Cross-talk between calcium signalling and protein phosphorylation at the thylakoid

The role of protein phosphorylation for adjusting chloroplast functions to changing environmental needs is well established, whereas calcium signalling in the chloroplast is only recently becoming appreciated. The work presented here explores the potential cross-talk between calcium signalling and protein phosphorylation in chloroplasts and provides the first evidence for targets of calcium-dependent protein phosphorylation at the thylakoid membrane. Thylakoid proteins were screened for calcium-dependent phosphorylation by 2D gel electrophoresis combined with phospho-specific labelling and PsaN, CAS, and VAR1, among other proteins, were identified repeatedly by mass spectrometry. Subsequently their calcium-dependent phosphorylation was confirmed in kinase assays using the purified proteins and chloroplast extracts. This is the first report on the protein targets of calcium-dependent phosphorylation of thylakoid proteins and provides ground for further studies in this direction.     

Differentiation of innate but not learnt responses to host‐habitat odours contributes to rapid host finding in a parasitoid genotype

Abstract In parasitoids, host‐habitat odour can influence host searching within the habitat. This is the case in Leptopilina sp. (Hymenoptera, Figitidae), a Drosophila parasitoid searching for larvae by ovipositor probes. This a behaviour can be conditioned to fruit odours. In a previous study, the latency of probing to a fruit odour is reported to have a genetic variability within a laboratory strain. This suggests a link between rapidity of host discovery and fitness. In the present study, this hypothesis is tested by comparing responses to host‐habitat odours between two genotypes of Leptopilina heterotoma Thomson, from the Mediterranean coast (Antibes) and from Burgundy (Tailly). The two genotypes present contrasting rhythms and levels of locomotor activity linked to contrasting interspecific competition in their area of origin. The high activity observed in the Mediterranean genotype is interpreted as an adaptive response to a limited time‐window to win against a competitor species absent in Burgundy. The present study finds differentiation in innate but not learnt responses to host‐habitat odours. The more active genotype (Antibes) has a higher probability and a shorter latency of innate probing to the odours than the less active genotype (Tailly); Antibes females also find larvae and complete infestations more rapidly. Learning equalizes the probability and the latency of probing to the odours in both strains, and increases the probing duration. Innate responses to host‐habitat odours would allow time‐limited insects to increase their reproductive rate, when host predictability is high in the habitat. Selection of faster innate responses to host and habitat cues without evolution of learnt responses indicates that the initial host discovery is more crucial to fitness than subsequent ones.     

Activation of Ca2+-independent membrane-damaging activity in Lys49-phospholipase A2 promoted by amphiphilic molecules

Association of class-II phospholipase A(2) (PLA(2)) with aggregated phospholipid substrate results in elevated levels of the Ca(2+)-dependent hydrolytic activity. The Asp49 residue participates in coordination of the Ca(2+) ion cofactor, however, in Lys49-PLA(2) homologues (Lys49-PLA(2)s), substitution of the Asp49 by Lys results in loss of Ca(2+) binding and lack of detectable phospholipid hydrolysis. Nevertheless, Lys49-PLA(2)s cause Ca(2+)-independent damage of liposome membranes. Bothropstoxin-I is a homodimeric Lys49-PLA(2) from the venom of Bothrops jararacussu, and in fluorescent marker release and dynamic light scattering experiments with DPPC liposomes we demonstrate activation of the Ca(2+)-independent membrane damaging activity by approximately 4 molecules of sodium dodecyl sulphate (SDS) per protein monomer. Activation is accompanied by significant changes in the intrinsic tryptophan fluorescence emission (ITFE) and near UV circular dichroism (UVCD) spectra of the protein. Subsequent binding of 7-10 SDS molecules results in further alterations in the ITFE and far UVCD spectra. Reduction in the rate of N-bromosuccinimide modification of Trp77 at the dimer interface suggests that initial binding of SDS to this region accompanies the activation of the membrane damaging activity. 1-anilinonaphthalene-8-sulphonic acid binding studies indicate that subsequent SDS binding to the active site is concomitant with the second structural transition. These results provide insights in the structural basis of amphiphile/protein coupling in class-II PLA(2)s.     

