Podcasts about Caspase

BUFFALO, NY- March 25, 2024 – A new #research paper was #published in Oncotarget's Volume 15 on March 14, 2024, entitled, “ABT199/venetoclax synergism with thiotepa enhances the cytotoxicity of fludarabine, cladribine and busulfan in AML cells.” ABT199/venetoclax, an inhibitor of the pro-survival BCL-2 protein, has improved AML treatment. Its efficacy in hematopoietic stem cell transplantation (HSCT), when combined with other chemotherapeutic drugs, has not been thoroughly investigated. In this new study, researchers Benigno C. Valdez, Bin Yuan, David Murray, Jeremy L. Ramdial, Uday Popat, Yago Nieto, and Borje S. Andersson from The University of Texas MD Anderson Cancer Center and the University of Alberta demonstrate the synergistic cytotoxicity of ABT199/venetoclax with the DNA alkylator thiotepa (Thio) in AML cells. “The results may provide relevant information for the design of clinical trials using these drugs to circumvent recognized drug-resistance mechanisms when used as part of pre-transplant conditioning regimens for AML patients undergoing allogenic HSCT.” Cleavage of Caspase 3, PARP1 and HSP90, as well as increased Annexin V positivity, suggest potent activation of apoptosis by this two-drug combination; increased levels of γ-H2AX, P-CHK1 (S317), P-CHK2 (S19) and P-SMC1 (S957) indicate an enhanced DNA damage response. Likewise, the increased level of P-SAPK/JNK (T183/Y185) and decreased P-PI3Kp85 (Y458) suggest enhanced activation of stress signaling pathways. These molecular readouts were synergistically enhanced when ABT199/venetoclax and Thio were combined with fludarabine, cladribine and busulfan. The five-drug combination decreased the levels of BCL-2, BCL-xL and MCL-1, suggesting its potential clinical relevance in overcoming ABT199/venetoclax resistance. Moreover, this combination is active against P53-negative and FLT3-ITD-positive cell lines. Enhanced activation of apoptosis was observed in leukemia patient-derived cell samples exposed to the five-drug combination, suggesting a clinical relevance. “The results provide a rationale for clinical trials using these two- and five-drug combinations as part of a conditioning regimen for AML patients undergoing HSCT.” DOI - https://doi.org/10.18632/oncotarget.28563 Correspondence to - Benigno C. Valdez - mbalasik@yahoo.com Sign up for free Altmetric alerts about this article - https://oncotarget.altmetric.com/details/email_updates?id=10.18632%2Foncotarget.28563 Subscribe for free publication alerts from Oncotarget - https://www.oncotarget.com/subscribe/ Keywords - cancer, acute myeloid leukemia, aml, pre-transplant regimens, venetoclax, thiotepa, busulfan About Oncotarget Oncotarget (a primarily oncology-focused, peer-reviewed, open access journal) aims to maximize research impact through insightful peer-review; eliminate borders between specialties by linking different fields of oncology, cancer research and biomedical sciences; and foster application of basic and clinical science. Oncotarget is indexed and archived by PubMed/Medline, PubMed Central, Scopus, EMBASE, META (Chan Zuckerberg Initiative) (2018-2022), and Dimensions (Digital Science). To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: Facebook - https://www.facebook.com/Oncotarget/ X - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/@OncotargetJournal LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Spotify - https://open.spotify.com/show/0gRwT6BqYWJzxzmjPJwtVh Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957

Kilder:Men succumb to manflu because women have stronger immune systems, claim scientistshttps://web.archive.org/web/20090517021219/http://www.telegraph.co.uk/health/healthnews/5313882/Men-succumb-to-manflu-because-women-have-stronger-immune-systems-claim-scientists.htmlSpread of an Inactive Form of Caspase-12 in Humans Is Due to Recent Positive Selectionhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1424700/Differential modulation of endotoxin responsiveness by human caspase-12 polymorphismshttps://pubmed.ncbi.nlm.nih.gov/15129283/Gender differences in expression of the human caspase-12 long variant determines susceptibility to Listeria monocytogenes infectionThe science behind “man flu”https://www.bmj.com/content/359/bmj.j5560.longhttps://videnskabeligtudfordret.dk/liveSupport the showHvis du vil være med til at optage live med os på Discord kan du støtte os på 10er og blive en af vores kernelyttere https://vudfordret.10er.appDu kan også tjekke vores webshop: bit.ly/vushop. Der er en hønsetrøje!Send os vanvittig videnskab eller stil et spørgsmål på facebook, Instagram eller vudfordret@gmail.comTak til Christian Eiming for disclaimer.Tak til Barometer-Bjarke for Gak-O-meteret.Husk at være dumme

A new research paper was published in Aging (Aging-US) Volume 15, Issue 14, entitled, “Inhibiting NLRP3 signaling in aging podocytes improves their life- and health-span.” The decrease in the podocyte's lifespan and health-span that typify healthy kidney aging cause a decrease in their normal structure, physiology and function. The ability to halt and even reverse these changes becomes clinically relevant when disease is superimposed on an aged kidney. NLRP3 [nod-like receptor protein 3] expression is increased in podocytes of mice with advanced age and contributes to their damage. “However, the functional consequence of increased levels of NLRP3 in aged podocytes is unknown.” In this new study, researchers Natalya Kaverina, R. Allen Schweickart, Gek Cher Chan, Joseph C. Maggiore, Diana G. Eng, Yuting Zeng, Sierra R. McKinzie, Hannah S. Perry, Adilijiang Ali, Christopher O'Connor, Beatriz Maria Veloso Pereira, Ashleigh B. Theberge, Joshua C. Vaughan, Carol J. Loretz, Anthony Chang, Neil A. Hukriede, Markus Bitzer, Jeffrey W. Pippin, Oliver Wessely, and Stuart J. Shankland from the University of Washington, Cleveland Clinic Foundation, National University Hospital Singapore, University of Pittsburgh, University of Michigan, and the University of Chicago hypothesized that reducing NLRP3 signaling earlier at middle-age improves overall podocyte health and slows down healthy podocyte aging in mice. “To this end, we performed a comprehensive analysis of inflammasome signaling including pharmacological and genetic NLRP3 loss-of-function approaches.” RNA-sequencing of podocytes from middle-aged mice showed an inflammatory phenotype with increases in the NLRP3 inflammasome, signaling for IL2/Stat5, IL6 and TNF, interferon gamma response, allograft rejection and complement, consistent with inflammaging. Furthermore, injury-induced NLRP3 signaling in podocytes was further augmented in aged mice compared to young ones. The NLRP3 inflammasome (NLRP3, Caspase-1, IL1β IL-18) was also increased in podocytes of middle-aged humans. Higher transcript expression for NLRP3 in human glomeruli was accompanied by reduced podocyte density and increased global glomerulosclerosis and glomerular volume. Pharmacological inhibition of NLRP3 with MCC950, or gene deletion, reduced podocyte senescence and the genes typifying aging in middle-aged mice, which was accompanied by an improved podocyte lifespan and health-span. Moreover, modeling the injury-dependent increase in NLRP3 signaling in human kidney organoids confirmed the anti-senescence effect of MC9950. Finally, NLRP3 also impacted liver aging. “In summary, our results demonstrate for the first time that aging podocytes acquire an inflammatory phenotype, which include the NLRP3 inflammasome and which is consistent with inflammaging.” DOI - https://doi.org/10.18632/aging.204897 Corresponding authors - Oliver Wessely - wesselo@ccf.org, and Stuart J. Shankland - stuartjs@uw.edu Keywords - aging, kidney, podocyte, NLRP3 inflammasome, reporter About Aging-US Launched in 2009, Aging-US publishes papers of general interest and biological significance in all fields of aging research and age-related diseases, including cancer—and now, with a special focus on COVID-19 vulnerability as an age-dependent syndrome. Topics in Aging-US go beyond traditional gerontology, including, but not limited to, cellular and molecular biology, human age-related diseases, pathology in model organisms, signal transduction pathways (e.g., p53, sirtuins, and PI-3K/AKT/mTOR, among others), and approaches to modulating these signaling pathways. Visit our website at https://www.Aging-US.com​​ and connect with us: Facebook - https://www.facebook.com/AgingUS/ Twitter - https://twitter.com/AgingJrnl Instagram - https://www.instagram.com/agingjrnl/ YouTube - https://www.youtube.com/@AgingJournal LinkedIn - https://www.linkedin.com/company/aging/ For media inquiries, please contact: MEDIA@IMPACTJOURNALS.COM

