMP synthase is a DNA sensor (Science) Pore-forming toxin (Wikipedia) Letters read on TWiV 222 Weekly Science Picks Kathy - Graphene batteriesAlan - How do we get cures? (YouTube)Rich - Explosive breach of Condit Dam (Vimeo)Vincent -  10 things you can do to support open access (Tree of Life) Listener Pick of the Week Mark - The real story of StuxnetJohn - D-connector virus Send your virology questions and comments (email or mp3 file) to twiv@twiv.tv

Thu, 5 Apr 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14310/ https://edoc.ub.uni-muenchen.de/14310/2/Jankl_Sarika.pdf Jankl, Sarika

The crista junction is a tubular element of the mitochondrial inner membrane connecting inner boundary membrane and cristae membrane. The molecular basis of its formation was so far largely unknown. The lack of the mitochondrial inner membrane protein mitofilin results in the absence of crista junctions in HeLa-cells. Moreover, deletion of Fcj1, the orthologue of mitofilin in S. cerevisiae, leads to concentric stacks of cristae membranes and the virtual absence of crista junctions. The aim of the present work was to investigate the function of Fcj1 as well as the molecular basis of the formation of cristae and crista junctions in S. cerevisiae. Fcj1 undergoes homotypic interactions and is part of a stable oligomeric protein complex. Using immuno-EM, Fcj1 has been shown to be specifically enriched at crista junctions. All other proteins, which have been studied so far, such as subunits Su e and Su g of the F1FO-ATP-Synthase, are rather underrepresented in this region. Overexpression of Fcj1 increases the number of crista junctions, enlarges the crista junction diameter and leads to internal branching of cristae. Upon downregulation of Fcj1, the mitochondrial ultrastructure progressively changes reflecting the deletion phenotype more and more. Considering the fact that the decrease of the number of crista junctions occurs at an early time point, Fcj1 seems directly involved in the formation of crista junctions. In order to investigate if cristae form a detached compartment in the absence of crista junctions, the accessibility of the intracristal space for the protein-modifying substance AMS was examined. In fcj1 mitochondria the modification of cytochrome c was temporally delayed and incomplete in comparison to wild-type. Hence, metabolite exchange between intracristal space and cristae space seems impaired though not entirely impossible in the absence of crista junctions. This might be due to some extent of fusion and fission tracing back to protein or lipid exchange within the inner membrane of mitochondria. Cryo-EM tomograms revealed regular zipper-like arrangements of F1FO-ATP-Synthase particles. An inverse correlation of the amount of Fcj1 and the oligomeric state of F1FO-ATP-Synthase was observed upon biochemical analysis: in fcj1 mitochondria, F1FO-ATP-Synthase oligomers have a higher molecular weight and are more stable than in wild-type mitochondria, whereas in mitochondria of cells overexpressing Fcj1 they are smaller and less stable. Subunits Su e and Su g are essential for the stabilization of dimers and oligomers of the F1FO-ATP-Synthase. As the influence of Su e/Su g on the formation of crista junctions has not been investigated so far, the mitochondrial ultrastructure of both deletion strains was analyzed concerning this matter. Enlargement of crista junctions and internal branching of cristae similar to the overexpression phenotype of Fcj1 have been observed in both strains. Furthermore, crista tips are virtually absent. Apart from this functional interaction, Fcj1 and Su e/Su g have also been shown to interact genetically. Based on the gained insights on the roles of Fcj1 and Su e/Su g in cristae formation the following model is proposed: the modulation of the inner membrane of mitochondria is based on an antagonistic behavior of Fcj1 and Su e/Su g. Fcj1 introduces negative membrane curvatures, possibly by impairing the oligomerisation of the F1FO-ATP-Synthase. The presence of Su e/Su g, in contrast, promotes positive curvatures of cristae membranes by stabilizing dimers and oligomers of the F1FO-ATP-Synthase. Taken together, relative amounts of Fcj1 and Su e/Su g locally modulate the oligomeric state of the F1FO-ATP-Synthase and thereby enable the formation of crista junctions and crista tips in mitochondria.