Cytochrome c release and mitochondria involvement in programmed cell death induced by acetic acid in Saccharomyces cerevisiae

Evidence is presented that mitochondria are implicated in the previously described programmed cell death (PCD) process induced by acetic acid in Saccharomyces cerevisiae. In yeast cells undergoing a PCD process induced by acetic acid, translocation of cytochrome c (CytC) to the cytosol and reactive oxygen species production, two events known to be proapoptotic in mammals, were observed. Associated with these events, reduction in oxygen consumption and in mitochondrial membrane potential was found. Enzymatic assays showed that the activity of complex bc(1) was normal, whereas that of cytochrome c oxidase (COX) was strongly decreased. This decrease is in accordance with the observed reduction in the amounts of COX II subunit and of cytochromes a+a(3). The acetic acid-induced PCD process was found to be independent of oxidative phosphorylation because it was not inhibited by oligomycin treatment. The inability of S. cerevisiae mutant strains (lacking mitochondrial DNA, heme lyase, or ATPase) to undergo acetic acid-induced PCD and in the ATPase mutant (knockout in ATP10) the absence of CytC release provides further evidence that the process is mediated by a mitochondria-dependent apoptotic pathway. The understanding of the involvement of a mitochondria-dependent apoptotic pathway in S. cerevisiae PCD process will be most useful in the further elucidation of an ancestral pathway common to PCD in metazoans.     

Fish diversity and community structure in two small tributaries of the Paraná River,Paraná State,Brazil

In eleven sites on two small tributaries of the Paraná River (North-West Paraná State), 6.8 and 4.0 km in length, 1263 fish specimens of 28 taxons and 14 families were collected using electrofishing. Up to twelve years ago the catchments of the two rivers were covered by tropical jungle; this has now been replaced by pasture and arable fields. Mean diversity indices of Simpson and Shannon indices were close to 0.6, which would indicate that human impact affected fish populations although the river beds have retained their original shape, except cleared of riparian trees. Despite their close location (about 18 km), the two streams differed from each other in their fish faunal composition. The distinctive nature of the fish communities in the two streams was a result of: conductivity, pH, also hiding places, riparian vegetation, submerged macrophytes and depth and width of the rivers.     

Involving society in science: Reflections on meaningful and impactful stakeholder engagement in fundamental research

Open Science calls for transparent science and involvement of various stakeholders. Here are examples of and advice for meaningful stakeholder engagement. Subject Categories: Economics, Law & Politics, History & Philosophy of Science

The concepts of Open Science and Responsible Research and Innovation call for a more transparent and collaborative science, and more participation of citizens. The way to achieve this is through cooperation with different actors or “stakeholders”: individuals or organizations who can contribute to, or benefit from research, regardless of whether they are researchers themselves or not. Examples include funding agencies, citizens associations, patients, and policy makers (https://aquas.gencat.cat/web/.content/minisite/aquas/publicacions/2018/how_measure_engagement_research_saris1_aquas2018.pdf). Such cooperation is even more relevant in the current, challenging times—even apart from a global pandemic—when pseudo‐science, fake news, nihilist attitudes, and ideologies too often threaten social and technological progress enabled by science. Stakeholder engagement in research can inform and empower citizens, help render research more socially acceptable, and enable policies grounded on evidence‐based knowledge. Beyond, stakeholder engagement is also beneficial to researchers and to research itself. In a recent survey, the majority of scientists reported benefits from public engagement (Burns et al, 2021). This can include increased mutual trust and mutual learning, improved social relevance of research, and improved adoption of results and knowledge (Cottrell et al, 2014). Finally, stakeholder engagement is often regarded as an important factor to sustain public investment in the life sciences (Burns et al, 2021).

Stakeholder engagement in research can inform and empower citizens, help render research more socially acceptable and enable policies grounded on evidence‐based knowledge

Here, we discuss different levels of stakeholder engagement by way of example, presenting various activities organized by European research institutions. Based on these experiences, we propose ten reflection points that we believe should be considered by the institutions, the scientists, and the funding agencies to achieve meaningful and impactful stakeholder engagement.     

Human topoisomerase IIalpha: targeting to subchromosomal sites of activity during interphase and mitosis

Mammalian topoisomerase IIalpha (topo IIalpha) plays a vital role in the removal of topological complexities left on DNA during S phase. Here, we developed a new assay to selectively identify sites of catalytic activity of topo IIalpha with subcellular resolution. We show that topo IIalpha activity concentrates at replicating heterochromatin in late S in a replication-dependent manner and at centric heterochromatin during G2 and M phases. Inhibitor studies indicate that this cell cycle-dependent concentration over heterochromatin is sensitive to chromatin structure. We further show that catalytically active topo IIalpha concentrates along the longitudinal axis of mitotic chromosomes. Finally, we found that catalytically inert forms of the enzyme localize predominantly to splicing speckles in a dynamic manner and that this pool is differentially sensitive to changes in the activities of topo IIalpha itself and RNA polymerase II. Together, our data implicate several previously unsuspected activities in the partitioning of the enzyme between sites of activity and putative depots.     