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.17.528998v1?rss=1 Authors: Scholz, N., Siebzehnrubl, F. A., Licchesi, J. D. F. Abstract: Programmed cell death is a complex and tightly regulated sequence of events that determines cell fate during tissue homeostasis, development, and pathogenesis. The small protein modifier ubiquitin mediates important regulatory functions during cell death by regulating the stability and activity of checkpoint proteins and the assembly of cell death signalling complexes. The caspase family of cysteine aspartases are essential effectors of apoptotic cell death. Components of the ubiquitin system including RING ubiquitin ligases XIAP, MDM2, RBX1; RBR E3 ubiquitin ligases Parkin and LUBAC; and HECT E3 ubiquitin ligases NEDD4 and Itch are also substrates of caspase-mediated cleavage. In the case of NEDD4 and Itch, the single cleavage event occurs outside of the catalytic HECT domain and it remains unclear whether such cleavage events impact on ubiquitin ligase activity and/or function. Here, we identified the E3 ubiquitin ligase HECTD1 as the third HECT E3 cleaved by caspase-mediated cleavage during apoptotic cell death, in a manner which does not affect the integrity of the catalytic C-ter HECT domain. We mapped the single cleavage event to DFLD1664{downarrow}S and showed that the cleaved C-ter product, which contains the HECT ligase domain, is as stable as the endogenous full length protein. We also found that HECTD1 transient depletion led to reduced caspase-3 activity, but not caspase 8 nor 9. Furthermore, we also identified caspase-3 as the protease responsible for HECTD1 cleavage at Asp1664 suggesting that HECTD1 and caspase-3 might be part of a novel feedback loop mechanism during apoptotic cell death. This study highlight novel crosstalk between cell death mechanisms and the ubiquitin system and raises important questions on whether proteolytic cleavage of E3 ubiquitin ligases might represent an underappreciated mode of regulation during cell death mechanisms. 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/2022.12.15.520548v1?rss=1 Authors: Martinez Lagunas, K., Savcigil, D. P., Zrilic, M., Carvajal Fraile, C., Craxton, A., Self, E., Uranga, I., de Miguel, D., Arias, M., Willenborg, S., Piekarek, M., Albert, M. C., Nugraha, K., Lisewski, I., Janakova, E., Igual, N., Tonnus, W., Hilendbrandt, X., Ibrahim, M., Ballegeer, M., Saelens, X., Kueh, A. J., Meier, P., Linkermann, A., Pardo, J., Eming, S., Walczak, H., MacFarlane, M., Peltzer, N., Annibaldi, A. Abstract: Cell death coordinates repair programs following pathogen attack and tissue injury. However, aberrant cell death can interfere with such programs and cause organ failure. cFLIP is a crucial regulator of cell death and a substrate of Caspase-8. Yet, the physiological role of cFLIP cleavage by Caspase-8 remains elusive. Here, we discovered an essential role for cFLIP cleavage in restraining cell death in different pathophysiological scenarios. Mice expressing a cleavage-resistant cFLIP mutant, CflipD377A, exhibited increased sensitivity to SARS-CoV-induced lethality, impaired skin wound healing and increased tissue damage caused by Sharpin deficiency. In vitro, abrogation of cFLIP cleavage sensitizes cells to TNF-induced necroptosis and apoptosis by favoring complex-II formation. Mechanistically, the cell death-sensitizing effect of the D377A mutation depends on Gln(Q)469. These results reveal a crucial role for cFLIP cleavage in controlling the amplitude of cell death responses occurring upon tissue stress, to ensure the execution of repair programs. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Having taken a short break from our Scripture & Science series, we are back. Today begins a series of three episodes exploring evolution. Whether you believe in evolution or not, it's important to understand what the theory claims and how evolutionists reason. In this episode, Will Barlow briefly explains how evolutionists explain the gradual change from one species to another. He dispels four common misconceptions and delves into the genetic insights popularized by Francis Collins who is both an evolutionist and a Christian. Listen to this episode on Spotify or Apple Podcasts https://www.youtube.com/watch?v=HDi6QRlyfSE&feature=emb_imp_woyt See below for notes. —— Links —— We are doing follow-up discussions to these episodes on YouTube. Check them out! See other episodes in this Scripture and Science Class Check out Barlow's previous podcast episodes Learn more about and support the church Barlow and his team are starting in Louisville, KY, called Compass Christian Church Find more articles and audios by Barlow on his website: Study Driven Faith Support Restitutio by donating here Designate Restitutio as your charity of choice for Amazon purchases Join our Restitutio Facebook Group and follow Sean Finnegan on Twitter @RestitutioSF Leave a voice message via SpeakPipe with questions or comments and we may play them out on the air Intro music: Good Vibes by MBB Attribution-ShareAlike 3.0 Unported (CC BY-SA 3.0) Free Download / Stream: Music promoted by Audio Library. Who is Sean Finnegan?  Read his bio here —— Notes —— Biochemistry and Evolution • Common misconceptions with evolution• General framework of evolution• Modern evolutionary theory Common misconceptions with evolution Misconception #1: “It's just a theory!” • Often, opponents of evolution will state that it is a theory (and use that term pejoratively)• However, evolution does account for much of the scientific evidence and has made many successful predictions Definition of “theory”“A scheme or system of ideas or statements held as an explanation or account of a group of facts or phenomena; a hypothesis that has been confirmed or established by observation or experiment, and is propounded or accepted as accounting for the known facts; a statement of what are held to be the general laws, principles, or causes of something known or observed.”-Richard Dawkins, The Greatest Show of Earth, pg. 9 Common misconceptions with evolution Misconception #2: It means to explain how life began • Often, opponents of evolution attack the beginning of life problem• However, evolution does not attempt to account for the beginning of life; this is the theory of abiogenesis Misconception #3: Evolution says that humans evolved from apes • This is a vast over-simplification of the theory• Evolution teaches that all life came evolved from a single organism, so apes and humans share an ancestor Misconception #4: Evolution is a progression from simpler organisms to more complex organisms • This is another over-simplification of the theory• No particular animal alive is more advanced than any prior animal in history• Current animals are simply better suited to survive than their ancestors General framework of evolution Thought experiment: Millions of years ago, there's an animal named “tom” that is a mammal • At one point, there is one population of “toms”• However, some time later, the population splits in two due to a geographical issue (let's say a mountain pass)• On one side of the mountain, there is more rain• On the other side of the mountain, there is less rain and more heat• Since the populations have been split (and are not sharing genetic information), random mutations occur in each population• Since the environments are different, the random mutations that lead to “better” alternatives are different• Over time, these mutations build up into noticeable differences• Eventually, two new species emerge: “tom a” and “tom b”Predictions of evolution Successful prediction #1: Scientists have demonstrated that DNA is more advanced as life is more advanced • For example, human DNA is 96% similar to chimpanzee DNA• Yet the number of protein-encoding genes is roughly the same for all animals and plants! Genetic Complexity “Our complexity must arise not from the number of separate instruction packets, but from the way they are utilized.”-Francis Collins, The Language of God, pg. 137 Predictions of evolution The concept of the “tree of life” is another important prediction of evolution.We should expect to see intermediate species in the fossil record and the genetic record as we study the world around us. Support for the tree of life: • Transition between fish and amphibians — we can find fish that could walk and breathe oxygen• Genetic sequencing has mostly confirmed scientists' predictions, with a few surprises! “At the level of the genome as a whole, a computer can construct a tree of life based solely upon the similarities of the DNA sequences of multiple organisms…. Bear in mind that this analysis does not utilize any information from the fossil record, or from anatomical observations of current life forms. Yet its similarity to c biology, biochemistry, evolution, what is evolution?, do Christians believe in evolution?, can a Christian believe in evolution?, common misconceptions about evolution, evolutionary theory, Richard Dawkins, The Greatest Show on Earth, ancestors, tree of life, Charles Darwin, speciation, macro evolution, micro evolution, predictions of evolution, DNA, chimpanzee, Francis Collins, The Language of God, fish, amphibians, junk DNA, human evolution, ancient repetitive elements, ARE, genetics, onclusions drawn from studies of comparative anatomy… is striking.” Successful prediction #2: Breeders and agriculture experts have demonstrated “micro-evolution” countless times. • Ex: dog breeds• Ex: Hybrid crops• Detractors of evolution sometimes say that micro-evolution is possible but “macro-evolution” is not• Francis Collins argues that the distinction is an artificial one Successful prediction #3: Accumulation of “junk DNA” • Darwin's theory predicts that mutations that do not affect function (located in “junk DNA”) will accumulate slowly over time• We see this, as well as “silent” differences in the genetic codeWhat about man? Does evolution successfully show that humans evolved? • Richard Dawkins demonstrates that the fossil record is full of so-called “missing” links• Perhaps even more powerful, Francis Collins gives evidence for human evolution from genetics What is Francis Collins' best piece of evidence? What happens when we compare the human genome to other animals' genomes?What about genes that appear to be non-functional in humans but functional in multiple species? Fact #1: Comparing human and mouse genomes • The overall size of the genome is roughly the same• The inventory of protein-encoding genes is remarkably similar• The order of genes is generally the same over substantial stretches of DNA• The existence of similar AREs (ancient repetitive elements) On AREs: “When one aligns sections of the human and mouse genomes, anchored by the appearance of gene counterparts that occur in the same order, one can usually also identify AREs in approximately the same location in these two genomes.” “The process of transposition often damages the jumping gene. There are AREs throughout the human and mouse genomes that were truncated when they landed, removing any possibility of their functioning. In many instances, one can identify a decapitated and utterly defunct ARE in parallel positions in the human and mouse genome.” Fact #2: Comparing human and chimpanzee chromosomal fusion “Recently… it has become possible to look at the precise location where this proposed chromosomal fusion must have happened…. But they are found right where evolution would have predicted, in the middle of our second fused chromosome.” Fact #3: Comparing human and chimpanzee genetic function • Caspase-12 is a functional gene in chimpanzees (and many other mammals, including mice)• The human version is found in the identical location as the chimp version, but lacks the function

This month, Drs. Juan Perez- Barcena and Juan Pablo de Rivero Vaccari are interviewed by Dr. Jon Rosenberg on their recent article, “Serum Caspase-1 as an Independent Prognostic Factor in Traumatic Brain Injured Patients.” NCS offers free CE credits for the NCS Podcast Series episodes. Listen to any of the posted episodes, complete a five question survey, and claim your credits here! Credits are available for physicians, pharmacists, nurses, and non-physicians. The NCS Podcast is the official podcast of the Neurocritical Care Society. Our senior producer is Bonnie Rossow. Our host is Fawaz Almufti, and our production staff includes Tareq Saad Almaghrabi, Andrew Bauerschmidt, Leonid Groysman, Atul Kalanuria, Lauren Koffman, Kassi Kronfeld, Holly Ledyard, Lindsay Marchetti, Alexandra Reynolds, Lucia Rivera Lara, Jon Rosenberg, Jason Siegel, Zachary Threlkeld, Teddy Youn, and Chris Zammit. Our administrative staff includes Bonnie Rossow. Music by Mohan Kottapally.

Aging-US published a Special Collection on Eye Disease which included "Role of Citicoline in an in vitro AMD model" which reported that citicoline is the exogenous form of the nootropic, Cytidine 5'-diphosphate-choline that exerts its neuroprotective effects in the brain as well as in the eye. Citicoline alleviates apoptotic effects as evidenced by diminished AnnexinV/PI and Caspase-3/7 staining, downregulation of apoptosis genes, enhanced cell viability, and reduced oxidative stress in AMD RPE cybrid cells. However, further studies are required to establish the merit of citrusoline as a cytoprotective molecule in AMD and to decipher the molecular underpinnings of its mechanism of action. Dr. M. Cristina Kenney from The University of California Irvine said, "Citicoline is the international nonproprietary name given to the exogenous pharmacological form of Cytidine 5'-diphosphate-choline (CDP-Choline, CDPCho), a naturally occurring endogenous nucleotide compound that is water-soluble and has a molecular weight of 488.32 g/mol" Citicoline maintains neuronal membrane integrity, influences neurotransmitter levels, increases norepinephrine and dopamine levels in the central nervous system, restores the activity of membrane sodium/potassium ATPase and mitochondrial ATPase, and enhances brain function. Owing to these mechanisms, it has been successfully used as a neuroprotective agent to prevent neuronal aging and improve memory and learning in vitro. It has been extensively used in preclinical studies and clinical trials for neurodegenerative diseases including Parkinson's disease and glaucoma. This in vitro study supports our hypothesis as Citicoline conferred significant protection against apoptotic cell death that was in-part mediated by damaged mtDNA from AMD patients. The Kenney Research Team concluded in their Aging-US Research Output, "although further studies with Citicoline/ AMD RPE cybrid cells are underway, these results present novel findings that identify Citicoline to be a potential protector that attenuates apoptotic cell death in AMD. Citicoline is available as an over-the-counter dietary supplement in the U.S. and offers the advantage of easy access that shortens considerably the transition from lab bench to clinic." Full Text - https://www.aging-us.com/article/103164/text Correspondence to: M. Cristina Kenney email: mkenney@uci.edu Keywords: Citicoline, age-related macular degeneration (AMD), neuroprotection, RPE, mitochondria About Aging-US Launched in 2009, Aging-US publishes papers of general interest and biological significance in all fields of aging research as well as topics beyond traditional gerontology, including, but not limited to, cellular and molecular biology, human age-related diseases, pathology in model organisms, cancer, signal transduction pathways (e.g., p53, sirtuins, and PI-3K/AKT/mTOR among others), and approaches to modulating these signaling pathways. To learn more about Aging-US, please visit http://www.Aging-US.com or connect with @AgingJrnl Aging-US is published by Impact Journals, LLC please visit http://www.ImpactJournals.com or connect with @ImpactJrnls Media Contact 18009220957x105 MEDIA@IMPACTJOURNALS.COM

The TWiM folk explore disruption of a Burkholderia intracellular niche by a cell death program, and an increase in Brucella infectiousness after intracellular passage. Become a patron of TWiM. Links for this episode: Disrupting intracellular niche (mBio) Intracellular passage increases infectiousness (Infect Immun) Type IV secretion systems (Front Micro) 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

