A novel checkpoint in the Bcl-2-regulated apoptotic pathway revealed by murine cytomegalovirus infection of dendritic cells

Infection with murine cytomegalovirus (MCMV) has contributed to understanding many aspects of human infection and, additionally, has provided important insight to understanding complex cellular responses. Dendritic cells (DCs) are a major target for MCMV infection. Here, we analyze the effects of MCMV infection on DC viability, and show that infected DCs become resistant to apoptosis induced by growth factor deprivation. The precise contribution of changes in the expression of Bcl-2 family proteins has been assessed and a new checkpoint in the apoptotic pathway identified. Despite their resistance to apoptosis, MCMV-infected DCs showed Bax to be tightly associated with mitochondria and, together with Bak, forming high molecular weight oligomers, changes normally associated with apoptotic cell death. Exposure of a constitutively occluded Bax NH2-terminal epitope was blocked after infection. These results suggest that MCMV has evolved a novel strategy for inhibiting apoptosis and provide evidence that apoptosis can be regulated after translocation, integration, and oligomerization of Bax at the mitochondrial membrane.     

Anti-herpes simplex virus 1 and immunomodulatory activities of a poly-γ- glutamic acid from Bacillus horneckiae strain APA of shallow vent origin

Applied Microbiology and Biotechnology - Herpes simplex virus type 1 (HSV-1) is responsible of common and widespread viral infections in humans through the world, and of rare, but extremely severe,...     

Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1

Dendritic cells (DCs) patrol the interstitial space of peripheral tissues. The mechanisms that regulate their migration in such constrained environment remain unknown. We here investigated the role of calcium in immature DCs migrating in confinement. We found that they displayed calcium oscillations that were independent of extracellular calcium and more frequently observed in DCs undergoing strong speed fluctuations. In these cells, calcium spikes were associated with fast motility phases. IP₃ receptors (IP₃Rs) channels, which allow calcium release from the endoplasmic reticulum, were identified as required for immature DCs to migrate at fast speed. The IP₃R1 isoform was further shown to specifically regulate the locomotion persistence of immature DCs, that is, their capacity to maintain directional migration. This function of IP₃R1 results from its ability to control the phosphorylation levels of myosin II regulatory light chain (MLC) and the back/front polarization of the motor protein. We propose that by upholding myosin II activity, constitutive calcium release from the ER through IP₃R1 maintains DC polarity during migration in confinement, facilitating the exploration of their environment.     

Large and dissimilar repertoire of Melan-A/MART-1-specific CTL in metastatic lesions and blood of a melanoma patient

It is widely accepted that the repertoire of Melan-A-specific T cells naturally selected in melanoma patients is diverse and mostly nonoverlapping among different individuals. To date, however, no studies have addressed the TCR profile in different tumor sites and the peripheral blood from the same patient. We compared the TCR usage of Melan-A-specific T cells from different compartments of a single melanoma patient to evaluate possible clonotype expansion or preferential homing over a 4-mo follow-up period. Using HLA-A2 peptide tetramers, CD8(+) T cells recognizing the modified Melan-A immunodominant ELAGIGILTV peptide were isolated from four metastatic lesions resected from a single melanoma patient, and their TCR repertoire was studied. A panel of T cell clones was generated by cell cloning of tetramer-positive cells. Analysis of the TCR beta-chain V segment and the complementarity-determining region 3 (CDR3) length and sequence revealed a large diversity in the TCR repertoire, with only some of the clones showing a partial conservation in the CDR3. A similar degree of diversity was found by analyzing a number of T cell clones obtained after sorting a Melan-A-specific population derived from PBLs of the same patient after in vitro culture with the immunodominant epitope. Moreover, clonotypes found at one site were not present in another, suggesting the lack of expansion and circulation of one or more clonotypes. Taken together, these results buttress the notion that the CTLs recognizing the immunodominant Ag of Melan-A comprise a high number of different clonotypic TCR, of which only some exhibit common features in the CDR3.     