Melanie Cushion holds down two jobs: she’s a research career scientist at the Veterans Administration Medical Center in Cincinnati, Ohio, and she’s also professor and associate chair for research in the department of internal medicine at the University of Cincinnati College of Medicine.  Dr. Cushion focuses her research on the fungus, Pneumocystis carinii, which is a harmless commensal for most people, but a deadly pathogen for others.  Pneumocystis carinii was shrouded in obscurity for many years until its fifteen minutes in the spotlight came in the 80’s, when, unfortunately, an outbreak of Pneumocystis pneumonia prefigured the AIDS epidemic.  Large numbers of previously healthy homosexual men in California became deathly ill with Pneumocystis pneumonia, and doctors knew something unusual (later found to be HIV) was going on.  Dr. Cushion says Pneumocystis pneumonia is an opportunistic infection: it strikes individuals with immune systems too weak to fend it off.  This explains why it was – and still is – a well-known sign that the patient is stricken with an active HIV infection or some other immune-suppressing disorder.  Dr. Cushion heads up the Pneumocystis genome project and she’s also looking into a new line of drugs called glucan synthase inhibitors, which have a profound effect on Pneumocystis’s life cycle and may offer new insights into managing the pathogen. In this interview, I talked with Dr. Cushion about some of the more surprising results to come out of her genomics work, why Pneumocystis is a tough nut to crack in the laboratory, and about why she’s not giving her young investigator award back to the Society of Protozoologists any time soon.

Einfluss der F1FO-ATP Synthase-Oligomerisierung auf den bioener-getischen Zustand von Mitochondrien Eine wichtige Funktion der Mitochondrien besteht in der Bereitstellung von ATP, dessen Synthese durch die OXPHOS-Komplexe der Innenmembran bewerkstelligt wird. Zusätzlich besitzt die F1FO-ATP Synthase eine strukturgebende Aufgabe. Dazu bildet diese Oligomere aus, die für die Ausbildung von Cristaestrukturen essentiell sind. Insbesondere die oligomer-spezifischen Untereinheiten Su e und Su g sind dafür, nicht jedoch für die enzymatische Aktivität der F1FO-ATP Synthase, notwendig. Der Einfluss der F1FO-ATP Synthase Assemblierung auf den bioenergetischen Zustand von Mitochondrien wurde in dieser Arbeit untersucht. Teildeletionen der C-terminalen ‚coiled-coil’-Domänen von Su e weisen eine verringerte Stabilität der Oligomere auf. Diese Destabilisierung geht mit einer Reduktion des mitochondrialen Membranpotentials und der Wachstumsrate einher, ist jedoch für die Ausbildung von Cristaestrukturen hinreichend. Des Weiteren sind die enzymatischen Aktivitäten der Atmungskettenkomplexe, die Integrität der Innenmembran sowie die Verlustrate der mtDNA in diesen Mutanten nicht beeinträchtigt. Der beobachtete Phänotyp ist daher nicht auf Sekundäreffekte zurückzuführen. Diese Arbeit unterstützt ein Modell, nach dem die Assemblierung der F1FO-ATP Synthase zu Oligomeren für die räumliche Anordnung auch von anderen Proteinkomplexen in Form von Mikrodomänen für einen effizienten Substratumsatz während der oxidativen Phosphorylierung oder für deren Regulation notwendig ist. Somit hat die Stabilität der F1FO-ATP Synthase-Oligomere einen Einfluss auf die bioenergetische Leistungsfähigkeit von Mitochondrien. Charakterisierung der mitochondrialen Rhomboidprotease Pcp1 Das Dynamin-ähnliche mitochondriale Protein Mgm1 kommt in zwei Isoformen vor, die möglicherweise an der Ausbildung von Cristaestrukturen beteiligt sind. Die kurze Isoform, s-Mgm1, entsteht in Abhängigkeit von der hochkonservierten Intramembran-Rhomboidprotease Pcp1. Zur genaueren Aufklärung dieser Prozessierung wurde die Topologie und Biogenese von Pcp1 ermittelt. Pcp1 durchspannt die mitochondriale Innenmembran mit sieben Transmembrandomänen und besitzt eine Nin-Cout-Topologie. Untersuchungen zum Import von Pcp1 zeigen, dass dessen Import in die innere Membran von der TIM23-Translokase vermittelt und die mitochondriale Signalsequenz schrittweise durch die zwei Proteasen MPP und MIP entfernt wird. Außerdem wird möglicherweise die C-terminale Transmembrandomäne von Pcp1, entsprechend einem ‚Stop-Transfer’-Mechanismus, in der TIM23-Translokase arretiert und anschließend lateral in die Innenmembran inseriert. Alternativ wird Pcp1 zunächst vollständig in die Matrix importiert und anschließend über den konservativen Sortierungsweg in die Innenmembran inseriert. Die bisherigen Ergebnisse lassen eine Unterscheidung zwischen diesen beiden Möglichkeiten nicht zu. Aufgrund von Sequenzvergleichen verschiedener Rhomboidproteasen wurden wie in anderen Serinproteasen drei konservierte Aminosäurereste als mögliche katalytische Triade postuliert. Um dies zu untersuchen, wurden diese Aminosäurereste jeweils gegen Alanin ausgetauscht. Zwei dieser Punktmutationen, in Serin-256 oder Histidin-313, führen zur vollständigen Inaktivierung von Pcp1, während die Aminosäure Asparagin-202 für die Aktivität nur partiell notwendig ist. Insgesamt zeigen diese Ergebnisse, dass die mitochondriale Rhomboidprotease Pcp1 mit einer katalytischen Diade eine besondere Stellung unter den Serinproteasen einnimmt.