The benefits of the Mediterranean diet pass on to the families of patients who follow it Hospital del Mar Medical Research Institute (Italy), March 9, 2021 People living with a patient undergoing an intensive weight loss treatment also benefit from this therapy. This has been demonstrated by a team of researchers from the Hospital del Mar Medical Research Institute (IMIM-Hospital del Mar) along with doctors from Hospital del Mar and the CIBER on the Physiopathology of Obesity and Nutrition (CIBERObn), in collaboration with IDIAPJGol, the Pere Virgili Health Research Institute (IISPV), IDIBELL, IDIBAPS and the Sant Joan de Reus University Hospital. The study has been published in the journal International Journal of Obesity. The study analysed data from 148 family members of patients included in the weight loss and lifestyle programme PREDIMED-Plus (PREVencióDIetaMEDiterranea Plus) over a two-year period. The researchers analysed whether these people also indirectly benefited from the programme, as they were not enrolled in the study and did not receive any direct treatment. PREDIMED-Plus is a multicentre study in which a group of patients follow an intensive weight reduction programme based on the Mediterranean diet and a plan promoting physical activity. Weight loss despite not being included in the programme The relatives (three out of four were the patient's partner and the rest were children, parents, siblings or had some other degree of kinship), lost an average of 1.25 kg of weight during the first year of the programme, compared to the relatives of the patients in the control group (those who did not follow the intensive treatment proposed by PREDIMED-Plus). This rose to almost 4 kg in the second year. These figures were better in cases where the family member ate with the patient and, above all, when it was the patient themselves who cooked. The treatment, aimed at achieving weight loss in people with obesity and high cardiovascular risk by following the Mediterranean diet, "Achieved effects beyond just weight loss in the patient, and this extended to their family environment", explains Dr. Albert Goday, the principal investigator on the project, head of section in the Department of Endocrinology and Nutrition at Hospital del Mar, researcher in the Cardiovascular Risk and Nutrition Research Group at the IMIM-Hospital del Mar and a CIBERobn researcher. "The effect was contagious, in this context it was, fortunately, a beneficial 'contagion', resulting in weight loss and improved dietary habits." Dr. Goday points out that "among the many possible dietary approaches to weight loss, the one based on the Mediterranean diet is the most easilt shared within a family environment." According to Dr. Olga Castañer, the final author of the study and a researcher in the Cardiovascular Risk and Nutrition Research Group at the IMIM-Hospital del Mar and CIBERobn, the good results can be explained "By an improved diet, since the same contagious effect was not observed in terms of physical activity among the patients and their relatives." Family members also showed increased commitment to the Mediterranean diet, according to a questionnaire assessing adherence to the dietary patterns of this regimen. But the same was not true in terms of physical activity. As Dr Castañer points out, "In addition to weight loss, there was greater adherence to the Mediterranean diet, which has intrinsic health benefits, such as protection against cardiovascular and neurodegenerative risks." The results of the study "Demonstrate the contagion effect, the halo effect, of a treatment programme in the relatives of participants involved in an intensive weight loss procedure, as well as increased adherence to the Mediterranean diet", stresses Dr. Albert Goday. "The beneficial effect of the programme on one member of the family unit can be extended to its other members, which is extremely significant in terms of reducing the burden of obesity on the public health system", he explains. The family members not only lost weight but also improved the quality of their diet. Effect of the programme on patients The study also analysed the results of the PREDIMED-Plus programme in 117 patients. Compared to participants in the control group, they lost 5.10 kg in the first year of intervention rising to 6.79 kg in the second year. They also significantly increased their physical activity levels, as well as their adherence to the Mediterranean diet.     CBD reduces plaque, improves cognition in model of familial Alzheimer's Medical College of Georgia at Augusta University, March 9, 2021 A two-week course of high doses of CBD helps restore the function of two proteins key to reducing the accumulation of beta-amyloid plaque, a hallmark of Alzheimer's disease, and improves cognition in an experimental model of early onset familial Alzheimer's, investigators report.  The proteins TREM2 and IL-33 are important to the ability of the brain's immune cells to literally consume dead cells and other debris like the beta-amyloid plaque that piles up in patients' brains, and levels of both are decreased in Alzheimer's.  The investigators report for the first time that CBD normalizes levels and function, improving cognition as it also reduces levels of the immune protein IL-6, which is associated with the high inflammation levels found in Alzheimer's, says Dr. Babak Baban, immunologist and associate dean for research in the Dental College of Georgia and the study's corresponding author.  There is a dire need for novel therapies to improve outcomes for patients with this condition, which is considered one of the fastest-growing health threats in the United States, DCG and Medical College of Georgia investigators write in the Journal of Alzheimer's Disease.  "Right now we have two classes of drugs to treat Alzheimer's," says Dr. John Morgan, neurologist and director of the Movement and Memory Disorder Programs in the MCG Department of Neurology. One class increases levels of the neurotransmitter acetylcholine, which also are decreased in Alzheimer's, and another works through the NMDA receptors involved in communication between neurons and important to memory. "But we have nothing that gets to the pathophysiology of the disease," says Morgan, a study coauthor.  The DCG and MCG investigators decided to look at CBD's ability to address some of the key brain systems that go awry in Alzheimer's.  They found CBD appears to normalize levels of IL-33, a protein whose highest expression in humans is normally in the brain, where it helps sound the alarm that there is an invader like the beta-amyloid accumulation. There is emerging evidence of its role as a regulatory protein as well, whose function of either turning up or down the immune response depends on the environment, Baban says. In Alzheimer's, that includes turning down inflammation and trying to restore balance to the immune system, he says. That up and down expression in health and disease could make IL-33 both a good biomarker and treatment target for disease, the investigators say.  CBD also improved expression of triggering receptor expressed on myeloid cells 2, or TREM2, which is found on the cell surface where it combines with another protein to transmit signals that activate cells, including immune cells. In the brain, its expression is on the microglial cells, a special population of immune cells found only in the brain where they are key to eliminating invaders like a virus and irrevocably damaged neurons. Low levels of TREM2 and rare variations in TREM2 are associated with Alzheimer's, and in their mouse model TREM2 and IL-33 were both low.  Both are essential to a natural, ongoing housekeeping process in the brain called phagocytosis, in which microglial cells regularly consume beta amyloid, which is regularly produced in the brain, the result of the breakdown of amyloid-beta precursor protein, which is important to the synapses, or connection points, between neurons, and which the plaque interrupts.  They found CBD treatment increased levels of IL-33 and TREM2 -- sevenfold and tenfold respectively.  CBD's impact on brain function in the mouse model of early onset Alzheimer's was assessed by methods like the ability to differentiate between a familiar item and a new one, as well as observing the rodents' movement.  People with Alzheimer's may experience movement problems like stiffness and an impaired gait, says Dr. Hesam Khodadadi, a graduate student working in Baban's lab. Mice with the disease run in an endless tight circle, behavior which stopped with CBD treatment, says Khodadadi, the study's first author.  Next steps include determining optimal doses and giving CBD earlier in the disease process. The compound was given in the late stages for the published study, and now the investigators are using it at the first signs of cognitive decline, Khodadadi says. They also are exploring delivery systems including the use of an inhaler that should help deliver the CBD more directly to the brain. For the published studies, CBD was put into the belly of the mice every other day for two weeks. A company has developed both animal and human inhalers for the investigators who also have been exploring CBD's effect on adult respiratory distress syndrome, or ARDS, a buildup of fluid in the lungs that is a major and deadly complication of COVID-19, as well as other serious illnesses like sepsis and major trauma. The CBD doses used for the Alzheimer's study were the same the investigators successfully used to reduce the "cytokine storm" of ARDS, which can irrevocably damage the lungs.  Familial disease is an inherited version of Alzheimer's in which symptoms typically surface in the 30s and 40s and occurs in about 10-15% of patients.  CBD should be at least equally effective in the more common, nonfamilial type Alzheimer's, which likely have more targets for CBD, Baban notes. They already are looking at its potential in a model of this more common type and moving forward to establish a clinical trial.  Plaques as well as neurofibrillary tangles, a collection of the protein tau inside neurons, are the main components of Alzheimer's, Morgan says. Beta-amyloid generally appears in the brain 15-20 years or more before dementia, he says, and the appearance of tau tangles, which can occur up to 10 years afterward, correlates with the onset of dementia. There is some interplay between beta amyloid and tau that decrease the dysfunction of each, Morgan notes.  The Food and Drug Administration is scheduled to make a ruling by early June on a new drug aducanumab, which would be the first to attack and help clear beta amyloid, Morgan says.     1,25-dihydroxyvitamin D supports bone marrow stem cell proliferation Cang-zhou Central Hospital (China), March 1, 2021 According to news originating from Hebei, People’s Republic of China, research stated, “Osteoporosis (OP) is a common clinical geriatric disease. Bone marrow mesenchymal stem cells (BMSCs) are widely applied in bone engineering. 1,25-dihydroxyvitamin D (1,25-(OH) 2D) deficiency involves in geriatric disease.” Our news journalists obtained a quote from the research from Cangzhou Central Hospital, “However, its effects on BMSCs differentiation and osteoporosis have not yet been elucidated. An OVX-induced OP rat model was constructed and treated with 10 mu M 1,25-(OH) 2D followed by analysis of bone mineral density and ALP activity. Rat BMSCs were isolated and divided into control group, OP group, OP rat BMSCs, and VD group (OP rats were injected with 1 mu M 1,25-(OH) 2D) followed by analysis of cell survival by MTT assay, Caspase 3 activity, type I collagen and Osterix expression by Real time PCR, Wnt5 expression by Western blot and TGF-beta secretion by ELISA. The bone density and ALP activity was significantly decreased in OP rats (P < 0.05). 1,25 (OH) 20 injected into OP rats significantly increased bone density and ALP activity (P < 0.05). The survival rate of BMSCs in OP group was significantly decreased and Caspase 3 activity was increased along with downregulated type I collagen and Osterix, TGF-beta secretion and Wnt5 expression (P < 0.05).” According to the news editors, the research concluded: “Adding 1,25-(OH) 2D to BMSCs cells in OP group could significantly reverse the above changes (P < 0.05). 1,25-dihydroxyvitamin D promotes BMSCs proliferation by regulating Wnt5/TGF-beta signaling, promotes differentiation into osteogenesis, increases bone density, and then improves osteoporosis.” This research has been peer-reviewed.     High-fat diets can cause normal liver tissue to behave like tumor tissue Flanders Institute of Biotechnology (Belgium), March 10, 2021 Normal, non-cancerous liver tissue can act like tumor tissue when exposed to a diet high in fat, linking diet and obesity to the development of liver cancer. The Laboratory of Cellular Metabolism and Metabolic Regulation headed by Prof. Sarah-Maria Fendt (VIB-KU Leuven Center for Cancer Biology), shows how the livers of mice on a high-fat diet used glucose in a way similar to aggressive cancer cells. This suggests that when the liver is exposed to excess fat, normal tissue could be primed to become cancerous. The study appeared in the journal Cancer Research. Cancer and obesity With global rates of obesity and liver cancerincreasing each year, understanding how excess fat availability can drive liver cancerdevelopment is important to understand how the disease starts and how it can be treated. To explore this, Prof. Fendt and her team tested the metabolic changes in liver tissue from mice fed a high-fat diet at an early time point when no tumors were present, and late time point when tumors had formed. They found that before there were any clues that cancer was developing, the liver tissue used glucose the same way that tumors would. This high use of glucose is one of the well-known hallmarks of cancer and is known as the Warburg effect. After finding these early changes to liver tissue, they investigated what happens when tumors have fully formed. One way they measured this was to test sensitivity to glucose, which is usually cleared away quickly by the body but is impaired in obesity-induced diabetic animals. Prof. Fendt describes what they found: "Strikingly, mice fed a high-fat diet who had a large tumor burden could remove glucose from their blood as easily as healthy mice despite being diabetic. Using state-of-the-art 13C6-glucose tracing technology, we could observe how glucose molecules are used in cells and tissues, and we found that that tumor tissue breaks down glucose in a consistent way, regardless of whether the mice were fed high-fat or normal diets." Alternative pathways These findings suggest that when cancer cells develop from normal liver cells, their metabolism consistently increases glucose use. Since a high-fat diet causes these changes before cancer is present, this may mean that—in a high-fat diet—non-cancer liver tissue could be more likely to become cancerous. The team also looked into deeper mechanisms for this effect. Dr. Lindsay Broadfield, one of the lead authors of the study, says: "We discovered that, before any cancer development, liver tissue exposed to high fat seemed to use an alternative pathway for fat breakdown in a cellular compartment called the peroxisome. Using cancer liver cells, we then confirmed that peroxisome metabolism increased cellular stress and glucose uptake." Fat can be used by cells in several ways—for energy, to stimulate growth pathways, or to be stored for later use. The scientists used the Lipometrix lipidomics platform at KU Leuven to see if there was anything unique about the fate of fat in tumor cells and found that the fat species and content in tumor cells were indeed different from non-cancerous liver tissue close to the tumors.     For teens, outdoor recreation during the pandemic linked to improved well-being North Carolina State University, March 9, 2021 A study from North Carolina State University found outdoor play and nature-based activities helped buffer some of the negative mental health impacts of the COVID-19 pandemic for adolescents.  Researchers said the findings, published in the International Journal of Environmental Research and Public Health, point to outdoor play and nature-based activities as a tool to help teenagers cope with major stressors like the COVID-19 pandemic, as well as future natural disasters and other global stressors. Researchers also underscore the mental health implications of restricting outdoor recreation opportunities for adolescents, and the need to increase access to the outdoors. "Families should be encouraged that building patterns in outdoor recreation can give kids tools to weather the storms to come," said Kathryn Stevenson, a study co-author and assistant professor of parks, recreation and tourism management at NC State. "Things happen in life, and getting kids outside regularly is an easy way to build some mental resilience." In the survey, conducted from April 30 to June 15, 2020, researchers asked 624 adolescents between the ages of 10 to 18 years to report their participation in outdoor recreation both before the pandemic and after social distancing measures were in effect across the United States. They also asked adolescents about their subjective well-being, a measure of happiness, and mental health. The findings revealed the pandemic had an impact on the well-being of many teens in the survey, with nearly 52 percent of adolescents reporting declines in subjective well-being. They also saw declines in teens' ability to get outside, with 64 percent of adolescents reporting their outdoor activity participation fell during the early months of the pandemic. Despite these declines in outdoor activity participation, nearly 77 percent of teens surveyed believed that spending time outside helped them deal with stress associated with the COVID-19 pandemic. "We know that a lot of outdoor activities that kids engage in happen during school, in youth sports leagues or clubs, and those things got put on hold during the pandemic," said the study's lead author Brent Jackson, a graduate student in the Fisheries, Wildlife and Conservation Biology Program at NC State. "Based on our study, they were getting outside less - we think not being in school and having those activities really contributed to that." When they broke down recreation by type, they saw participation in outdoor play activities such as sports, biking, going for walks, runs or skating declined by 41.6 percent, nature-based activities such as camping, hiking, fishing, hunting, and paddling dropped by 39.7 percent, and outdoor family activities declined by 28.6 percent. In those early months of the pandemic, about 60 percent of teens said they were able to get outside once a week or less. "We saw declines in all three types of outdoor recreation participation," Jackson said. "Nature-based activities had the lowest participation before and during the pandemic, which may point to the need for more access to natural spaces in general." Results showed that well-being and outdoor recreation trends were linked, and the negative trends they saw during the pandemic for well-being and participation in outdoor recreation were seen regardless of teens' race, gender, age, income community type or geographic region. Kids who did not get outside as much saw declines in well-being, but those who got outside both before and during the pandemic were able to maintain higher levels of well-being.  "This tells us that outdoor recreation can promote well-being for kids when it happens, and can potentially take away from well-being when it doesn't," Stevenson said. Teens who had high rates of outdoor play before the pandemic were more resistant to negative changes in social well-being. Those who got outside frequently before the pandemic were more likely to experience a lesser decline in well-being, regardless of participation during the pandemic. And, for teens who were able to play outside or get involved in nature-based activities during the pandemic, their well-being was on par with pre-pandemic levels. "Kids who were able to continue participating in outdoor play and nature-based activities had subjective well-being levels that were similar to what they were before the pandemic, but kids who weren't able to participate saw much greater declines," Jackson said. The study's findings also point to strategies to help kids navigate future global stressor events, as well as the importance of ensuring access to outdoor recreation. They help define the risks associated with policies that reduce kids' ability get outside. "Going outside and participating in activities that provide exposure to nature, physical activity and safe social interaction during the pandemic were really powerful in terms of improving kids' resilience," Jackson said.     Study finds two servings of fish per week can help prevent recurrent heart disease McMaster University (Ontario), March 8, 2021 An analysis of several large studies involving participants from more than 60 countries, spearheaded by researchers from McMaster University, has found that eating oily fish regularly can help prevent cardiovascular disease (CVD) in high-risk individuals, such as those who already have heart disease or stroke. The critical ingredient is omega-3 fatty acids, which researchers found was associated with a lower risk of major CVD events such as heart attacks and strokes by about a sixth in high-risk people who ate two servings of fish rich in omega-3 each week. "There is a significant protective benefit of fish consumption in people with cardiovascular disease," said lead co-author Andrew Mente, associate professor of research methods, evidence, and impact at McMaster and a principal investigator at the Population Health Research Institute.  No benefit was observed with consumption of fish in those without heart disease or stroke. "This study has important implications for guidelines on fish intake globally. It indicates that increasing fish consumption and particularly oily fish in vascular patients may produce a modest cardiovascular benefit."  Mente said people at low risk for cardiovascular disease can still enjoy modest protection from CVD by eating fish rich in omega-3, but the health benefits were less pronounced than those high-risk individuals.  The study was published in JAMA Internal Medicine on March 8.  The findings were based on data from nearly 192,000 people in four studies, including about 52,000 with CVD, and is the only study conducted on all five continents. Previous studies focused mainly on North America, Europe, China and Japan, with little information from other regions. "This is by far the most diverse study of fish intake and health outcomes in the world and the only one with sufficient numbers with representation from high, middle and low income countries from all inhabited continents of the world," said study co-lead Dr. Salim Yusuf, professor of medicine at the Michael G. DeGroote School of Medicine and executive director of the PHRI.  This analysis is based in data from several studies conducted by the PHRI over the last 25 years. These studies were funded by the Canadian Institutes for Health Research, several different pharmaceutical companies, charities, the Population Health Research Institute and the Hamilton Health Sciences Research Institute.     COVID-19 or something else? Learn how COVID-19 symptoms compare to other illnesses, and when you should call the doctor. Harvard University, March 10, 2021   Before 2020, you might not have worried much about a tickle in your throat or a little tightness in your chest. But that's changed. Now even slight signs of a respiratory bug might make you wonder if it's the start of COVID-19, the illness that has become a pandemic. How do you distinguish one illness from another? It's complicated. "Many of the symptoms overlap. For example, it's very hard for me clinically, as a physician, to be able to look at someone and say it's COVID-19 or it's influenza," says Dr. Ashish Jha, former director of the Harvard Global Health Institute and now dean of the Brown University School of Public Health. Don't jump to conclusions if you start to feel sick. Learn the hallmarks of common illnesses and how they differ from COVID-19, so you can take the appropriate action. COVID-19 COVID-19 is an extremely contagious respiratory illness caused by a type of virus (a coronavirus) called SARS-CoV-2. It's a cousin of the common cold, but its potential consequences are far more serious: hospitalization, lasting complications, and death. Hallmarks: Loss of taste and smell (in the absence of nasal congestion), fever, cough, shortness of breath, and muscle aches. Other potential symptoms: Sore throat, diarrhea, congestion, runny nose, chills, shivering, headache, fatigue, and loss of appetite. Note: Some infected people don't have any symptoms of COVID-19, but they're still contagious. Influenza Influenza (flu) is a highly contagious respiratory infection caused by the influenza A, B, or C virus. The U.S. flu season typically lasts from October to March, but flu is present year-round. Hallmarks: Fever, muscle aches, and cough. Other potential symptoms: Sore throat, diarrhea, congestion, runny nose, chills, shivering, headache, fatigue, loss of appetite. Different from COVID-19: Flu usually does not cause shortness of breath. Common cold The common cold (viral rhinitis) is an upper respiratory infection that can be caused by any of hundreds of different viruses (including coronaviruses or rhinoviruses). It's usually mild and resolves within a week. Hallmarks: Congestion, runny nose, cough, and sore throat. Other potential symptoms: Fever, muscle aches, and fatigue. Different from COVID-19: A cold does not cause shortness of breath, body aches, chills, or loss of appetite, and it usually doesn't cause fever. Seasonal allergies A seasonal allergy isn't a virus; it's caused when the immune system responds to a harmless non-human substance, like tree pollen, as if it were a dangerous threat. Allergies are typically seasonal, lasting for weeks or months, depending on the allergen in the air (mold is the common allergen in the fall and winter). Hallmarks: Runny nose, itchy eyes, sneezing, congestion. Other potential symptoms: Loss of smell from congestion. Different from COVID-19: Allergies do not cause fevers, coughing, shortness of breath, muscle aches, sore throat, diarrhea, chills, headaches, fatigue, or loss of appetite. Asthma Asthma is a chronic lung condition caused by inflammation in the air passages. Airways narrow and make it harder to breathe, which can cause concern that it might be COVID-19. "Asthma can be triggered by a cold or influenza, but it's a separate condition," Dr. Jha says. Hallmarks: Wheezing (a whistling sound as air is forcibly expelled), difficulty breathing, chest tightness, and a persistent cough. Other potential symptoms: A severe asthma attack can cause sudden, extreme shortness of breath; chest tightness; a rapid pulse; sweating; and bluish discoloration of the lips and fingernails. Different from COVID-19: Asthma does not cause a fever, muscle aches, sore throat, diarrhea, congestion, loss of taste or smell, runny nose, chills, shivering, headache, fatigue, or loss of appetite.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.08.288043v1?rss=1 Authors: Cosker, K., Mallach, A., Limaye, J., Piers, T. M., Staddon, J., Neame, S. J., Hardy, J., Pocock, J. M. Abstract: The R47H variant of the microglial membrane receptor TREM2 is linked to increased risk of late onset Alzheimers disease. Human induced pluripotent stem cell derived microglia, iPS-Mg, from patient iPSC lines expressing the AD-linked R47Hhet TREM2 variant, common variant, Cv, or an R47Hhom CRISPR edited line and its isogeneic control, demonstrated that R47H-expressing iPS-Mg expressed a deficit in signal transduction in response to the TREM2 endogenous ligand phosphatidylserine with reduced pSYK-pERK1/2 signalling and a reduced NLRP3 inflammasome response, including ASC speck formation, Caspase-1 activation and IL-1beta secretion. Apoptotic cell phagocytosis and soluble TREM2 shedding were unaltered, suggesting a disjoint between these pathways and the signalling cascades downstream of TREM2 in R47H-expressing iPS-Mg, whilst metabolic deficits in glycolytic capacity and maximum respiration were reversed when R47H expressing iPS-Mg were exposed to PS+ expressing cells. These findings suggest that R47H-expressing microglia are unable to respond fully to cell damage signals such as phosphatidylserine, which may contribute to the progression of neurodegeneration in late-onset AD. Copy rights belong to original authors. Visit the link for more info