The dynamics of parental care in Choughs(Pyrrhocorax pyrrhocorax)

Summary The dynamics of parental investment throughout the nestling stage and the factors affecting it were studied in the Chough(Pyrrhocorax pyrrhocorax), a species whose patterns of apportioning parental care are largely unknown. The occurrence of important trade-offs between the sexes, among the different activities of parental care and between parents' survival and current offspring survival were estimated. The parental contributions of both sexes were assessed mainly in terms of food provisioning rate and nest attendance time. Only the female brooded young nestlings while the two sexes contributed equally in food deliveries and nest sanitation. Nestling age greatly affected nest attendance time. The female spent a long time brooding in the first 10 days after hatching. Both sexes increased attendance towards the end of the nestling stage. Conversely, feeding rate and feeding rate per nestling remained approximately constant throughout the nestling period. Nestlings in smaller broods received more feeding visits than those in larger broods. The shape of the per-nestling feeding rate curve was concave-up, supporting Nur's (1984) trade-offs model rather than the Lack-Gibb hypothesis. Maintaining a high feeding frequency in broods already above the modal value might be disadvantageous, implying few benefits and large energy costs (i.e. the reduction of the parents' residual reproductive value). Female brooding time in relation to brood size showed the same decreasing concave-up trend line. Short-term trade-offs proved to be important determinants of the dynamics of parental care. Specifically, the distance from the feeding areas greatly affected the delivery rate: pairs spent a disproportionately longer time foraging in more distant patches than in closer ones. Diurnal variations and changes owing to weather conditions were also examined.

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Die Dynamik elterlicher Investition bei der Alpenkrähe(Pyrrhocorax pyrrhocorax)

Zusammenfassung Der elterliche Aufwand und die geschlechtliche Verteilung des Brutaufwandes bei Alpenkrähen ist weitgehend unbekannt. Ziel der Arbeit war es deshalb, die verschiedenen Aktivitäten der elterlichen Brutversorgung und deren Konsequenzen für die Überlebensverhältnisse der Eltern und des Nachwuchses näher zu untersuchen. Die Fütterung der Brut und die Anwesenheit und Betreuung am Nest standen im Mittelpunkt. Während nur das Weibchen brütete, teilten sich die Eltern die Jungenaufzucht und die Pflege des Nestes etwa gleichmäßig, wobei das Nestlingsalter einen erheblichen Einfluß auf die Nestversorgung hatte. In den ersten 10 Tagen huderte das Weibchen intensiv. Beide Eltern steigerten ihre Brutpflege zum Ende der Nestlingszeit. Dagegen blieben die Fütterungsrate und die Anzahl Fütterungen je Nestling über die gesamte Nestlingszeit in etwa konstant. Junge in kleineren Bruten erhielten mehr Fütterungen als solche in großen. Der Verlauf der Abhängigkeit der Fütterungen je Nestling von der Brutgröße stützt mehr die Hypothese von Nur (1984) als die von Lack und Gibb. Die Aufrechterhaltung einer hohen Fütterungsrate auch bei großen Bruten dürfte nachteilig sein, da sie nur wenig Nutzen bei einem hohen Aufwand (Beeinträchtigung der späteren Brutmöglichkeiten) bringt. Der Huderaufwand des Weibchens zeigt in etwa denselben Zusammenhang mit der Brutgröße. Kurzzeitige elterliche Entscheidungen scheinen eine wichtige Rolle in der Regulation der elterlichen Brutpflege zu spielen. Dabei kommt gerade der räumlichen Lage der Nahrungsplätze eine große Bedeutung zu: an weiter entfernt gelegenen Nahrungsplätzen verbrachten die Eltern unverhältnismäßig mehr Zeit als an nahen Futterplätzen. Daneben haben die Tageszeit und das Wetter einen Einfluß auf die elterliche Brutfürsorge der Alpenkrähen.

