Ricardo Weinlich

Necroptosis in development, inflammation and disease.

In the early 2000s, receptor-interacting serine/threonine protein kinase 1 (RIPK1), a molecule already recognized as an important regulator of cell survival, inflammation and disease, was attributed an additional function: the regulation of a novel cell death pathway that came to be known as necroptosis. Subsequently, the related kinase RIPK3 and its substrate mixed-lineage kinase domain-like protein (MLKL) were also implicated in the necroptotic pathway, and links between this pathway and apoptosis were established. In this Timeline article, we outline the discoveries that have helped to identify the roles of RIPK1, RIPK3, MLKL and other regulators of necroptosis, and how they interact to determine cell fate.

Comparison of the anti-apoptotic effects of Bcr-Abl, Bcl-2 and Bcl-xL following diverse apoptogenic stimuli

Ectopic expression of Bcr‐Abl, Bcl‐2 or Bcl‐xL in HL‐60 cells conferred resistance to apoptosis against a variety of death‐inducing agents. Bcr‐Abl‐mediated interference with mitochondrial events was confirmed by the analysis of the loss of mitochondrial transmembrane potential and cytochrome c release. HL‐60.Bcr‐Abl cells were extremely resistant to all apoptogenic stimuli tested, even in circumstances where HL‐60.Bcl‐2 or HL‐60.Bcl‐xL cells were only partially protected from apoptosis. The levels of Mcl‐1, Bax, Bid, Akt, c‐IAP‐1, c‐IAP‐2, XIAP and c‐FLIP were compared in all HL‐60 lines. Our findings show that Bcr‐Abl is a more powerful anti‐apoptotic molecule than Bcl‐2 or Bcl‐xL.

Melatonin Protects CD4+ T Cells from Activation-Induced Cell Death by Blocking NFAT-Mediated CD95 Ligand Upregulation

Over the past 20 y, the hormone melatonin was found to be produced in extrapineal sites, including cells of the immune system. Despite the increasing data regarding the biological effects of melatonin on the regulation of the immune system, the effect of this molecule on T cell survival remains largely unknown. Activation-induced cell death plays a critical role in the maintenance of the homeostasis of the immune system by eliminating self-reactive or chronically stimulated T cells. Because activated T cells not only synthesize melatonin but also respond to it, we investigated whether melatonin could modulate activation-induced cell death. We found that melatonin protects human and murine CD4+ T cells from apoptosis by inhibiting CD95 ligand mRNA and protein upregulation in response to TCR/CD3 stimulation. This inhibition is a result of the interference with calmodulin/calcineurin activation of NFAT that prevents the translocation of NFAT to the nucleus. Accordingly, melatonin has no effect on T cells transfected with a constitutively active form of NFAT capable of migrating to the nucleus and transactivating target genes in the absence of calcineurin activity. Our results revealed a novel biochemical pathway that regulates the expression of CD95 ligand and potentially other downstream targets of NFAT activation.

Hypoxia Inducible Factor–Dependent Regulation of Angiogenesis by Nitro–Fatty Acids

Objective—Nitro-fatty acids (NO2-FAs) are emerging as a new class of cell signaling mediators. Because NO2-FAs are found in the vascular compartment and their impact on vascularization remains unknown, we aimed to investigate the role of NO2-FAs in angiogenesis. Methods and Results—The effects of nitrolinoleic acid and nitrooleic acid were evaluated on migration of endothelial cell (EC) in vitro, EC sprouting ex vivo, and angiogenesis in the chorioallantoic membrane assay in vivo. At 10 &mgr;mol/L, both NO2-FAs induced EC migration and the formation of sprouts and promoted angiogenesis in vivo in an NO-dependent manner. In addition, NO2-FAs increased intracellular NO concentration, upregulated protein expression of the hypoxia inducible factor-1&agr; (HIF-1&agr;) transcription factor by an NO-mediated mechanism, and induced expression of HIF-1&agr; target genes, such as vascular endothelial growth factor, glucose transporter-1, and adrenomedullin. Compared with typical NO donors such as spermine-NONOate and deta-NONOate, NO2-FAs were slightly less potent inducers of EC migration and HIF-1&agr; expression. Short hairpin RNA–mediated knockdown of HIF-1&agr; attenuated the induction of vascular endothelial growth factor mRNA expression and EC migration stimulated by NO2-FAs. Conclusion—Our data disclose a novel physiological role for NO2-FAs, indicating that these compounds induce angiogenesis in an NO-dependent mechanism via activation of HIF-1&agr;.

Sustained activation of p53 in confluent nucleotide excision repair-deficient cells resistant to ultraviolet-induced apoptosis.