ENCODEプロジェクトにおけるサイレンサー探索の論文紹介と現代生物学の複雑性と多様性について話しました。Shownotes ENCODE…ENCODEとはEncyclopedia of DNA Elementsの略！ ENCODE (Wikipedia) ヒトゲノムの機能解明に向けたENCODEの試み (pdf) Initial sequencing and analysis of the human genome. Nature 2001…ヒトゲノム解読の論文。 The Sequence of the Human Genome. Science 2001…ヒトゲノム解読の論文。 ENCODE 4…ENCODEのPhase 4についてはここにaimなどが書いてある。 ENCODE3勉強会…Sohも論文読みをしました。 ENCODE 3 paper collection…ENCODE 3の論文リストはここにあります。 DNase I hypersensitive site (Wikipedia) Systematic identification of silencers in human cells. Nature Genetics, 2020…今回Sohが解説する論文はこれです。　 転写制御因子 (脳科学辞典) … プロモーターやエンハンサー、サイレンサーまわりについて。遺伝子発現とは、遺伝子というDNA上にコードされた情報がいかにRNA、タンパク質として実体を伴って機能するかということであるのだが、この量を調節する機能、とくにDNA->RNAの段階(転写)で調節することを転写制御という。ここで重要となるのがプロモーターやエンハンサー、サイレンサー、さらにはインスレーターなどである。 遺伝子発現制御機構: クロマチン, 転写制御, エピジェネティクス (Amazon) … tadasuはこの本で一度転写周りを勉強しなおしたが、自分が思っていたプロモーターやエンハンサーと実際の定義はかなりズレがあったことを思い知らされた。 STARR-seq (Wikipedia)…ゲノム中に含まれるエンハンサーやプロモーター配列をバーコードと次世代シーケンサーを用いて大量にアッセイする方法の一つ。オリジナルの論文はここ: Genome-Wide Quantitative Enhancer Activity Maps Identified by STARR-seq, Science 2013 Caspase-9 (Wikipedia)…Caspase9はアポトーシスを担う重要なタンパク質の一つ。 EF1aプロモーター…ヒトやマウスの細胞の中で高い活性を持つ恒常発現プロモーターのうちの一つ。CMVプロモーターも使われることが多い。 サイレンサー…哺乳動物細胞における転写を制御する配列のうち、転写活性に対して抑制的に働く配列を指しています。恒常発現プロモーターの下流にあるとそのさらに下流にある遺伝子発現を強く抑制する配列（200bp程度）を指しています。 ハウスキーピング遺伝子…細胞機能を維持するために常に発現している遺伝子。これを「恒常的な遺伝子発現」と呼んだりします。 K562…ヒト慢性骨髄性白血病由来の培養細胞株。 293T…ヒト胎児腎由来の培養細胞株。 HepG2…ヒト肝癌由来の細胞株。 ルシフェラーゼアッセイ pGL4.51[luc2/CMV/Neo] Vector…スクリーニングで見つかったサイレンサーはこのベクターを用いてルシフェラーゼアッセイによってその活性が調べられた。 ヒストン修飾 (Abcam) ヒストン (Wikipedia) Purification of Proteins Associated with Specific Genomic Loci. Cell, 2009.…PICh法を用いるとゲノムに結合したタンパク質を同定することができる。 Hi-C…Hi-Cについてはエピソード9などで話しました。 ChiA-PET (Wikipedia) FANTOM…“FANTOMは、理化学研究所のマウスゲノム百科事典プロジェクトで収集された完全長cDNAのアノテーション（機能注釈）を行うことを目的に、林崎良英博士が中心となり2000年に結成された国際研究コンソーシアムです” NIH Roadmap Epigenomics Mapping Consortium…ヒトのエピジェネティクスに関連する大量のデータを計測しアトラスを作ることを目的とした国際コンソーシアム。 Illumina Body Map 2.0 Human Cell Atras…ヒトの体を構成する全ての細胞の分類とマッピングを目指す国際共同プロジェクト。 4D Nucleome Project…染色体構造のマッピングのためのコンソーシアム。 The 4D nucleome project. Nature, Dekker at al 2017 The Cancer Genome Atlas Program (TCGA)…がんゲノムの大量のデータを収集している国際プロジェクト。 ゲノム編集とはなにか (ブルーバックス) (Amazon)…山本先生が書かれたゲノム編集について非常にわかりやすい本が出ております。ぜひ。 ep47, Researchat.fm…エピソード47ではCas13を用いた微量の核酸検出SHERLOCKやDETECTRについて紹介しています。 Advances in Chromatin and Chromosome Research: Perspectives from Multiple Fields. Molecular Cell, 2020 … tadasuが参加しているBoston Chromatin Clubで書いた筆頭著者17人のレビュー。 ep37, Biological Enigma, Researchat.fm…セントラルドグマを含む分子生物学の基礎についても話しました。 ep7, In the golden age of molecular biology … 遺伝暗号解明周りの研究について話しました。 ep9, One-shot beautiful experiment … 細胞系譜について ep2, An emerging technology is always not perfect … CRISPRとゲノム編集周辺について話しました。 Gene drive (Wikipedia) … 遺伝子ドライブ ワトソンの遺伝子の分子生物学 (Amazon) 後成説 (Wikipedia) … エピジェネシス。アリストテレスから始まる発生の理論。 前成説 (Wikipedia) 分子進化のほぼ中立説 偶然と淘汰の進化モデル (ブルーバックス) (Amazon) ep36, DNA-of-things … DNAを練りこんだ3Dプリンティング技術について話しました。 情報量 … 情報量、エントロピー シャノンの情報理論入門 (ブルーバックス) (Amazon) ファインマンの計算機科学 (Amazon) … tadasuおすすめの情報理論の教科書。ゲノムやセントラルドグマの話についての情報理論についても記述がある(あった気がする)。 Developmental Biology (Amazon) … 発生のGilbert本 Principles of Development (Amazon) … 発生のWolpert本 Molecular Biology of the Cell (NCBI) Shownotes 正直、プロモーターとかエンハンサーとかサイレンサーって言っていますが、実際にプロモーターとエンハンサーの違いなどよく考えるとわからなくなってきます。とても難しい。雰囲気でやっています。(soh) データは山のようにある。あとは君たち次第だ！(いいのかそれで？)(tadasu) 作業が楽しくても3日以上寝ないと空から奴らが襲ってくるので要注意です(coela)