ATP synthase is one of the major photosynthetic complexes that represents one of the smallest molecular motors known in nature. The rotating γ subunit is a key feature of this enzyme. It contains features specific for the chloroplast ATP synthase. In this work the γ subunit has been functionally analyzed in Arabidopsis thaliana. - The nuclear gene atpC1 encoding the γ subunit of the plastid ATP synthase has been inactivated by T-DNA insertion mutagenesis. In the seedling-lethal dpa1 mutant the absence of detectable amounts of the γ subunit destabilizes the entire ATP synthase complex and consequently photophosphorylation is abolished. However, in vivo protein labelling analysis suggests that assemαβ bly of the ATP synthase and subunits into the thylakoid membrane still occurs in dpa1. Further effects of the mutation include an increased light sensitivity of the plants and an altered photosystem II activity. A high non-photochemical quenching develops with increasing actinic light intensity. It has been shown that a high proton gradient is responsible for most quenching (qE). The photoprotective role of qE was further demonstrated in the double mutant dpa1 x psbS in which PsbS, essential factor for qE, is missing. - The expression of a second gene copy, atpC2, is unaltered in dpa1 and is not sufficient to compensate for the lack of atpC1 expression. The two proteins, AtpC1 and AtpC2, share less similarity than AtpC1 of Arabidopsis with γ subunits of other plant species suggesting that the γ subunits so far isolated in other plant species are AtpC1 orthologs. It has been established that AtpC2 is also imported into the chloroplast. Therefore, it is likely that the chloroplast ATP synthase complexes contain both atpC1 and atpC2 encoded γ subunits. However, the atpC2 gene is expressed more than hundred times at a lower level than atpC1 and array data show the differential and tissue specific expression of the two genes. The function of AtpC2 could not be revealed by inactivating the gene. Overexpression of atpC2 in dpa1 generated viable lines with an ATP synthase complex containing only γ2, although wild type phenotype is not completely restored. The second part of this work regarded the optimization of conditions for plastid transformation in Arabidopsis thaliana. An efficient and fast regeneration system from cotyledon protoplasts was established for Arabidopsis thaliana accessions C24, Columbia, and Wassilewskija. Culture conditions and media compositions were optimized for the development of protoplasts embedded in thin alginate layers. The protocol is reproducible, efficient, extremely fast, and regenerated plants are fertile. Thus, this cotyledon-based system could prove useful for studying plant cell and molecular biology in A. thaliana. - The sul gene appeared to be a potential novel candidate as selectable marker for plastid transformation. However, genetic and molecular studies demonstrated that sul can not be used for this purpose. On the other hand a new function of sul appeared. The gene could be the missing marker for mitochondria transformation in higher plants.

Fri, 15 Jul 2005 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/4338/ https://edoc.ub.uni-muenchen.de/4338/1/Bertmann_Jasmin.pdf Bertmann, Jasmin