P53 activation is one of the main signals after DNA damage, controlling cell cycle arrest, DNA repair and apoptosis. We have previously shown that confluent nucleotide excision repair (NER)-deficient cells are more resistant to apoptosis induced by ultraviolet irradiation (UV). Here, we further investigated the effect of cell confluence on UV-induced apoptosis in normal and NER-deficient (XP-A and XP-C) cells, as well as the effects of treatments with the ATM/ATR inhibitor caffeine, and the patterns of p53 activation. Strong p53 activation was observed in either proliferating or confluent cells. Caffeine increased apoptosis levels and inhibited p53 activation in proliferating cells, suggesting a protective role for p53. However, in confluent NER-deficient cells no effect of caffeine was observed. Transcription recovery measurements showed decreased recovery in proliferating XPA-deficient cells, but no recovery was observed in confluent cells. The levels of the cyclin/Cdk inhibitor, p21(Waf1/Cip1), correlated well with p53 activation in proliferating cells. Surprisingly, confluent cells also showed similar activation of p21(Waf1/Cip1). These results indicate that reduced apoptosis in confluent cells is associated with the deficiency in DNA damage removal, since this effect is not clearly observed in NER-proficient cells. Moreover, the strong activation of p53 in confluent cells, which barely respond to apoptosis, suggests that this protein, under these conditions, is not linked to UV-induced cell death signaling.

An oligonucleotide primer set for PCR amplification of the complete honey bee mitochondrial genome

Mitochondrial DNA markers have been widely used to address population and evolutionary questions in the honey bee Apis mellifera. Most of the polymorphic markers are restricted to few mitochondrial regions. Here we describe a set of 24 oligonucleotides that allow PCR amplification of the entire mitochondrial genome of the honey bee A. mellifera in 12 amplicons. These fragments have important applications for the study of mitochondrial genes in different subspecies of A. mellifera and as heterospecific probes to characterize mitochondrial genomes in other bee species.ZusammenfassungDer Informationsgehalt des mitochondrialen Genoms (mtDNA) ist in weitem Mass von Bedeutung für populationsgenetische und evolutive Studien. Bei der Honigbiene, Apis mellifera, trug die Aufdeckung von Polymorphismen in diesem Molekül zu wichtigen Einsichten in die Populationsstruktur und zur Phylogenie bei. Obwohl A. mellifera die Hymenopterenart ist, für die als erste das mitochondriale Genom komplett sequenziert wurde, beschränken sich selbst bei dieser Art die meisten Populations- und Evolutionsstudien auf nur einige wenige mitochondriale Regionen. Für die Nutzung weiterer mtDNA Regionen ist die Entwicklung geeigneter Primer von zentraler Bedeutung für erfolgreiche PCR-Amplifikationen. Die Verwendung genereller Oligonukleotidprimer (universelle Primer) für das mitochondriale Genom von Insekten führte bei der Honigbiene oft zu Fehlschlägen oder lieferte keine hochwertigen PCR-Produkte. Wir beschreiben in der vorliegenden Arbeit einen Satz bestehend aus 24 Oligonukleotiden, mit denen es möglich war, das gesamte mitochondriale Genom der Honigbiene A. mellifera in Form von 12 Fragmenten zu amplifizieren (Abb. 1, Tab. I). Die Amplifikationsbedingungen für diese Primerkombinationen wurden etabliert und sind in Tabelle II zusammengestellt. Diese Fragmente können von Wichtigkeit sein für die Untersuchung mitochondrialer Gene bei verschiedenen Unterarten von A. mellifera und ebenso als heterospezifische Sonden zur Charakterisierung des mitochondrialen Genoms anderer Bienenarten dienen. Ausserdem kann die Verwendung eines Teilsatzes dieser Primer für die Amplifizierung homologer Regionen bei anderen Bienen genutzt werden (Tab. III) und damit künftige Populationsuntersuchungen und evolutive Studien innerhalb der Hymenopteren vorantreiben.

Pattern Recognition Receptors and the Host Cell Death Molecular Machinery

Pattern Recognition Receptors (PRRs) are proteins capable of recognizing molecules frequently found in pathogens (the so-called Pathogen-Associated Molecular Patterns—PAMPs), or molecules released by damaged cells (the Damage-Associated Molecular Patterns—DAMPs). They emerged phylogenetically prior to the appearance of the adaptive immunity and, therefore, are considered part of the innate immune system. Signals derived from the engagement of PRRs on the immune cells activate microbicidal and pro-inflammatory responses required to eliminate or, at least, to contain infectious agents. Molecularly controlled forms of cell death are also part of a very ancestral mechanism involved in key aspects of the physiology of multicellular organism, including the elimination of unwanted, damaged or infected cells. Interestingly, each form of cell death has its particular effect on inflammation and on the development of innate and adaptive immune responses. In this review article, we discuss some aspects of the molecular interplay between the cell death machinery and signals initiated by the activation of PRRs by PAMPs and DAMPs.