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.07.241620v1?rss=1 Authors: Yao, L., Swartz, P. D., Hamilton, P., Clark, C. Abstract: Caspase enzymes play important roles in apoptosis and inflammation, and the non-identical but overlapping specificity profiles direct cells to different fates. Although all caspases prefer aspartate at the P1 position of the substrate, the caspase-6 subfamily shows preference for valine at the P4 position, while caspase-3 shows preference for aspartate. In comparison to human caspases, caspase-3a from zebrafish has relaxed specificity and demonstrates equal selection for either valine or aspartate at the P4 position. In the context of the caspase-3 conformational landscape, we show that changes in hydrogen bonding near the S3 subsite affect selection of the P4 amino acid. Swapping specificity with caspase-6 requires accessing new conformational space, where each landscape results in optimal binding of DxxD (caspase-3) or VxxD (caspase-6) substrate and simultaneously disfavors binding of the other substrate. Within the context of the caspase-3 conformational landscape, substitutions near the active site result in nearly equal activity against DxxD and VxxD by disrupting a hydrogen bonding network in the substrate binding pocket. The converse substitutions in zebrafish caspase-3a result in increased selection for P4 aspartate over valine. Overall, the data show that evolutionary neofunctionalization resulting in a dual function protease, as in zebrafish caspase-3a, requires fewer amino acid substitutions compared to those required to access new conformational space for swapping substrate specificity, such as between caspases-3 and -6. Copy rights belong to original authors. Visit the link for more info

Vincent and Daniel solve the case of the Panamanian Farmer with Three Weeks of Diarrhea, and discuss how microbes egested during bites of sand flies exacerbate the severity of leishmaniasis. Hosts: Vincent Racanielloand Daniel Griffin Become a patron of TWiP. Links for this episode: Sand fly microbesand leishmaniasis severity (Cell Host Micr) Letters read on TWiP 153 Case Study for TWiP 153 Man in clinic, in city recently, developed rash in groin area, concerned about this. Lives in open relationship with male partner, had sexual encounter with another male. Few weeks. Rash is in the pubic hair, is very itchy. Mostly around umbilicus, can see blue spots in this area. These are skin changes. Buys magnifying glass to look and describes seeing things which we see as well. No notable medical history. Send your case diagnosis, questions and comments to twip@microbe.tv Music by Ronald Jenkees

Synaptic activity shifts dendritic lysosomes Invading pathogens or other toxic agents can trigger the assembly of the inflammasome adaptor ASC into large, intracellular specks that activate caspase-1 to initiate a proinflammatory cell death called pyroptosis. Kuri et al. follow the dynamics of ASC speck formation in live zebrafish, revealing their lethal effects on epidermal keratinocytes and their subsequent engulfment and degradation by macrophages. This biosights episode presents the paper by Kuri et al. from the September 4th, 2017, issue of The Journal of Cell Biology and includes an interview with two of the paper's authors, Paola Kuri and Maria Leptin (EMBL). Produced by Caitlin Sedwick and Ben Short. See the associated paper in JCB for details on the funding provided to support this original research.

Host: Vincent Racaniello Guests: Rollie Clem and Lorena Passarelli Vincent visits the 'Little Apple' and speaks with Rollie and Lorena about their work on mosquito-born viruses and baculoviruses. Links for this episode Apoptosis inhibition in A. aegypti (Apop) Initiator caspase in armyworm (Insect Bioc Mol Biol) Manipulating apoptosis in A. aegypti (J Virol) Viral inhibitors of apoptosis (Sem Cell Dev Biol) Baculoviruses: Sophisticated pathogens (PLoS Path) Baculovirus morphogenesis (Virol) Barriers to baculovirus success (Virol) Baculovirus endonuclease (J Virol) Baculovirus DNA damage response (J Virol) Video of this episode - view at YouTube Send your virology questions and comments (email or mp3 file) to twiv@twiv.tv

Der TNF-verwandte Apoptose induzierende Ligand TRAIL ist in der Lage, spezifisch Apoptose in Tumorzellen zu induzieren ohne normales Gewebe zu schädigen und gilt deswegen als vielversprechender Kandidat für den Einsatz in der Tumortherapie. Neben der Induktion von Apoptose ist TRAIL allerdings auch in der Lage den Transkriptionsfaktor NF-κB („nuclear factor 'kappa-light-chain-enhancer' of activated B-cells") zu aktivieren, was wiederum die Induktion von Zelltod durch TRAIL vermindert. Für die Tumortherapie wäre es daher wünschenswert, durch TRAIL selektiv den Apoptosesignalweg und nicht NF-κB zu aktivieren. Dazu ist ein Verständnis beider Signalwege unerlässlich. Im Gegensatz zum Signalweg der TRAIL-induzierten Apoptose ist der Signalweg der TRAIL-induzierten Aktivierung von NF-κB aber bisher wenig erforscht. Das Ziel der vorliegenden Arbeit war es daher, das rezeptornahe Adaptorprotein und die ersten, rezeptornahen Signalschritte der NF-κB Aktivierung durch TRAIL zu identifizieren. Die Überexpression konstitutiv aktiver Fusionsproteine der beiden TRAIL-Rezeptoren 1 und 2 war in der Lage, NF-κB zu aktivieren. Fusionsproteine mit verkürztem C-Terminus, der die Apoptose-vermittelnde Todesdomäne enthält, waren dabei nicht im Stande den Signalweg zu aktivieren. Dadurch konnte die entscheidende Funktion der Todesdomäne für TR1 und TR2 bei der Induktion von NF-κB gezeigt werden. Um Untersuchungen des TRAIL-Signalweges mit dem Liganden durchführen zu können, wurde die bakterielle Produktion und Aufreinigung von rekombinantem humanem TRAIL, sowie einem hochaktiven ILZ (Isoleucin Zipper)-TRAIL und einer inaktiven Kontrollvariante etabliert. Die Untersuchung von FADD („fas associated death domain“)-defizienten JURKAT-Zellen, zeigte, dass diese nicht in der Lage waren, NF-κB auf TRAIL zu aktivieren. Die transiente und auch stabile Reexpression von FADD stellte dabei die Aktivierbarkeit von NF-κB durch TRAIL wieder her. Somit konnte FADD als Adaptorprotein für die TRAIL-induzierte Aktivierung von NF-κB identifiziert werden. Die Expression von FADD-Varianten mit je zwei Punktmutationen in der Todeseffektordomäne war hingegen nicht in der Lage, TRAIL-induzierte Aktivierung von NF-κB wiederherzustellen, was auf die Notwendigkeit der Multimerisierung von FADD hinweist. Des Weiteren aktivierten auch JURKAT-Zellen, die defizient für Caspase 8 („cysteinyl-aspartate specific protease 8“) waren, den TRAIL-induzierten NF-κB-Signalweg nicht. Auch die Verminderung der Expression von Caspase 8 in HEK 293T führte zu einer verminderten NF-κB Aktivierung durch die Überexpression des konstitutiv aktiven Fusionsproteins des TRAIL-Rezeptors 2. Im Rahmen dieser Arbeit war es somit gelungen, die Todesdomäne der TRAIL-Rezeptoren 1 und 2, das Adaptorprotein FADD mit seiner Todeseffektordomäne und wahrscheinlich Caspase 8 als rezeptornahe Signalschritte der TRAIL-induzierten Aktivierung von NF-κB zu identifizieren. Folglich sind die Signalwege von Apoptose und Aktivierung von NF-κB durch TRAIL im rezeptornahen Signalweg identisch. Damit ist es nicht möglich, an Hand der gezielten Aktivierung der rezeptornahen Signalschritte TRAIL-induzierte Apoptose selektiv zu aktivieren. Hierzu muss eine Untersuchung weiterer distaler Signalschritte erfolgen.

Kaposi's sarcoma herpesvirus (KSHV) encodes a cluster of twelve micro (mi)RNAs, which are abundantly expressed during both latent and lytic infection. Previous studies reported that KSHV is able to inhibit apoptosis during latent infection; we thus tested the involvement of viral miRNAs in this process. We found that both HEK293 epithelial cells and DG75 cells stably expressing KSHV miRNAs were protected from apoptosis. Potential cellular targets that were significantly down-regulated upon KSHV miRNAs expression were identified by microarray profiling. Among them, we validated by luciferase reporter assays, quantitative PCR and western blotting caspase 3 (Casp3), a critical factor for the control of apoptosis. Using site-directed mutagenesis, we found that three KSHV miRNAs, miR-K12-1, 3 and 4-3p, were responsible for the targeting of Casp3. Specific inhibition of these miRNAs in KSHV-infected cells resulted in increased expression levels of endogenous Casp3 and enhanced apoptosis. Altogether, our results suggest that KSHV miRNAs directly participate in the previously reported inhibition of apoptosis by the virus, and are thus likely to play a role in KSHV-induced oncogenesis.

In der vorliegenden Arbeit wurde die Wirkung der Farnesyltransferase-Inhibitoren hinsichtlich der antitumorösen Wirkung auf gastrointestinale Tumore in verschiedenen in vitro Experimenten untersucht. Als Marker für die therapeutische Wirkung wurde die Induktion der Apoptose gewählt. Durchgeführt wurden unsere Versuche mit den Zelllinien HepG2, gewonnen aus einem hepatozellulären Karzinom, sowie HT29, einem kolorektalen Adenokarzinom, unter Verwendung von BMS-214662 und SCH66336 als Vertreter der Farnesyltransferase-Inhibitoren. Diese werden derzeit in klinischen Studien getestet. BMS-214662 befindet sich in Phase II, im Rahmen von SCH66336 sind bereits Phase III Studien abgeschlossen worden. Anhand von Apoptose-Assays waren wir in der Lage einen antitumorösen Effekt durch Induktion der Apoptose aufzuzeigen. Hierbei war die Apoptose-Auslösung dosisabhängig von dem verwendeten Farnesyltransferase-Inhibitor. Im Gegensatz zu SCH66336 induzierte BMS-214662 bereits unter Verwendung von 1µM in 24h die Apoptose, wohingegen eine Konzentration von 25µM SCH66336 notwendig war, eine signifikante Apoptose hervorzurufen. Die Induktion der Apoptose war dabei unabhängig von einer Ras-Mutation. Sowohl in den HT29-Zellen mit Mutation des Ras-Proteins, als auch in den mutationsfreien HepG2-Zellen erfolgte ein Anstieg der Apoptoserate. Zusätzlich zeigten wir einen möglichen synergistischen Effekt für den Antikörper anti-APO in Verbindung mit BMS-214662. Mit Hilfe des Western Blots konnte als ein möglicher Induktionsfaktor eine gesteigerte Rekrutierung der Caspase 8 dargestellt werden. Dies bestätigte sich durch eine messbare Erhöhung der Caspase 3, welche durch Caspase 8 aktiviert wird und den apoptotischen Tod der Zelle hervorruft. Insgesamt läuft der Mechanismus Rezeptor- bzw. Liganden-unabhängig ab, da in der durchgeführten rt-PCR weder vermehrt CD95-Rezeptor bzw. dessen Ligand nachgewiesen werden konnte.