     

Cnidarian chemical neurotransmission, an updated overview

The ultrastructural, histochemical, immunocytochemical, biochemical, molecular, behavioral and physiological evidence for non-peptidergic and peptidergic chemical neurotransmission in the Anthozoa, Hydrozoa, Scyphozoa and Cubozoa is surveyed. With the possible exception of data for the catecholamines and peptides in some animals, the set of cumulative data - the evidence from all methodologies - is incomplete. Taken together, the evidence from all experimental approaches suggests that both classical fast (acetylcholine, glutamate, GABA, glycine) and slow (catecholamines and serotonin) transmitters, as well as neuropeptides, are involved in cnidarian neurotransmission. Ultrastructural evidence for peptidergic, serotonergic, and catecholaminergic synaptic localization is available, but the presence of clear and dense-cored synaptic vesicles also suggests both fast and slow classical transmission. Immunocytochemical studies, in general, reveal a continuous, non-localized distribution of neuropeptides, suggesting a neuromodulatory role for them. Immunocytochemical and biochemical studies indicate the presence of glutamate, GABA, serotonin, catecholamines (and/or their receptors), RFamides, nitric oxide and eicosanoids in cnidarian neurons and tissues. Gene sequences for peptidergic preprohormones have been reported; putative gene homologies to receptor proteins for vertebrate transmitters have been found in Hydra. Behavioral and physiological studies implicate classical transmitters, neuropeptides, eicosanoids and nitric oxide in the coordination of the neuroeffector systems.     

Tau dephosphorylation and microfilaments disruption are upstream events of the anti‐proliferative effects of DADS in SH‐SY5Y cells

Garlic organosulphur compounds have been successfully used as redox anti-proliferative agents. In this work, we dissect the effects of diallyl disulphide (DADS) focusing on the events upstream of cell cycle arrest and apoptosis induced in neuroblastoma SH-SY5Y cells. We demonstrate that DADS is able to cause early morphological changes, cytoskeleton oxidation, microfilaments reduction and depolymerization of microtubules. These events are attenuated in cells stably overexpressing the antioxidant enzyme SOD1, suggesting that superoxide plays a crucial role in destabilizing cytoskeleton. Moreover, we evidence that the main microtubules-associated protein Tau undergoes PP1-mediated dephosphorylation as demonstrated by treatment with okadaic acid as well as by immunoreaction with anti-Tau-1 antibody, which specifically recognizes its dephosphorylated forms. Tau dephosphorylation is inhibited by the two-electron reductants NAC and GSH ester but not by SOD1. The inability of DADS to induce apoptosis in neuroblastoma-differentiated cells gives emphasis to the anti-proliferative activity of DADS, which can be regarded as a promising potent anti-neuroblastoma drug by virtue of its widespread cytoskeleton disrupting action on proliferating cells.     

The p13 protein of human T cell leukemia virus type 1 (HTLV-1) modulates mitochondrial membrane potential and calcium uptake

Human T cell leukemia virus type 1 (HTLV-1) encodes p13, an 87-amino-acid protein that accumulates in the inner mitochondrial membrane. Recent studies performed using synthetic p13 and isolated mitochondria demonstrated that the protein triggers an inward potassium (K⁺) current and inner membrane depolarization. The present study investigated the effects of p13 on mitochondrial inner membrane potential (Δψ) in living cells. Using the potential-dependent probe tetramethyl rhodamine methyl ester (TMRM), we observed that p13 induced dose-dependent mitochondrial depolarization in HeLa cells. This effect was abolished upon mutation of 4 arginines in p13's α-helical domain that were previously shown to be essential for its activity in in vitro assays. As Δψ is known to control mitochondrial calcium (Ca²⁺) uptake, we next analyzed the effect of p13 on Ca²⁺ homeostasis. Experiments carried out in HeLa cells expressing p13 and organelle-targeted aequorins revealed that the protein specifically reduced mitochondrial Ca²⁺ uptake. These observations suggest that p13 might control key processes regulated through Ca²⁺ signaling such as activation and death of T cells, the major targets of HTLV-1 infection.     

Pyrano[2,3-e]isoindol-2-ones,new angelicin heteroanalogues

A convenient synthesis of the pyrano[2,3-e]isoindol-2-one ring system, an heteroanalogue of angelicin, is reported. Our synthetic approach consists of the annelation of the pyran ring on the isoindole moiety using 5-dialkylamino- or 5-hydroxymethylene intermediates as building blocks. The photoantiproliferative activity of the new derivatives was studied. Some of them bearing the benzyl group at the 8 position were active with IC₅₀ in the micromolar range. Cell cytotoxicity involves apoptosis, alteration of cell cycle profile and membrane photodamage.