Ziel dieser Arbeit war die Untersuchung des Einflusses verschiedener Naturstoffe auf die Expression und Aktivität des menschlichen eNOS Proteins in endothelialen Zellen. Zum einen wurde untersucht, ob die Sojaisoflavone Formononetin, Biochanin A, Genistein und Daidzein die Expression und Substartumsatz der eNOS erhöhen können. Zum anderen wurde die Wirkung einer großen Anzahl von Rotweinpolyphenolextrakten auf die eNOS Expression und Enzymaktivität untersucht. Damit sollte die Frage geklärt werden, ob Anbaugebiet, Rebsorte und Vinifikationsprozeß das Ausmaß der Steigerung von eNOS Expression und Aktivität durch Rotweinpolyphenolextrakte beeinflussen und ein Hinweis auf wirksamkeitsbastimmende Substanzen erhalten werden. Das Sojaisoflavon Genistein erhöht die eNOS Enzym Aktivität und die eNOS vermittelte NO Produktion nach 48-96 h Stimulation. Diese Effekte sind durch den Estrogen Rezeptor Antagonisten ICI 182,780 nicht hemmbar. Durch den Vergleich einer großen Anzahl Rotweinpolyphenolextrakte aus verschiedenen Rotweinen konnte in dieser Arbeit gezeigt werden, daß Rebsorte und Anbaugebiet einen Einfluß auf das Ausmaß der Wirkung von Rotwein auf die Expression des eNOS Gens haben. Weißweinpolyphenolextrakte steigern in vergleichbarem Maß die Aktivität des eNOS Promotors. Aus der unterschiedlichen Zusammensatzung von wirksamen und weniger wirksamen Rotweinpolyphenolextrakten und Weißweinpolyphenolextrakten könnten künftig Rückschlüsse auf die wirksamkeitsbestimmenden Inhaltsstoffe gezogen werden. Bereits in den Trauben liegen Substanzen vor, die die eNOS Expression verstärken. Der Vinifikationsprozeß scheint die Aktivität jedoch weiter zu steigern. Für den Rotweininhaltsstoff Resveratrol konnte ein Effekt auf die eNOS Expression nachgewiesen werden. Resveratrol steigerte die eNOS Expression jedoch erst in höheren Konzentrationen, als bei der Stimulation endothelialer Zellen mit RWPE erreicht werden konnten. Resveratrol scheidet somit als alleiniger Stimulus der eNOS Expression aus. Zusammen mit anderen Inhaltstoffen kann es aber möglicherweise essentiell zum Gesamteffekt beitragen. Mit dem Beginn der Evaluierung von Einsatzmöglichkeiten der CARS Mikroskopie zur Detektion biologisch interessanter Moleküle, insbesondere von NO, in lebenden Zellen wurde ein grundlegender Beitrag zur Erweiterung der Methodenpalette innerhalb der biomedizinischen Forschung geleistet.

Pathogenetische Bedeutung der induzierbaren Stickstoffmonoxid-Synthase für den akuten hyperoxischen Lungenschaden Die längere Applikation von Sauerstoff in höheren Konzentrationen führt zu einer oxidativen Schädigung der Lunge. In den letzten Jahren gab es auch Hinweise darauf, dass Stickstoffmonoxid (NO), produziert durch die induzierbare NO-Synthase (iNOS), an der Pathogenese des hyperoxischen Lungenschadens beteiligt sein könnte. Ziel der Studie war es, die Beteiligung von NO an der Entstehung des akuten Lungenschadens unter Hyperoxie in vivo zu untersuchen. Dazu wurden unter standardisierten, experimentellen Bedingungen iNOS-defiziente und Wildtyp-Mäuse erhöhten Sauerstoffkonzentrationen (60 und 100 %)über 24 bzw. 72 h ausgesetzt. Die Ergebnisse zeigen, dass nach 72 h unter 100 % Sauer-stoff sowohl in der Wildtyp- als auch in der iNOS-defizienten Gruppe ein deutlich vermehrter Zellschaden im Sinne einer Abnahme der Zellvitalität und einer Zunahme der LDH-Aktivität auftritt. Als Zeichen einer verstärkten inflammatorischen Reaktion kam es zu einer Erhöhung der Gesamtzellzahl und -proteinkonzentration in der Bronchiallavageflüssigkeit sowie zu einer vermehrten TNF-alpha-Expression, welche durch die Transkriptionsfaktoren NF-Kappa B und Aktivatorprotein 1 (AP1) vermittelt wurde. Diese Auswirkungen waren auch in den histologischen Schnitten zu beobachten. Als Zeichen vermehrten oxidativen Stresses war eine erhöhte Lipidperoxidation zu verzeichnen. Die genannten Beobachtungen waren bei den Wildtyp-Mäusen wesentlich ausgeprägter als bei den iNOS-defizienten Tieren, was auf die erhöhte iNOS-Expression zurückgeführt werden könnte. Aus der Gesamtheit der Daten lässt sich schliessen, dass unter akuter Hyperoxie die proinflam-matorische Komponente der iNOS zu überwiegen scheint, dadurch wird das Ausmaß der inflammatorischen Reaktion und die resultierende Lungenschädigung potenziert.