IL-1β and IL-18 are proinflammatory cytokines that contribute to renal immune complex disease, but whether IL-1β and IL-18 are mediators of intrinsic glomerular inflammation is unknown. In contrast to other cytokines the secretion of IL-1β and IL-18 requires a second stimulus that activates the inflammasome-ASC-caspase-1 pathway to cleave pro-IL-1β and -IL-18 into their mature and secretable forms. As the NLRP3 inflammasome and caspase-1 were shown to contribute to postischemic and postobstructive tubulointerstitial inflammation, we hypothesized a similar role for NLRP3, ASC, and caspase-1 in glomerular immunopathology. This concept was supported by the finding that lack of IL-1R1 reduced antiserum-induced focal segmental necrosis, crescent formation, and tubular atrophy when compared to wildtype mice. Lack of IL-18 reduced tubular atrophy only. However, NLRP3-, ASC- or caspase-1-deficiency had no significant effect on renal histopathology or proteinuria of serum nephritis. In vitro studies with mouse glomeruli or mesangial cells, glomerular endothelial cells, and podocytes did not reveal any pro-IL-1β induction upon LPS stimulation and no caspase-1 activation after an additional exposure to the NLRP3 agonist ATP. Only renal dendritic cells, which reside mainly in the tubulointerstitium, expressed pro-IL-1β and were able to activate the NLRP3-caspase-1 axis and secrete mature IL-1β. Together, the NLRP3-ASC-caspase-1 axis does not contribute to intrinsic glomerular inflammation via glomerular parenchymal cells as these cannot produce IL-1β during sterile inflammation.

Herpesviren exprimieren Micro-(mi)RNAs, welche die Expression von zellulären und viralen Genen beeinflussen. Das Genom des Kaposi Sarkom Assoziierten Herpesvirus (KSHV) kodiert ein Cluster von insgesamt 12 miRNAs, welche sowohl während der latenten, als auch während der lytischen Infektion exprimiert werden. Da bisher nur sehr wenige zelluläre Zielgene für KSHV miRNAs bekannt sind, war es das Ziel dieser Studie, Gene zu identifizieren, deren Expression durch virale miRNAs von KSHV beeinflusst wird. Zu diesem Zweck wurden KSHV miRNAs mit Hilfe eines lentiviralen Transduktionssystems in B-Zellen und in Endothelzellen exprimiert. Diese sind beide natürliche Wirtszellen für KSHV. Die dabei entstandenen Zelllinien wurden mit Hilfe von zwei unterschiedlichen experimentellen Ansätzen untersucht: Beim ersten Ansatz wurde das gesamte Expressionsprofil dieser Zellen mit Hilfe von Microarrays analysiert und, nach Filterung durch bioinformatische Methoden, wurden Kandidaten für eine Regulation durch virale miRNAs identifiziert. Das Ergebnis wurde anhand biochemischer Methoden validiert und zwei zelluläre Transkripte als Zielgene bestätigt. In funktionellen Analysen konnte gezeigt werden, dass KSHV miRNAs die Caspase 3 inhibieren und dadurch die Zellen vor Apoptose schützen. Im zweiten, weitaus effizienteren Ansatz, wurden die sogenannten RISC-Komplexe mit Hilfe von AGO2-spezifischen Antikörpern sowohl aus den KSHV miRNA exprimierenden Zellen als auch aus latent KSHV infizierten Zellen isoliert und die daran gebundenen mRNAs identifiziert. Der RISC-Komplex spielt die entscheidende Rolle bei der miRNA-induzierten Regulation und enthält neben Proteinkomponenten (u.a. Argonauten (AGO)-Proteinen) sowohl die aktiven miRNAs als auch die regulierten mRNAs. Nach Isolierung der gebundenen RNAs konnten mit dieser Methode 72 Gene als Zielgene für KSHV miRNAs identifiziert werden. Viele davon spielen eine wichtige Rolle in unterschiedlichen Prozessen wie Zellzykluskontrolle, in der Apoptose oder der mRNA-Prozessierung. Insgesamt 11 identifizierte Zielgene wurden mit Hilfe von real-time PCRs untersucht und 10 bestätigt. Mittels 3’UTR-Luciferase-Assays wurden 6 davon weiter analysiert und bestätigt. Für die Gene LRRC8D, NHP2L1 und GEMIN8 konnten die zuständigen KSHV miRNAs und die dazugehörigen Bindungsstellen auf dem Transkript identifiziert werden. Bei den letzteren beiden lagen diese interessanterweise nicht wie erwartet in der 3’UTR, sondern in dem kodierenden Bereich.

Background: Programmed cell death (PCD) is essential for development and homeostasis of multicellular organisms and can occur by caspase-dependent apoptosis or alternatively, by caspase-independent PCD (ciPCD). Bcl-2, a central regulator of apoptosis, localizes to both mitochondria and the endoplasmic reticulum (ER). Whereas a function of mitochondrial and ER-specific Bcl-2 in apoptosis has been established in multiple studies, corresponding data for ciPCD do not exist. Methods: We utilized Bcl-2 constructs specifically localizing to mitochondria (Bcl-2 ActA), the ER (Bcl-2 cb5), both (Bcl-2 WT) or the cytosol/nucleus (Bcl-2 Delta TM) and determined their protective effect on ceramide-mediated ciPCD in transiently and stably transfected Jurkat cells. Expression of the constructs was verified by immunoblots. Ceramide-mediated ciPCD was induced by treatment with human recombinant tumor necrosis factor and determined by flow cytometric measurement of propidium iodide uptake as well as by optical analysis of cell morphology. Results: Only wildtype Bcl-2 had the ability to efficiently protect from ceramide-mediated ciPCD, whereas expression of Bcl-2 solely at mitochondria, the ER, or the cytosol/nucleus did not prevent ceramide-mediated ciPCD. Conclusion: Our data suggest a combined requirement for both mitochondria and the ER in the induction and the signaling pathways of ciPCD mediated by ceramide.

Thu, 26 Jul 2007 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/7689/ https://edoc.ub.uni-muenchen.de/7689/1/Wittmann_Stefanie.pdf Wittmann, Stefanie Christina ddc:600, ddc:

Ziel dieser Arbeit war es, den Einfluss der Organtemperatur während der Ischämie auf den mikrovaskulären Ischämie-Reperfusionsschaden systematisch zu analysieren. Um dies zu ermöglichen wurde im ersten Abschnitt der vorliegenden Arbeit ein geeignetes Tiermodell etabliert. Das entwickelte Modell ermöglichte eine reversible in situ Ischämie mit anschliessender Reperfusion des linken Leberlappens der Maus. Während der Ischämie konnte der ischämische Leberlappen genau auf die gewünschte Temperatur eingestellt und konstant gehalten werden. Abgesehen von der unterschiedlich eingestellten Organtemperatur, herrschten bei allen Versuchsgruppen gleiche Bedingungen. Weder bei der Körperkerntemperatur noch bei dem mittleren arteriellen Blutdruck gab es signifikante Unterschiede zwischen den Versuchsgruppen. Nach darauffolgender Reperfusion ermöglichte das Modell eine intravitalmikroskopische Analyse der Mikrozirkulation und anschliessende ex vivo Messungen vieler Schadensparameter. Damit ist es gelungen, ein geeignetes Tiermodell zur systematischen in vivo Analyse des singulären Einflusses verschiedener Organtemperaturen auf den hepatischen Ischämie-Reperfusionsschaden der Maus, zu etablieren. Die Ergebnisse wiesen, in Übereinstimmung mit der Literatur, einen ausgeprägten Ischämie-Reperfusionsschaden nach normothermer Ischämie auf. Dabei war die sinusoidale Perfusionsrate signifikant reduziert, es trat eine deutliche Zunahme der Leukozyten-Endothelzell-Interaktion auf und es folgte eine massive Freisetzung der Leberenzyme AST/GOT und ALT/GPT. Erstaunlicherweise konnten bereits durch eine milde Hypothermie von 26°C sämtliche Schadensparameter auf das Niveau der Shamgruppe reduziert werden. Entgegen der Erwartung scheint es keinen linearen Zusammenhang zwischen Organtemperatur und Ausmaß des mikrovaskulären Schadens zu geben. Vergleichbar mit den Ergebnissen der Mikrozirkulation war nach normothermer Ischämie auch eine Vielzahl apoptotischer Zellen zu verzeichnen. Nahezu 17% der Hepatozyten waren TUNEL-positiv und die Aktivität der apoptose-spezifischen Caspase-3 nahm um etwa das vierfache zu. Auch hier bewirkte bereits ein mildes Abkühlen des ischämischen Leberlappens auf 31,5°C eine vollständige Protektion der Leberzellen vor Apoptose. Es zeigte sich damit auch hier kein linearer Zusammenhang zwischen Organtemperatur und Ausmaß einer Apoptoseinduktion. Der Verdacht auf eine durch Kälte induzierte Apoptose konnte sich in diesem Versuchsaufbau nicht erhärten. Nach 90-minütiger Ischämie bei 4°C waren, wie auch in der Shamgruppe, weniger als 1% TUNEL-positive Zellen zu verzeichnen und ein Anstieg der Caspase-3-Aktivität blieb vollständig aus. Bei der Betrachtung, ob es sich bei der Zellschädigung um nekrotischen oder um apoptotischen Zelltod handelt, ergaben die verwendeten Messmethoden deutliche Hinweise für beide Arten des Zelltodes.

Die Exposition von Makrophagen gegenüber Bakterien oder LPS führt über die Aktivierung zellulärer Signalkaskaden zu einer vermehrten Expression von Genen, deren Proteinprodukte zelluläres Überleben unter Infektionsbedingungen ermöglichen. Die Aktivierung des Transkriptionsfaktors NF - kB spielt dabei eine wichtige Rolle. Pathogene Yersinia enterocolitica - Stämme hemmen die Aktivierung von NF - kB und induzieren Apoptose bei Makrophagen. In dieser Arbeit konnte gezeigt werden, dass das Yersinia - Effektorprotein YopP maßgeblich an der Apoptoseinduktion beteiligt ist. Bei Infektionsversuchen konnten lediglich die Yersinienstämme bei Makrophagen Apoptose induzieren, die über einen funktionstüchtigen Typ III - Sekretionsapparat und ein dadurch transloziertes YopP verfügen. Eine transiente Überexpression der transkriptionell aktiven NF - kB - Untereinheit p65 schützt Makrophagen spezifisch vor durch Yersinien, nicht jedoch vor durch Salmonellen induzierter Apoptose. Das weist darauf hin, dass YopP durch die Blockierung des NF - kB aktivierenden Signalwegs Apoptose bei Makrophagen vermittelt. Die Transfektion von J774A.1 Makrophagen mit YopP induzierte bei 40 - 50% der transfizierten Zellen Apoptose. Durch die zusätzliche Stimulation mit LPS konnte die Apoptoserate auf 80 - 90% gesteigert werde. Dieser synergistische, proapoptotische Effekt ist direkt auf durch LPS induzierte Signaltransduktions -prozesse zurückzuführen. Aus Transfektionsversuchen mit dominant - negativen Signalmolekülen der TLR - Signalkaskade ergaben sich Hinweise auf eine Beteiligung der Transmitter MyD88 und IRAK2 an der Apoptoseeinleitung. IRAK1 und TRAF6 scheinen dagegen eher ein antagonistisches, NF - kB aktivierendes LPS - Signal zu bedienen, welches unter dem Einfluss von YopP unterdrückt wird. Dadurch überwiegt das durch LPS induzierte, proapoptotische Signal, welches den apoptotischen Zelltod einleitet. Die Aktivierung des Apoptoseprogramms selbst erfolgt über FADD und Caspase - 8.

