cGMP potentiates receptor-stimulated Ca2+ influx in Dictyostelium discoideum

AbstractBinding of extracellular cAMP to surface receptors induces at least two responses in Dictyostelium discoideum, the G-protein-dependent activation of guanylyl cyclase, and the opening of a plasma membrane Ca2+ channel. Some experiments suggest that intracellular cGMP opens the Ca2+ channel, while others demonstrate that the channel can open in the absence of functional G-proteins (and thus in the absence of cGMP formation). We have analysed 45Ca2+ uptake in three mutants with altered cGMP formation. Mutant stmF shows a prolonged cGMP response due to deletion of an intracellular phosphodiesterase. Uptake of receptor-stimulated 45Ca2+ is enhanced about two-fold in this mutant if compared to wild-type cells, suggesting that cGMP regulates the opening of the channel. Mutant KI-7 has very low levels of surface cAMP receptors, but nevertheless an enhanced receptor-stimulated cGMP response due to a defect in the turn-off of guanylyl cyclase. This mutant shows poor receptor-stimulated 45Ca2+ uptake, suggesting that cGMP alone is not sufficient to open the Ca2+ channel. Finally, mutant KI-8 has no cGMP due to the absence of nearly all guanylyl cyclase activity. The mutant shows significant but reduced 45Ca2+ uptake (19% of wild-type; 60% if corrected for the reduced level of surface cAMP receptors), suggesting that the channel can open in the absence of cGMP. Taken together, the results demonstrate that receptor-stimulated Ca2+ influx is not directly induced by cGMP formation; it can occur in the absence of cGMP, but is potentiated two- to four-fold by cGMP

Chemoattractants and chemorepellents act by inducing opposite polarity in phospholipase C and PI3-kinase signaling

During embryonic development, cell movement is orchestrated by a multitude of attractants and repellents. Chemoattractants applied as a gradient, such as cAMP with Dictyostelium discoideum or fMLP with neutrophils, induce the activation of phospholipase C (PLC) and phosphoinositide 3 (PI3)-kinase at the front of the cell, leading to the localized depletion of phosphatidylinositol 4,5-bisphosphate (PI[4,5]P(2)) and the accumulation of phosphatidylinositol-3,4,5-trisphosphate (PI[3,4,5]P(3)). Using D. discoideum, we show that chemorepellent cAMP analogues induce localized inhibition of PLC, thereby reversing the polarity of PI(4,5)P(2). This leads to the accumulation of PI(3,4,5)P(3) at the rear of the cell, and chemotaxis occurs away from the source. We conclude that a PLC polarity switch controls the response to attractants and repellents

MRI based preterm white matter injury classification: the importance of sequential imaging in determining severity of injury

The evolution of non-hemorrhagic white matter injury (WMI) based on sequential magnetic resonance imaging (MRI) has not been well studied. Our aim was to describe sequential MRI findings in preterm infants with non-hemorrhagic WMI and to develop an MRI classification system for preterm WMI based on these findings.Eighty-two preterm infants (gestation ≤35 weeks) were retrospectively included. WMI was diagnosed and classified based on sequential cranial ultrasound (cUS) and confirmed on MRI.138 MRIs were obtained at three time-points: early (<2 weeks; n = 32), mid (2-6 weeks; n = 30) and term equivalent age (TEA; n = 76). 63 infants (77%) had 2 MRIs during the neonatal period. WMI was non-cystic in 35 and cystic in 47 infants. In infants with cystic-WMI early MRI showed extensive restricted diffusion abnormalities, cysts were already present in 3 infants; mid MRI showed focal or extensive cysts, without acute diffusion changes. A significant reduction in the size and/or extent of the cysts was observed in 32% of the infants between early/mid and TEA MRI. In 4/9 infants previously seen focal cysts were no longer identified at TEA. All infants with cystic WMI showed ≥2 additional findings at TEA: significant reduction in WM volume, mild-moderate irregular ventriculomegaly, several areas of increased signal intensity on T1-weighted-images, abnormal myelination of the PLIC, small thalami.In infants with extensive WM cysts at 2-6 weeks, cysts may be reduced in number or may even no longer be seen at TEA. A single MRI at TEA, without taking sequential cUS data and pre-TEA MRI findings into account, may underestimate the extent of WMI; based on these results we propose a new MRI classification for preterm non-hemorrhagic WMI