Mon, 26 Apr 2004 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/2109/ https://edoc.ub.uni-muenchen.de/2109/1/Conrad_Nicole.pdf Conrad, Nicole

Background: Hyperoxic exposures are often found in clinical settings of respiratory insufficient patients, although oxygen therapy (>50% O-2) can result in the development of acute hyperoxic lung injury within a few days. Upon hyperoxic exposure, the inducible nitric oxide synthase (iNOS) is activated by a variety of proinflammatory cytokines both in vitro and in vivo. In the present study, we used a murine hyperoxic model to evaluate the effects of iNOS deficiency on the inflammatory response. Methods: Wild-type and iNOS-deficient mice were exposed to normoxia, 60% O-2 or >95% O-2 for 72 h. Results: Exposure to >95% O-2 resulted in an increased iNOS mRNA and protein expression in the lungs from wild-type mice. No significant effects of iNOS deficiency on cell differential in bronchoalveolar lavage fluid were observed. However, hyperoxia induced a significant increase in total cell count, protein concentration, LDH activity, lipid peroxidation, and TNF-alpha concentration in the bronchoalveolar lavage fluid compared to iNOS knockout mice. Moreover, binding activity of NF-kappaB and AP-1 appeared to be higher in wild-type than in iNOS-deficient mice. Conclusion: Taken together, our results provide evidence to suggest that iNOS plays a proinflammatory role in acute hyperoxic lung injury.

Die Chlorophyll Synthase ist ein äußerst hydrophobes Transmembranprotein und sitzt in der Thylakoidmembran im Chloroplasten. Sie verestert Chlorophyllid und Phytylpyrophosphat zu Chlorophyll. In dieser Arbeit sind Untersuchungen zur Lage der Transmembranbereiche, zum aktiven Zentrum bzw. zu essentiellen Aminosäuren und zum Reaktionsmechanismus dargestellt.

Die vorliegende Arbeit beschäftigt sich mit dem Einfluss von Drogenextrakten und Naturstoffen auf die endotheliale NO-Synthase (eNOS) in humanen Endothelzellen (Primärkulturen und Zelllinien). Insbesondere wurde dabei ihr Einfluss auf den verschiedenen zellulären Ebenen (Transkription, Proteinexpression, NO-Synthese) untersucht. Da es in dieser Arbeitsgruppe die erste Arbeit auf dem Gebiet der eNOS war, mussten zunächst Methoden etabliert und entwickelt werden, mit denen die eNOS auf den verschiedenen zellulären Ebenen detektiert werden kann. Etabliert wurden ein Luciferase-Reportergen-Assay zur Messung der eNOS Promotoraktivität, eine Northern Blot-Methode zur Bestimmung der eNOS-mRNA und eine Western Blot-Methode zur Messung der eNOS Proteinmenge. Entwickelt wurden ein L-Arginin/L-Citrullin Umwandlungsassay zur Bestimmung der eNOS-bedingten L-Citrullin-produktion und ein DAF-2 Fluoreszenzassay zur Messung der eNOS-bedingten NO-Produktion. Getestet wurden Extrakte und Naturstoffe, bei denen bereits positive kardiovaskuläre Eigenschaften wie Vasodilatation bekannt bzw. in der Diskussion waren. Keinen Einfluss auf die eNOS hatten: •Knoblauchextrakte und schwefelhaltige Knoblauchextraktinhaltsstoffe •Der Weißdornblüten- und Blätterextrakt WS1442 •Die Catechinderivate Epicatechin-3-gallat und Epigallocatechin-3-gallat •Die Rotweinpolyphenole Delphinidin, Quercetin, Epicatechin und Rutin Dagegen konnten Isoflavone der Sojabohne, wie Genistein, Daidzein, Formononetin, Biochanin A und Equol die eNOS Promotoraktivität konzentrationsabhängig erhöhen. Genistein (stellvertretend für alle Isoflavone im Western Blot getestet) erhöhte auch die eNOS Proteinmenge. Allerdings bewirkte Genistein, trotzt der Erhöhung der Proteinmenge, keine Erhöhung der eNOS abhängigen Bildung an L-Citrullin und NO. Positive Ergebnisse brachten die Tests mit einem Rotweinpolyphenolextrakt (RWPE). Dieser Extrakt erhöhte signifikant die NO-Produktion in den beiden getesteten Endothelzellarten (EA.hy926 Zellen und HUVECs). Um den molekularen Mechanismus der NO-Produktionserhöhung durch RWPE aufzuklären, wurden verschiedene Ebenen der eNOS Regulation untersucht. Dabei konnte in dieser Arbeit zum ersten mal gezeigt werden, dass RWPE sowohl die eNOS Promotoraktivität als auch die eNOS Proteinexpression erhöht. Die nächste Frage war, ob die NO-Produktionserhöhung kausal mit der gemessenen Transkriptionserhöhung zusammenhängt. Zeitabhängige Untersuchungen auf den verschiedenen Ebenen der eNOS Regulation ergaben ähnliche Ergebnisse mit einer signifikant messbaren Beeinflussung stets nach ca. 10 h. Dies deutet darauf hin, dass RWPE die eNOS-abhängige NO-Produktion über eine Erhöhung der eNOS Transkription/Translation erhöht. Allerdings konnte dies auf Grund fehlender Experimente über eine posttranslationelle eNOS Beeinflussung nicht eindeutig bewiesen werden. Abschließend sollte mit der Suche nach den wirksamen Bestandteilen im RWPE begonnen werden. Auch wenn im Verlauf dieser Arbeit die wirksamen Verbindungen noch nicht gefunden wurden, konnten zumindest einige Substanzen als wirksamkeitsbestimmend oder -mitbestimmend ausgeschlossen werden. Neben einigen nicht wirksamen Rotweinpolyphenolen (Delphinidin, Rutin, Quercetin, Epicatechin) geben die durchgeführten Experimente Hinweise darauf, dass auch Anthocyane, Tannine und oligomere Procyanidine unwirksam sind. Das Stilbenderivat Resveratrol, welches oft als eine kardiovaskulär aktive Komponente im Rotwein angesehen wird, hatte nur einen sehr geringen und auf der Ebene der NO-Produktion nicht signifikanten Effekt auf die eNOS. Zusammenfassend wurde in dieser Arbeit ein Modell zur Messung von Einflüssen auf die eNOS aufgebaut. Von den getesteten Extrakten und Naturstoffen beeinflusste nur RWPE signifikant die eNOS. Es konnte erstmalig gezeigt werden, dass RWPE in Endothelzellen nach Langzeitstimulation (20 h) die eNOS Transkription, eNOS Expression und eNOS bedingte NO-Produktion erhöht. Dieses Ergebnis ist physiologisch äußerst interessant. Denn bisher bekannte, die eNOS Expression erhöhende Substanzen (z.B: Östradiol, Cyclosporin A, Insulin, Phorbolester, Wasserstoffperoxid, Staurosporin, Angiotensin II) sind auf Grund ihrer vielseitigen physiologischen/toxischen Wirkungen therapeutisch zur Prophylaxe und Behandlung von kardiovaskulären Erkrankungen kaum einsetzbar. Die Aufgabe zukünftiger Arbeiten wird es sein, die Wirkung von RWPE in vivo zu untersuchen und die für die Wirkung verantwortlichen Bestandteile des RWPE zu finden.