Untersuchungen strahleninduzierter Änderungen der Proliferation und des Zelltodes stellen Schwerpunkte der radiobiologischen Forschung dar. Aus strahlentherapeutischer Sicht interessieren hier im Besonderen die in Tumoren nach Strahlenexposition zu findenden Genexpressionsänderungen, die assoziiert mit strahleninduzierten Änderungen der Proliferation und des Zelltods auftreten. Detaillierte Kenntnisse der diesen biologischen Prozessen zugrundeliegenden Änderungen auf Genexpressionsebene könnten dazu beitragen, die Effizienz der Strahlentherapie humaner und tierischer Tumoren zu verbessern. So ist eine große Anzahl an für Proliferation und Apoptose kodierenden Genen bekannt. Es sind bisher jedoch nicht alle an der Proliferationskontrolle beteiligten Gene gefunden worden. Ebenso wird postuliert, dass auch andere Formen des Zelltodes als Apoptose auf Genexpressionsebene reguliert werden. Deshalb wurde mithilfe eines Mikroarrays mit 11.835 Genen ein Screening nach differentiell exprimierten Genen an strahlenexponierten A549 Zellen (humanen Lungenkarzinomzellen) vorgenommen. Hierzu wurden die Zellen synchronisiert, in der S-Phase mit 5 Gy bestrahlt und an den Zeitpunkten, die der Ausbildung des G2-Blocks und dem Anstieg mikrokernhaltiger und abnormaler Zellen zeitlich vorausgingen, das Screening durchgeführt. Die geeigneten Zeitpunkte wurden zuvor anhand durchflusszytometrischer Zellzyklusuntersuchungen und der Messung des Zelltodes (MAA-Assay) bestimmt. Die hybridisierten Mikroarrays wurden nach dem Digitalisieren unter Zuhilfenahme einer geeigneten Software interaktiv ausgewertet. Es konnten maximal 987 exprimierte Gene gefunden werden, was 12 % aller Gene des Mikroarrays entsprach. Setzte man die Genexpression der mit 5 Gy bestrahlten Zellen ins Verhältnis zu der Kontrolle, konnten 101 Gene als differentiell exprimiert ermittelt werden. Die Anzahl der herunterregulierten differentiell exprimierten Gene übertraf die Anzahl der hochregulierten differentiell exprimierten Gene zu jedem gemessenen Zeitpunkt immer ca. um den Faktor 4. Des Weiteren wurden die differentiellen Genexpressionen relativ zur Kontrolle der unterschiedlichen Zeitpunkte miteinander verglichen, wobei eine auffällige homogene Herunterregulation der Gene festzustellen war. Nach Einteilung der differentiell exprimierten Gene in funktionelle Gruppen konnten viele Gene, die für den Aufbau des Zytoskeletts kodierten, ermittelt werden. Hierbei standen im Vordergrund vor allem Gene für Tubulinproteine und Aktin. Des Weiteren konnten 8 Gene, die für ribosomale Proteine kodieren, identifiziert werden. Der Anteil bekannter, die Proliferation ("cyclin-dependent kinase inhibitor 1A" (p21, Cip1), "prothymosin, alpha") bzw. die Apoptose ("Caplain" und "TNF receptor-associated factor 1", "Caspase recruitment domain protein 14") regulierender Gene war gering. In Übereinstimmung mit zuvor durchgeführten Untersuchungen in anderen in vitro Modellen konnte eine aktive Herunterregulation bestimmter biologischer Funktionen (z.B. Zytoskelett, Proteinbiosynthese) bei gleichzeitiger Inhibition anderer Funktionen (Proliferation)gezeigt werden ("active silencing"). Da die Aussagen des Mikroarrays nur semiquantitativ sind, müssen die Ergebnisse noch durch ein quantitatives Verfahren (RTQ-PCR) validiert und ergänzt werden. Die vorläufigen Ergebnisse dieser Arbeit geben Hinweise darauf, dass neben den bekannten Zellproliferation und Zelltod kodierenden Genen in einem erheblichen Maß auch andere funktionelle Gengruppen wie z.B. Zytoskelett- und ribosomale Proteine kodierende Gene beteiligt sind und die Zelle im Sinne eines "active silencings" durch Abschalten verschiedener Zellfunktionen ihren eigenen Untergang vorbereitet.

Sat, 16 Jul 2005 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/4323/ https://edoc.ub.uni-muenchen.de/4323/1/Heimann_Kerstin.pdf Heimann, Kerst

Der programmierte Zelltod, oder Apoptose, ist ein physiologischer Vorgang mit zentraler Bedeutung für die Entwicklung und Homöostase mehrzelliger Organismen. Die Auslösung der Apoptose führt zur Aktivierung von Caspasen. Dies sind Cysteinproteasen, die gezielte Substratproteine spalten und so die koordinierte Zerstörung der Zelle herbeiführen. Apoptose wird durch pro- und antiapoptotische Proteine reguliert, die die Aktivierung von Caspasen stimulieren bzw. unterdrücken. Da diese Proteine sich gegenseitig beeinflussen, wird das Schicksal einer Zelle durch die relative Aktivität pro- und antiapoptotischer Faktoren bestimmt. BRUCE (BIR repeat-containing ubiquitin-conjugating enzyme) ist ein konserviertes, 528 kDa großes, peripheres Membranprotein des trans-Golgi Netzwerks. Seine charakteristischen Merkmale sind eine N-terminale BIR-Domäne und eine C-terminale UBC-Domäne, die dem Molekül Ubiquitin-Konjugationsaktivität verleiht. Diese Arbeit demonstriert, dass BRUCE Zellen vor der Apoptose schützt und als ein inhibitor of apoptosis protein (IAP) wirkt, indem es an aktive Caspasen bindet und diese inhibiert. Die Verwendung von Wildtyp und Mutanten des BRUCE Proteins zeigt, dass diese Aktivitäten von der BIR-Domäne abhängen. Während der Apoptose wird BRUCE durch unterschiedliche Mechanismen blockiert. Zum einen wird BRUCE durch Caspasen und HtrA2 proteolytisch gespalten und dadurch inaktiviert. Zum anderen bindet der mitochondriale IAP-Antagonist Smac an die BIR-Domäne von BRUCE und unterdrückt dessen Caspase-inhibitorische Aktivität, indem es die Bindung von BRUCE an Caspasen verhindert. Aufgrund seiner Lokalisierung an Membranen des trans-Golgi Netzwerks könnte BRUCE ein spezialisiertes, antiapoptotisches Protein mit räumlich begrenzter Aktivität sein. Wie die stark negative Regulation verdeutlicht, scheint außerdem die Entfernung von BRUCE aus der Zelle während der Apoptose wichtig zu sein. Räumlich und zeitlich begrenzte Inhibition von Caspasen während der Apoptose könnte daher für einen geordneten Ablauf apoptotischer Prozesse, wie zum Beispiel den Abbau des Golgi-Apparates und seine Verpackung in apoptotische Vesikel, notwendig sein.

Effect of apoptosis-inhibition by protein p35 on hemodynamics and infarct size in a rat myocardial infarct model Numerous in vitro studies showed the ability of the baculoviral protein p35 to effectively inhibit apoptosis in cells. Furthermore ex vivo Langendorff-experiments proved a reduction of infarct size by p35-intervention. The aim of the present study is to investigate the p35 dependent inhibition of apoptosis after adenoviral gene transfer into the myocardium with respect to infarct size as well as to cardiac function and hemodynamics. Myocardial infarction is induced by ligation of the left anterior descending artery (LAD) for 30 minutes followed by a 24 h reperfusion period. The following measurements are performed during the examination period: echocardiography (EF), left ventricular pressure measurement (LVPsys, LVPedp, dLVP/dtmax, dLVPdtmin), planimetry (infarct size, AR, I/AR). The evaluated echocardiographical data show a significant improvement of ejection fraction (EF: p35-G 50,35% ± 0,76, GFP-CG 46,54 ± 1,18) in the p35-group (n=18) in comparison to the control group (n=15). Likewise left ventricular pressure parameters showed a significant improvement except for LVPedp which was not significantly different. (LVPsys: p35-G 106,48 mmHg ± 4,48, GFP-CG 91,58 mmHg ± 3,86, LVPedp: p35-G 6 mmHg ± 3, GFP-CG 9,03 mmHg ± 4, dLVP/dtmax: p35-G 5659 mmHg/s ± 584,8, GFP-CG 4634 mmHG/s ± 256,21, dLVP/dtmin: p35-G -3882 mmHg/s ± 256,43, GFP-CG -3193 mmHg/s ± 191,15). No significant differnce was seen for the mean heart weight of the p-35 group measured 1,325 g ± 0,04 compared to the mean heart weight of 1,317 g ± 0,06 in the GFP-control group. In agreement with previous studies infarct size showed a significant reduction in the p35 transfected hearts, in opposition to the control animals with AR remaining constant in both groups. (Absolute infarct size: p35-G 0,11 cm3 ± 0,03, GFP-CG 0,33 cm3 ± 0,06, I/AR %: p35-G 6% ± 0,0 , GFP- CG 17% ± 0,02). The present study demonstrates that inhibition of apoptosis after adenoviral gene transfer of p35 reduces infarct size, and further improves cardiac function in a rat model of myocardial infarction. This confirms that Caspase-inhibition helps to maintain the function of the contractile apparatus in infarcted Cardiomyocytes. Thus, not only securing survival, but maintaining the function of Cardiaomyocytes in myocardial infarction.

Den überwiegend in den Mitochondrien lokalisierten Membranproteinen der Bcl-2-Familie wird eine besonders kritische Bedeutung beim Schutz der Zelle gegen den apoptotischen Zelltod beigemessen. Der genaue Wirkungsmechanismus dieser Proteine ist bis dato unbekannt. Sie sind aber in der Lage, in der mitochondrialen Membran Kanäle zu bilden und dadurch den funktionellen Veränderungen in den Mitochondrien im Verlauf der Apoptose sowie ihrer Schwellung entgegenzuwirken. Eine Störung des mitochondrialen Elektronentransports und die Öffnung der sogenannten „permeability transition“-Pore sind als frühe Ereignisse im Verlauf des apoptotischen Zelltodes bekannt. Der Entkoppler mitochondrialer Atmung und Phosphorylierung, FCCP, verursacht durch eine Erhöhung der Permeabilität der inneren mitochondrialen Membran für Protonen ähnliche Störungen der mitochondrialen Funktion. Ziel dieser Arbeit war es, den Effekt des mitochondrialen Entkopplers FCCP alleine oder in Kombination mit bekannten Apoptoseinduktoren, wie dem Proteinkinase C-Inhibitor Che und dem Serin/Threonin-Proteinkinasehemmer Sts zu untersuchen. Desweiteren sollten biochemische Mechanismen aufgeklärt werden, die der Modulation der Apoptose durch FCCP in Leukämiezellinien des lymphatischen (CCRF-CEM) und myeloischen (HL-60) Ursprungs zugrunde liegen. Der Effekt mitochondrialer Entkoppler auf die durch Che und Sts induzierte Apoptose sollte ausserdem mit der Wirkung bekannter Modulatoren des programmierten Zelltodes wie Caspasehemmer zVAD und Ca2+Mg2+-Endonuklease-Inhibitor ATA in gleichem Modellsystem verglichen werden, um weitere Hinweise über die Stärke und Dauer der apoptosemodulierenden Wirkung von FCCP zu erhalten. Die durchgeführten in vitro Zellkulturuntersuchungen zeigten, dass eine Inkubation der CCRF-CEM- und HL-60-Zellen mit FCCP dosisabhängig den verzögerten Zelltod in beiden Leukämiezellinien induzierte. Im FCS-haltigen Zellkulturmedium wurde in beiden Zellinien eine Apoptose nach 18-stündiger Behandlung mit 4 µM FCCP in 25% der Population beobachtet. 50% der HL-60-Zellen und 85% der CCRF-CEM-Zellen waren apoptotisch oder tot, wenn 20 µM FCCP über einen Zeitraum von 18 Stunden eingesetzt wurden. Ein FCS-Entzug resultierte in der Sensibilisierung der CCRF-CEM- und HL-60-Zellinien gegenüber FCCP: mehr als die Hälfte der Population in beiden Leukämiezellinien waren bereits 12 Studen nach Behandlungsbeginn mit FCCP apoptotisch bzw. tot. Die im Westernblot demonstrierte Caspase-3-abhängige proteolytische Spaltung von PARP, sowie die Reduktion des intrazellulären CPP-32-Spiegels (Procaspase-3) zeigte sich bereits 6 Stunden nach Behandlungsbeginn mit FCCP, statt, verglichen mit 24 Stunden unter normalen Inkubationsbedingungen. Im Verlauf der durch FCCP induzierten Apoptose konnten wir mittels konventioneller DNA-Agarose-Gelelektrophorese keine oligonukleosomale DNA-Fragmentierung (180-200 bp) nachweisen, mit Hilfe der pulsed field-Gelelektrophorese wurden lediglich große DNA-Fragmente (15-40 kbp) aufgezeigt. Nach zweistündiger Inkubation mit 10 µM Che bzw. achtstündiger Behandlung mit 300 nM Sts starben 90% CCRF-CEM-Zellen, während 60% der HL-60-Zellen nach zweistündiger Einwirkung von Che apoptotisch bzw. tot waren. Überraschenderweise war die proteolytische Spaltung von PARP nach Behandlung beider Zellinien mit einer niedrigeren Konzentration von Che (10 µM) ausgeprägter als mit der höheren Konzentration des Proteinkinase C-Hemmers (20 µM), obwohl die Anzahl der toten Zellen direkt proportional zur eingesetzten Che-Konzentration war. Die Zugabe von FCCP bzw. von Caspasen-Inhibitor zVAD verzögerten den durch Che und Sts induzierten apoptotischen Zelltod: 20-40% mehr Zellen überlebten innerhalb der ersten sechs Stunden der Inkubation, wenn 4-20 µM FCCP zum Inkubationsmedium zugegeben wurden, während nur 15-20% mehr Zellen bei Zugabe von 50 µM zVAD am Leben blieben. Der protektive Effekt von zVAD und ATA war jedoch nur vorübergehend: sechs Stunden nach Behandlungsbeginn mit Che oder Sts gab es keinen statistisch signifikanten Unterschied im Überleben der Zellen. Die Vorbehandlung mit ATA verhinderte komplett eine Apoptose in beiden Zellinien, so daß diese mindestens einige Tage nach Behandlungsbeginn mit Serin/Threonin-Proteinkinase-Hemmern intakt blieben. Alle eingesetzten Modulatoren hemmten das Auftreten der durch Che bzw. Sts ausgelösten biochemischen Zeichen der Apoptose, wie oligonukleosomale DNA-Degradation, Abfall der PARP-Aktivität und Aktivierung der Caspase-3. Eine 3-stündige Inkubation der CCRF-CEM- und HL-60-Zellen mit 10 µM Che führte in beiden Zellinien zu einem deutlichen Abfall der intrazellulären NAD+-, NADH-, NADPH- und ATP-Konzentrationen. Insbesondere in der CCRF-CEM-Zellinie stand die Senkung des intrazellulären Gehaltes an Pyridinnukleotiden im Vordergrund, in den myeloischen HL-60-Zellen war die ATP-Depletion ausgeprägter. Während FCCP oder zVAD den Abfall der Energie- und Redoxäquivalente lediglich partiell verhinderten, war ATA in der Lage, die Depletion von NAD+, NADH, NADPH und ATP komplett zu inhibieren. Da FCCP und zVAD lediglich die mit der Apoptose assoziierten biochemischen Phänomene, wie die Aktivierung der Caspase-3 oder der Ca2+-Mg2+-Endonuklease und nicht die Depletion der Energie- und Redoxäquivalente in Leukämiezellen aufhoben, waren die durch die Einwirkung von Che aufgetretenen Störungen des Energiestoffwechsels ein möglicher Grund, weshalb die protektive Wirkung von FCCP und zVAD nur vorübergehend war.