The Dictyostelium discoideum Rap1 signalling cascade and its functions during growth and development

Elk levend organisme, of het nu een enkele cel is of een verzameling van miljoenen cellen, kan per definitie basale processen uitvoeren; deze processen moeten nauwkeurig gecoördineerd worden in tijd en ruimte. Coördinatie wordt gereguleerd door signaleringsroutes in de cel die snel aan- of uitgeschakeld worden als reactie op interne of externe stimuli. Ras is een verzamelnaam voor een familie van signaleringseiwitten die in vrijwel alle eukaryoten voorkomt en evolutionair sterk geconserveerd is. Een Ras eiwit is gebonden aan óf een GTP- óf een GDP-molecuul, wat de actieve, respectievelijk inactieve toestand weerspiegelt. Alleen in de actieve vorm bindt Ras aan zijn effectoren en zet daarmee de onderliggende signaleringscascades aan. Het doel van dit proefschrift was om de functie van één van deze Ras eiwitten, Rap1, in meer detail te onderzoeken. Wij hebben Dictyostelium gebruikt als modelsysteem voor ons onderzoek omdat in dit organisme het Rap1 eiwit en de onderliggende signaleringroutes grotendeels evolutionair geconserveerd zijn met de menselijke variant, terwijl het technisch veel makkelijker is om experimenten te doen met Dictyostelium dan met humane cellen. De data in dit proefschrift laten zien dat Rap1 een belangrijke regulerende functie heeft bij een veelheid van cellulaire processen. Tijdens vegetatieve groei reguleert het cel-beweging, voedselopname, substraatadhesie en celdeling, terwijl in gehongerde cellen het Rap1 betrokken is bij het dirigeren van processen als chemotaxis en ontwikkeling naar meercellig organisme. Onze conclusie is dat Rap1 beschouwd kan worden als centrale component in verscheidene essentiële signaleringscascades in Dictyostelium

End stage renal disease and survival in people with diabetes:a national database linkage study

© The Author 2014. Published by Oxford University Press on behalf of the Association of Physicians. Funding This work was supported by the Wellcome Trust through the Scottish Health Informatics Programme (SHIP). The SHIP is collaboration between the Universities of Aberdeen, Dundee, Edinburgh, Glasgow and St Andrews and the Information Services Division of National Health Service National Service Scotland. Funding for diabetes register linkage and data extraction was provided by the Chief Scientist’s Office of the Scottish Government. The Scottish Diabetes Research Network receives financial support from National Health Services Research Scotland. The Scottish Renal Registry is funded by the Information Services Division of National Health Service National Services Scotland but relies heavily on the goodwill of the contributing renal units who spent a large amount time working with Scottish Renal Registry staff to ensure that the data held within the register are accurate and complete.Peer reviewedPublisher PD

The very question of future development of extremely preterm infants

Non peer reviewe

Investigating ‘rhomboid’ membrane proteases’ roles in development and signalling in Dictyostelium

The rhomboid family of intramembrane serine proteases is almost ubiquitous across all kingdoms of life. The enzymes are poorly conserved at sequence level but are similar in their structures and active-site motifs. They cleave disparate substrates, and it is the role of rhomboids across evolution that seems to be better conserved, since a membrane location means they are ideally placed for signalling and proteolytic activation events. Thus, we are investigating rhomboid function in the development and chemotaxis of Dictyostelium discoideum, the microbial, biomedical model organism that is capable of unicellular vegetative growth and multicellular development. A small group, including four apparently enzymatically active, rhomboids was identified in Dictyostelium. We found that development was unaltered following deletion of rhmC, whereas attempts to knock out the putative mitochondrial rhmD proved lethal. rhmA and rhmB null mutants give rise to changes in development, the lack of RhmA altering the response to chemoattractants and decreasing motility of the multicellular ‘slug’. rhmB null cells have lower viability, a smaller spore-sorus and a decreased response to folic acid stimulation. These results correspond with qRTPCR analysis, in which rhmA and rhmB transcript levels are highest during the multicellular growth phase. TEM intriguingly suggests a role in mitochondrion ultrastructure for RhmA, supported by bioinformatic interaction networks. These reveal Dictyostelium RhmA cotranscription with homologues of the Saccharomyces cerevisiae mitochondrial rhomboid’s substrates, which also regulate mitochondrial morphology