Glucans, with the (1-3)-b-glucosidic linkage as major feature, are present in most of the higher plants, in many lower plants, as well as in microorganisms (Stone and Clarke, 1992). The synthesis of (1-3)-b-glucan in vivo is catalysed by the enzyme (1-3)-b-glucan synthase (EC 2.4.1.34; UDP-glucose:1,3-b-D-glucan 3-b-D-glucosyl transferase) using UDP-glucose as substrate. The (1-3)-b-glucan synthase was characterised in a number of fungi and plants, but not much work was done with oomycetes (Stone and Clarke, 1992), even though one of the earliest successful in vitro assays for glucan synthase activity was done using Phytophthora cinnamomi (Wang and Bartnicki-Garcia, 1976, Selitrennikoff 1995). In this work, the glucan synthase of the oomycete Phytophthora sojae was characterised, solubilized, and partially purified, and the cDNA for a protein co-purifying with the glucan synthase activity was cloned. The glucan synthase of P. sojae had several features that distinguish it from what is known for glucan synthases from fungi and plants (callose synthases). Its apparent Km value for UDP-glucose was higher than reported for other glucan synthases. The activity was GTP-independent and shown not to be activated by divalent cations like Mg2+ or Ca2+, and shown to be inhibited by some others, like Cu2+ or Zn2+. Some of these properties are shared with the glucan synthase from Achlya ambisexualis (Cabib and Kang, 1987), an organism that belongs to the same kingdom as P. sojae: the Chromista. It was also demonstrated by NMR analysis and enzymatic degradation that the sole product of the CHAPS-solubilized glucan synthase of P. sojae was composed of long linear (1-3)-b-glucan chains. The glucan synthase was purified by product entrapment. Two proteins, with apparent molecular masses of 108 and 50 kDa, were enriched and microsequenced. With the degenerated oligonucleotides derived from the sequenced peptides, PCR experiments were performed using as a template a cDNA library of actively growing P. sojae mycelium. No positive result could be obtained by using the oligonucleotides derived from the 108 kDa protein. In contrast, a full length cDNA (named Ps-P50) was cloned, using the oligonucleotides derived from the 50 kDa protein (P50). The deduced amino acid sequence of Ps-P50 cDNA contains sequence motifs homologous to the peptides sequenced from P50. This cDNA encodes a protein with a molecular mass of 49.991 Da with no homology found in the data bases. Diversity between the PCR product and the cDNA clone, and various different homologous ESTs indicates that Ps-P50 is a member of a gene family.