CNG-Kanäle sind elementare Bestandteile der Seh- und Riechkaskade. Es existieren sechs verschiedene Gene, die für unterschiedliche CNG-Untereinheiten kodieren. Im Rahmen dieser Arbeit wurde zunächst die Expression und Funktion der CNG-Kanäle im ZNS der Maus untersucht. RT-PCR-Untersuchungen zeigten, dass CNGA3 in der Amygdala, dem Cerebellum und dem Hippocampus die am stärksten exprimierte CNGA-Untereinheit war. Auch mittels in situ-Hybridisierung und Immunhistochemie konnte CNGA3 im Hippocampus der Maus nachgewiesen werden. Um zu untersuchen, ob CNGA3 eine funktionelle Rolle im Hippocampus der Maus besitzt, wurde die synaptische Plastizität in der CA1-Region des Hippocampus CNGA3-defizienter Mäuse gemessen. Bei CNGA3 -/- Mäusen konnte eine signifikant erhöhte Langzeitpotenzierung bei normal erhaltener Langzeitdepression beobachtet werden. Mit zwei unabhängigen Lernversuchen wurde untersucht, ob dieser Befund Auswirkungen auf die Funktion des Hippocampus für das räumliche Lernvermögen dieser Mäuse besitzt. Die Leistungsfähigkeit sowohl bei einem water-maze Versuch als auch bei der kontextuellen Angstkonditionierung zeigte sich jedoch durch die Deletion von CNGA3 nicht beeinträchtigt. Da CNGA3 auch in der Amygdala nachgewiesen werden konnte, wurden die CNGA3 -/- Mäuse auf eine Beeinträchtigung der physiologischen Funktion dieser Gehirnregion getestet. Zu diesem Zweck wurde eine akustische Angstkonditionierung durchgeführt. Bei diesem klassischen Test der Amygdalafunktion zeigten die CNGA3-defizienten Mäuse überraschenderweise ein signifikant weniger stark ausgeprägtes Angstverhalten. Detailliertere Untersuchungen sind notwendig, um die genaue Funktion von CNG-Kanälen in der Amygdala aufzuklären. Im zweiten Teil der Arbeit sollte die bei CNGA3-defizienten Mäusen zu beobachtende retinale Degeneration untersucht werden. Die Konsequenzen der Deletion von CNGA3 sollten auf molekularer Ebene beschrieben werden. Interessanterweise zeigte die Degeneration der Seh-Zapfen keine gleichmäßige Verteilung über die gesamte Netzhaut. Im oberen Teil der Netzhaut war ein verlangsamter Verlauf des Seh-Zapfen-Verlustes zu erkennen. Selbst bei über 1 Jahr alten CNGA3-defizienten Mäusen war in der dorsalen Retina eine Persistenz von etwa 50 % der Seh-Zapfen zu beobachten. Dagegen waren in der unteren Retina viel früher schon fast keine Zapfen-Außensegmente mehr zu sehen. Auf molekularer Ebene zeigte sich schon während der ersten Wochen der postnatalen Entwicklung ein Verlust elementarer Proteine der Phototransduktionskaskade. Sehr früh war für die zwei Zapfen-Opsine der Maus, wie für die meisten weiteren untersuchten Proteine der Seh-Kaskade der Zapfen, ein Verlust der Immunreaktivität in den Zapfen-Außensegmenten zu beobachten. Die Transkription der untersuchten Gene war, mit Ausnahme des SWS-Opsins, bei 3 Monate alten CNGA3 -/- Mäusen vergleichbar der bei Wildtyp Mäusen. Das Ausmaß und der Beginn des degenerativen Prozesses in der CNGA3 -/- Retina wurde durch den Nachweis einer sehr früh beginnenden und stark ausgeprägten Induktion von Müller-Gliazellen deutlich. Charakteristische Merkmale die für die Aktivierung einer Apoptose in Photorezeptoren sprechen konnten bei CNGA3 -/- Mäusen ausgemacht werden. Mittels TUNEL-Analyse konnte eine erhöhte DNS-Fragmentation beobachtet werden mit einem Maximum zwischen der dritten und vierten postnatalen Woche. Im gleichen Zeitraum war ebenfalls eine Aktivierung der Caspase 3 und eine Freisetzung von Cytochrom c zu erkennen. Zusätzlich zu den Veränderungen in den Photorezeptoren konnten subtile Veränderungen in der inneren Netzhaut der CNGA3 -/- Mäuse gezeigt werden. Zum einen entwickelten die Horizontalzellen neuronale Ausläufer, die in die äußere Körnerschicht hineinwuchsen. Zum anderen wurde ein Verlust der Immunreaktivität für das G-Protein Goa in ON-Bipolarzellen CNGA3-defizienter Mäuse festgestellt.

Fri, 7 Feb 2003 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/1167/ https://edoc.ub.uni-muenchen.de/1167/1/Matz_Susanne.pdf Matz, Susanne

Die vorliegende Arbeit wurde durch den SFB 369 (Teilprojekt B7) unterstützt. Sie sollte auf Basis der bereits vorliegenden Erkenntnisse klären, welcher Mechanismus der Apoptoseinduktion durch Ajoen in HL-60 Zellen zu Grunde liegt. Dirsch et al. 5 zeigten bereits 1998, dass Ajoen Apoptose in humanen akut-myeloischen Leukämiezellen (HL-60) induziert. Weiterhin zeigte sich eine dosis- und zeitabhängige Produktion der reaktiven Sauerstoffspezies (ROS). N-Acetylcystein (NAC), ein Antioxidans, verhinderte partiell die Ajoeninduzierte ROS-Produktion und die Apoptose. Auf dieser Grundlage konnten folgende Ergebnisse erarbeitet werden: 1) Ajoen verursacht in HL-60 Zellen die Aktivierung der Caspase-3 sowie der Caspase-8. Eine generelle Aktivierung von Caspasen ist für die Ajoen-induzierte Apoptose nötig, da der Breitbandcaspaseinhibitor z-VAD-fmk die durch Ajoen provozierte DNA-Fragmentation völlig verhindert. 2) Die Ajoen-induzierte Apoptose wird nicht durch den Todesrezeptor CD95 vermittelt. Dafür sprechen folgende Ergebnisse: a) Unsere HL-60 Zellen exprimieren den CD95-Rezeptor, jedoch kann der natürliche CD95-Ligand (CD95L) keine Apoptose hervorrufen. Wahrscheinlich ist der CD95-Rezeptor inaktiv. b) Außerdem kann für die Caspase-8, die für die Signalweiterleitung vom CD95-Rezeptor u.a. mit verantwortlich ist, durch Einsatz eines spezifischen Caspase-8 Inhibitors keine Bedeutung für die Ajoeninduzierten Apoptose gezeigt werden. c) CD95-resistente Jurkat Zellen (JurkatR) sind auf Ajoen genauso empfindlich wie die Kontrollzellen. 3) Es konnte bewiesen werden, dass Ajoen Apoptose über den mitochondrialen Signalweg induziert: a) Ajoen verursacht sowohl den Verlust des mitochondrialen Membranpotentials der inneren Membran als auch eine Cytochrom c- Freisetzung aus dem intermembranären Spalt. b) Die Ajoen-induzierte Apoptose hängt von der provozierten mitochondrialen Dysfunktion ab: HL-60 Zellen, die das anti-apoptotische Protein Bcl-xL überexprimieren, sind vor Apoptose geschützt. c) Die höhere Sensitivität der HL-60/neo bzw. die niedrigere Sensitivität der HL-60/bcl-xL Zellen auf Ajoen konnte auch morphologisch durch TEM-Untersuchungen untermauert werden. Zusammenfassend konnte also für die Ajoen-induzierte Apoptose eine von Mitochondrien abhängige Signalweiterleitung gezeigt werden. 4) Untersuchungen zur Frage, wie es zur Ajoen-induzierten mitochondrialen Dysfunktion kommt, brachten folgende Erkenntnisse: a) Eine Aktivierung von Caspasen ist für die Auslösung der mitochondrialen Ereignisse nicht notwendig. Die abgeschwächte und verzögerte Caspaseaktivierung in HL-60/bcl-xL Zellen beweist: Caspasen werden „downstream“ der mitochondrialen „Aktivierung“ gespalten. b) Die ROS-Entstehung ist ein Ereignis vor („upstream“) der mitochondrialen Dysfunktion. c) Die folgerichtige Untersuchung der MAPK JNK, p38 und ERK1/2 (die Aktivierung des Transkriptionsfaktors AP-1 war bereits bekannt), ergab deren Aktivierung, jedoch ist diese für die Signalvermittlung nicht nötig. Nur für die ERK1/2 Kinase konnte eine direkte Beteiligung, und zwar als „survival“-Faktor, festgestellt werden, während die Akt als „survival“- Faktor keine Bedeutung hat. Ergebnisübersicht: CD95 Caspase-8 ROS p 38 MAPK ERK1/2 JNK Akt DNA- Fragmentierung Apoptose Caspase-3 Caspase-8 Aktivierung Caspase-3- ähnlicherCaspasen z-VAD-fmk Cytochrom c- Freisetzung z-VAD-fmk AP-1 Extrinsischer intrinsischer Signalweg Signalweg Bcl-xL Ajoen Abb. 61: Ergebnisübersicht Rot:-- Involvierung im Signalweg, blau:-- kein kausaler Zusammenhang gegeben; –I = Hemmung.