Adenosine modulation of D-[3H]aspartate release in cultured retina cells exposed to oxidative stress

In this study we evaluated the role of adenosine receptor activation on the K+-evoked D-[3H]aspartate release in cultured chick retina cells exposed to oxidant conditions. Oxidative stress, induced by ascorbate (3.5 mM)/Fe2+ (100 microM), increased by about fourfold the release of D-[3H]aspartate, evoked by KCl 35 mM in the presence and in the absence of Ca2+. The agonist of A1 adenosine receptors, N6-cyclopentyladenosine (CPA; 10 nM), inhibited the K+-evoked D-[3H]aspartate release in control in oxidized cells. The antagonist of A1 adenosine receptor, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 50 nM), potentiated the release of D-[3H]aspartate in oxidized cells, and reverted the effect observed in the presence of CPA 10 nM. However, in oxidized cells, when DPCPX was tested together with CPA 100 nM the total release of D-[3H]aspartate increased from 5.1 +/- 0.4% to 11.4 +/- 1.0%, this increase being reverted by 3,7-dimethyl-1-propargylxanthine (DMPX; 100 nM), an antagonist of A2A adenosine receptors. In cells of both experimental conditions, the K+-evoked release of D-[3H]aspartate was potentiated by the selective agonist of A2A adenosine receptors, 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosin e (CGS 21680; 10 nM), whereas the antagonist of these receptors, DMPX (100 nM), inhibited the release of D-[3H]aspartate in oxidized cells, but not in control cells. Adenosine deaminase (ADA; 1 U/ml), which is able to remove adenosine from the synaptic space, reduced the K+-evoked D-[3H]aspartate release, from 5.1 +/- 0.4% to 3.1 +/- 0.3% in oxidized cells, and had no significant effect in control cells. The extracellular accumulation of endogenous adenosine, upon K+-depolarization, was higher in oxidized cells than in control cells, and was reduced by the inhibitors of adenosine transporter (NBTI) and of ecto-5'-nucleotidase (AOPCP). This suggests that adenosine accumulation resulted from the outflow of adenosine mediated by the transporter, and from extracellular degradation of adenine nucleotide. Our data show that both inhibitory A1 and excitatory A2A adenosine receptors are present in cultured retina cells, and that the K+-evoked D-[3H]aspartate release is modulated by the balance between inhibitory and excitatory responses. Under oxidative stress conditions, the extracellular accumulation of endogenous adenosine seems to reach levels enough to potentiate the release of D-[3H]aspartate by the tonic activation of A2A adenosine receptors.     

Chromosomal G-dark Bands Determine the Spatial Organization of Centromeric Heterochromatin in the Nucleus

Gene expression can be silenced by proximity to heterochromatin blocks containing centromeric alpha-satellite DNA. This has been shown experimentally through cis-acting chromosome rearrangements resulting in linear genomic proximity, or through trans-acting changes resulting in intranuclear spatial proximity. Although it has long been been established that centromeres are nonrandomly distributed during interphase, little is known of what determines the three-dimensional organization of these silencing domains in the nucleus. Here, we propose a model that predicts the intranuclear positioning of centromeric heterochromatin for each individual chromosome. With the use of fluorescence in situ hybridization and confocal microscopy, we show that the distribution of centromeric alpha-satellite DNA in human lymphoid cells synchronized at G(0)/G(1) is unique for most individual chromosomes. Regression analysis reveals a tight correlation between nuclear distribution of centromeric alpha-satellite DNA and the presence of G-dark bands in the corresponding chromosome. Centromeres surrounded by G-dark bands are preferentially located at the nuclear periphery, whereas centromeres of chromosomes with a lower content of G-dark bands tend to be localized at the nucleolus. Consistent with the model, a t(11; 14) translocation that removes G-dark bands from chromosome 11 causes a repositioning of the centromere, which becomes less frequently localized at the nuclear periphery and more frequently associated with the nucleolus. The data suggest that "chromosomal environment" plays a key role in the intranuclear organization of centromeric heterochromatin. Our model further predicts that facultative heterochromatinization of distinct genomic regions may contribute to cell-type specific patterns of centromere localization.