Im Mittelpunkt der vorliegenden Arbeit steht die Funktion der Untereinheit e (Sue) der mitochondrialen F1FO-ATP Synthase von Saccharomyces cerevisi-ae. Anhand der Resultate der durchgeführten Experimente wurden folgende Schlussfolgerungen gezogen: (1) Sue ist der Lage, ein Sue-Homodimeres zu bilden. Das Protein spielt eine zentrale Rolle bei der Assemblierung der F1FO-ATP Synthase zu einem stabilen Dimer. Sue-Disruptanten bilden entsprechend kein stabiles ATP Synthase-Dimeres aus. Die C-terminalen 36 Aminosäurereste von Sue, die gegenüber Untereinheiten e aus Säugerzellen zusätzlich vorhanden sind, sind für die Dimerisierung von Sue und der F1FO-ATP Synthase ohne Bedeutung. (2) Zwischen den Untereinheiten e und k, die beide im FO-Sektor der ATP Synthase lokalisiert sind, besteht eine enge räumliche Beziehung. Für die In-teraktion von Sue mit Suk ist der Bereich von Sue, der anderen Untereinheiten e aus Säugerzellen ähnelt, ausreichend. (3) Im Hefegenom wurde ein der Sequenz von Suk nahe verwandtes Le-seraster gefunden. Die Sequenzähnlichkeit auf Aminosäureebene beträgt 45%. Ein entsprechendes hypothetisches Protein wurde als Suk hom bezeichnet. Eine Deletion dieser Sequenz allein oder gemeinsam mit dem Gen für Suk blieb ohne Auswirkungen auf die oxidative Phosphorylierung, die Assemblie-rung der F1FO-ATP Synthase und die Expression von Untereinheiten des FO-Sektors der F1FO-ATP Synthase. (4) Die Dimerisierung der F1FO-ATP Synthase und somit auch die Funkti-on von Sue als Dimerisierungsfaktor erwies sich als essentiell für die Funkti-on weiterer Komponenten der Atmungskette: der Cytochrom c-Oxidase und des Cytochrom bc1-Komplexes. Die Assemblierung der ATP Synthase wirkt sich auf die Aktivitäten der beiden Komponenten des Cytochrom bc1-COX-Suprakomplexes aus. Sie beeinflusst auch deren Assemblierung in den Supra-komplex beziehungsweise seine Stabilität. Die Anwesenheit der Region von Sue, die anderen Untereinheiten e aus Säugetierzellen ähnelt, reicht für die Bildung des Cytochrom bc1-COX-Suprakomplexes aus. Das Dimer der ATPase Synthase ist demzufolge in den Suprakomplex einge-bunden. Allerdings hat der Assemblierungszustand des Cytochrom bc1-COX-Suprakomplexes keinerlei Auswirkungen auf die Assemblierung der ATP Synthase. Dies deutet auf einen hierarchisch ablaufenden Prozeß der Bildung eines Suprakomplexes aus Cytochrom bc1-Komplex, Cytochrom c-Oxidase und F1FO-ATP Synthase hin. Die ATP Synthase nutzt zur Bildung von ATP den elektrochemischen Gra-dienten über die innere Mitochondrienmembran, an dessen Aufbau der Cy-tochrom bc1-Komplex und die Cytochrom c-Oxidase beteiligt sind. Eine Ein-bindung dieser Enzyme in einen Suprakomplex würde eine koordinierte Re-gulation der oxidativen Phosphorylierung ermöglichen. (5) Zwischen der Untereinheit Sue der F1FO-ATP Synthase und der Unter-einheit Cox2 der Cytochrom c-Oxidase konnte eine enge räumliche Bezie-hung nachgewiesen werden. Diese ist von der Funktionalität der F1FO-ATP Synthase abhängig. Anhand der Ergebnisse der vorliegenden Arbeit konnte somit folgende Erklä-rung für die Funktion der Untereinheit e der mitochondrialen F1FO-ATP Synthase gefunden werden: Sue dient als Dimerisierungsfaktor der F1FO-ATP Synthase. Die Dimerisie-rung von Sue und damit die Dimerisierung der ATP Synthase ist essentiell für die Stabilisierung des Cytochrom bc1-COX-Suprakomplexes und die Funkti-on seiner Komponenten.

