Mass spectrometric identification of increased C16 ceramide levels during apoptosis.

A variety of molecular changes occur during the process of apoptosis. Much of the recent work has focused on changes in critical cellular proteins, proteins necessary for the initiation and continuation of the apoptotic process. Given the fact that numerous membrane changes occur throughout the apoptotic process, we initiated an investigation aimed at determining the major lipid changes that occurred during programmed cell death. When ionizing radiation was used to initiate the apoptotic process in Jurkat cells, one of the major changes that occurred within 24 h was an increase in a species with a m/z of 572 as determined by negative ion electrospray mass spectrometry. This particular mass ion displayed high performance liquid chromatography characteristics of a neutral lipid species. Further analysis by collision-induced-dissociation tandem mass spectrometry indicated only one daughter species indicative of a Cl adduct and therefore a parental mass of 537. Comparison to a commercial C16 ceramide yielded identical spectra by mass spectrometry (MS) and MS/MS analysis in the negative ion mode. Increases in C16 ceramide levels occurred 2 h after initiation of apoptosis by ionizing radiation, and its accumulation paralleled apoptosis as determined by cellular morphology. Interestingly, radiation-sensitive Jurkat cells displayed increased levels of long term C16 ceramide accumulation, whereas radiation-resistant K562 cells did not. These findings were supported by increases in caspase-3 activity in Jurkat cells, whereas caspase-3 activity in K562 cells remained unchanged. C16 ceramide accumulation and sensitivity to ionizing radiation was investigated further in a melanoma cell line. Only those cells that were radiation sensitive (approximately 70-75%) displayed increases in long term ceramide accumulation. Taken together, these results indicated a correlation between increases in C16 ceramide accumulation and radiation sensitivity. Increases in long term C16 ceramide accumulation were also seen in Fas-induced apoptosis, which occurred at time points greater than 2 h. Analysis of mitochondrial modifications using the mitochondrial probe nonyl acridine orange (NAO) indicated that initial increases in C16 ceramide levels closely paralleled the decrease in mitochondrial mass during Fas or radiation-induced apoptosis. Taken together, these results support a role for C16 ceramide in the effector (mitochondrial) phase of apoptosis.     

Control of macroalgal blooms at early developmental stages: Pilayella littoralis versus Enteromorpha spp.

Although blooms of opportunistic fast-growing macroalgae now occur frequently in coastal ecosystems affected by eutrophication, their initiation and control is little understood. Most previous studies have focused on the ecophysiology of adult algae only. We show that spores and/or germlings may represent critical stages in the life cycles and mass-developments of co-occurring opportunistic macroalgae in the Baltic (Pilayella littoralis and Enteromorpha spp.). We investigated the overwintering of spores, timing of germination, subsequent growth, and grazing on spores and germlings, in order to explain the initiation of mass blooms and species dominance patterns. In the field, Enteromorpha spp. showed 10- to 50-fold higher abundances of overwintering microscopic forms (up to 330 individuals cm⁻²) than P. littoralis. Moreover, we found continuous production of spores (up to 1.2 million settling spores m⁻² h⁻¹) from April to October in Enteromorpha spp., while there was evidence of only a short reproductive period in Pilayella. However, in spring, germlings and adults of P. littoralis appeared earlier in the field and reached a 10-fold higher biomass than Enteromorpha spp. In factorial laboratory experiments including temperature and light, there were clear differences in timing of germination. P. littoralis germinated at 5°C whereas Enteromorpha spp. required temperatures of 10–15°C for germination. In contrast, we detected only minor differences in growth response among adults of P. littoralis and Enteromorpha spp. Germination, not growth of adults, appeared to be the ecophysiological bottleneck for initiating mass spring development. Following the spring Pilayella bloom, Enteromorpha germlings occurred massively in the field (April–September), but rarely developed into adults. In laboratory feeding experiments we tested whether crustacean mesograzers common in summer may control development of Enteromorpha germlings. Both germination of settled spores and growth of germlings were reduced by 93–99% in the presence of grazers (Idotea chelipes and Gammarus locusta). Thus in addition to ecophysiological constraints, grazers, if present, may play a decisive role in the early life stages of macroalgal mass developments. These results mirror patterns of overwintering of seeds, germination control, seed and seedling predation in terrestrial plant communities. Received: 7 March 1998 / Accepted: 18 November 1998     

Incorporating climate change adaptation into marine protected area planning

Climate change is increasingly impacting marine protected areas (MPAs) and MPA networks, yet adaptation strategies are rarely incorporated into MPA design and management plans according to the primary scientific literature. Here we review the state of knowledge for adapting existing and future MPAs to climate change and synthesize case studies (n = 27) of how marine conservation planning can respond to shifting environmental conditions. First, we derive a generalized conservation planning framework based on five published frameworks that incorporate climate change adaptation to inform MPA design. We then summarize examples from the scientific literature to assess how conservation goals were defined, vulnerability assessments performed and adaptation strategies incorporated into the design and management of existing or new MPAs. Our analysis revealed that 82% of real‐world examples of climate change adaptation in MPA planning derive from tropical reefs, highlighting the need for research in other ecosystems and habitat types. We found contrasting recommendations for adaptation strategies at the planning stage, either focusing only on climate refugia, or aiming for representative protection of areas encompassing the full range of expected climate change impacts. Recommendations for MPA management were more unified and focused on adaptative management approaches. Lastly, we evaluate common barriers to adopting climate change adaptation strategies based on reviewing studies which conducted interviews with MPA managers and other conservation practitioners. This highlights a lack of scientific studies evaluating different adaptation strategies and shortcomings in current governance structures as two major barriers, and we discuss how these could be overcome. Our review provides a comprehensive synthesis of planning frameworks, case studies, adaptation strategies and management actions which can inform a more coordinated global effort to adapt existing and future MPA networks to continued climate change.     

Recovery of marine animal populations and ecosystems

Many marine populations and ecosystems have experienced strong historical depletions, yet reports of recoveries are increasing. Here, we review the growing research on marine recoveries to reveal how common recovery is, its magnitude, timescale and major drivers. Overall, 10-50% of depleted populations and ecosystems show some recovery, but rarely to former levels of abundance. In addition, recovery can take many decades for long-lived species and complex ecosystems. Major drivers of recovery include the reduction of human impacts, especially exploitation, habitat loss and pollution, combined with favorable life-history and environmental conditions. Awareness, legal protection and enforcement of management plans are also crucial. Learning from historical recovery successes and failures is essential for implementing realistic conservation goals and promising management strategies.     

Task-dependent interaction between parietal and contralateral primary motor cortex during explicit versus implicit motor imagery

Both mental rotation (MR) and motor imagery (MI) involve an internalization of movement within motor and parietal cortex. Transcranial magnetic stimulation (TMS) techniques allow for a task-dependent investigation of the interhemispheric interaction between these areas. We used image-guided dual-coil TMS to investigate interactions between right inferior parietal lobe (rIPL) and left primary motor cortex (M1) in 11 healthy participants. They performed MI (right index-thumb pinching in time with a 1 Hz metronome) or hand MR tasks, while motor evoked potentials (MEPs) were recorded from right first dorsal interosseous. At rest, rIPL conditioning 6 ms prior to M1 stimulation facilitated MEPs in all participants, whereas this facilitation was abolished during MR. While rIPL conditioning 12 ms prior to M1 stimulation had no effect on MEPs at rest, it suppressed corticomotor excitability during MI. These results support the idea that rIPL forms part of a distinct inhibitory network that may prevent unwanted movement during imagery tasks.     

Damage associated molecular pattern molecule-induced microRNAs (DAMPmiRs) in human peripheral blood mononuclear cells

Endogenous damage associated molecular pattern molecules (DAMPs) released from necrotic, damaged or stressed cells are associated with an inflammatory response. Whether the microRNA (miR) expression signature of this response is different from that of a pathogen associated molecular pattern (PAMP)-stimulated inflammatory response is unknown. We report here that miR-34c and miR-214 are significantly expressed in fresh human peripheral blood mononuclear cells (PBMCs) exposed to DAMP-containing freeze-thaw lysates, or to conditioned media from serum-starved and glucose-deprived cells (p<6×10(-4) and p<3.7×10(-3)), respectively. Interestingly, only miR-34c expression was differentially expressed in PBMCs exposed to freeze-thaw lysates or conditioned media from wildtype High Mobility Group B1 (HMGB1(+/+)) mouse embryonic fibroblast (MEF) cells, when compared to cultures exposed to lysates or conditioned media from HMGB1(-/-) MEFs. miR-155 expression in these cultures was negligible, but was significantly expressed in PBMCs stimulated with Lipopolysaccahride (LPS) or most other Toll-like receptor (TLR) ligands, making it the prototypic "PAMPmiR". Exposure to a damaged human colorectal carcinoma cell line lysate (HCT116) similarly resulted in increased miR-34c and miR-214 levels. When PBMCs were pre-transfected with anti-miR-34c and then exposed to lysate, expression levels of IKKγ mRNA, a putative target of miR-34c, increased, while protein levels of IKKγ in cultures transfected with a pre-miR-34c were abrogated. Levels of miR-34c expression (as well as pro-inflammatory cytokines, IL-1β and TNFα) decreased when PBMC cultures were briefly pre-incubated with the K(+) channel (inflammasome) inhibitor, glybenclamide, suggesting that inflammasome activation is upstream of miR-34c expression in response to DAMPs. Our findings demonstrate that a specific microRNA expression signature is associated with the inflammatory response to damaged/injured cells and carries implications for many acute and chronic inflammatory disorders.