Production of nitric oxide by the inducible NO synthase (iNOS) is known to be enhanced in chronic joint inflammation and osteoarthritis as well as aseptic loosening of joint prostheses. Initial studies yielded promising results after inhibition of the nitric oxide synthase (NOS). However, the effect of NOS inhibition has not been studied at the site of the primary function of NO, the microcirculation of the synovium in vivo. Using our recently developed model for the in vivo study of synovial microcirculation in the mouse knee joint, the effects of selective versus nonselective inhibition of iNOS were investigated by means of intravital fluorescence microscopy. After resection of the patella tendon, the synovial fatty tissue was exposed for intravital microscopy. Diameter of arterioles, functional capillary density (FCD), diameter of venules, venular red blood cell velocity and leukocyte-endothelial cell interaction were quantitatively analyzed before, and 10 and 60 min after intravenous injection of NOS inhibitors {[}selective iNOS inhibitor N-iminoethyl-L-lysine (L-NIL), and nonselective NOS inhibitor N-G-nitro-L-arginine methyl ester (L-NAME)]. Our results demonstrate that L-NAME causes a significant decrease in the arteriolar diameter and FCD associated with an increase in the leukocyte accumulation in the synovium in vivo. In contrast, L-NIL neither altered the microhemodynamics nor the leukocyte-endothelial cell interaction in the synovium, indicating its potential use for selective inhibition of iNOS in joint inflammation. Using our method, further studies will provide new insights into the unknown effect of NOS inhibition on the synovial microvasculature in inflammatory joint disease in vivo. Copyright (C) 1999 S. Karger AG, Basel.

An enzyme system catalysing the formation of the methylenedioxy bridge at ring A of (S)-canadine [ = (S)-tetrahydroberberine] from (S)-tetrahydrocolumbamine has been detected in microsomal preparations from different Ranunculaceae and Berberidaceae cell cultures. The cytochrome P-450 enzyme complex has been partly characterized from a protoberberine alkaloid producing Thalictrum tuberosum L. cell line. The enzyme complex consisting of a microsomal associated oxidase with a cytochrome P-450 reductase has a pH optimum at pH 8.5 and a temperature optimum of 40°. The apparent Km values are 33 μM for NADPH and 11.5 μM for tetrahydrocolumbamine.

Sat, 1 Jan 1983 12:00:00 +0100 http://epub.ub.uni-muenchen.de/3634/ http://epub.ub.uni-muenchen.de/3634/1/3634.pdf Schumacher, H. M.; Rüffer, Martina; Nagakura, Naotaka; Zenk, Meinhart H. Schumacher, H. M.; Rüffer, Martina; Nagakura, Naotaka und Zenk, Meinhart H. (1983): Partial purification and properties of (S)-norlaudanosoline synthase from Eschscholtzia tenuifolia cell cultures. In: Planta Medica, Vol. 48: pp. 212-220.

Thu, 1 Jan 1981 12:00:00 +0100 http://epub.ub.uni-muenchen.de/3636/ http://epub.ub.uni-muenchen.de/3636/1/017.pdf Rüffer, Martina; El-Shagi, Hannemarie; Nagakura, Naotaka; Zenk, Meinhart H. Rüffer, Martina; El-Shagi, Hannemarie; Nagakura, Naotaka und Zenk, Meinhart H. (1981): (S)norlaudanosoline synthase. the first enzyme in the benzylisoquinoline biosynthetic pathway. In: FEBS Letters, Vol. 129, Nr. 1: pp. 5-9.

Sat, 1 Dec 1979 12:00:00 +0100 http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T36-44DSR1F-B5&_user=616146&_coverDate=12%2F15%2F1979&_rdoc=18&_fmt=high&_orig=browse&_srch=doc-info(%23toc%234938%231979%23998919997%23272157%23FLP%23display%23Volume)&_cdi=4938&_sort=d&_docancho https://epub.ub.uni-muenchen.de/7342/1/Neupert_Walter_7342.pdf Neupert, Walter; Harmey, Matthew A. ddc:610, Medizin