Haemocytes and blastogenetic cycle in the colonial ascidian <Emphasis Type="Italic">Botryllus schlosseri</Emphasis>: a matter of life and death

A recurrent blastogenetic cycle characterizes colonies of the ascidian Botryllus schlosseri. This cycle starts when a new zooid generation opens its siphons and ends with take-over, when adult zooids cease filtering and are progressively resorbed and replaced by a new generation of buds, reaching functional maturity. During the generation change, massive apoptosis occurs in the colony, mainly in the tissues of old zooids. In the present study, we have investigated the behaviour of haemocytes during the colonial blastogenetic cycle, in terms of the occurrence of cell death and the expression of molecules involved in the induction of apoptosis. Our results indicate that, during take-over, caspase-3 activity in haemocyte lysates increases. In addition, about 20%–30% of haemocytes express phosphatidylserine on the outer leaflet of their plasma membrane, show DNA fragmentation and are immunopositive for caspase-3. Senescent cells are quickly ingested by circulating phagocytes that frequently, having once engulfed effete cells, in turn enter apoptosis. Dying cells and corpses are replaced by a new generation of cells that appear in the circulation during the generation change. This research was supported by the Italian M.I.U.R. (PRIN 2006)     

BS-cadherin in the colonial urochordate Botryllus schlosseri: one protein, many functions

Botryllus schlosseri is a colonial urochordate composed of coexisting modules of three asexually derived generations, the zooids and two cohorts of buds, each at disparate developmental stage. Functional zooids are replaced weekly by the older generation of buds through a highly synchronized developmental cycle called blastogenesis (which is, in turn, divided into four major stages, A to D). In this study, we examined the mode of expression of BS-cadherin, a 130-kDa transmembrane protein isolated from this species, during blastogenesis. BS-Cadherin is expressed extensively in internal organs of developing buds, embryos, ampullae and, briefly, in the digestive system of zooids at early blastogenic stage D (in contrast to low mRNA expression at this stage). In vitro trypsin assays on single-cell suspensions prepared from blastogenic stage D zooids, confirmed that BS-cadherin protein is expressed on cell surfaces and is, therefore, functional. BS-Cadherin expression is also upregulated in response to various stress conditions, such as oxidative stress, injury and allorecognition. It plays an important role in colony morphogenesis, because siRNA knockdown during D/A blastogenic transition causes chaotic colonial structures and disrupts oocytes homing onto their bud niches. These results reveal that BS-cadherin protein functions are exerted through a specific spatiotemporal pattern and fluctuating expression levels, in both development/regular homeostasis and in response to various stress conditions.     

Acetylsalicylic acid induces programmed cell death in Arabidopsis cell cultures

Acetylsalicylic acid (ASA), a derivative from the plant hormone salicylic acid (SA), is a commonly used drug that has a dual role in animal organisms as an anti-inflammatory and anticancer agent. It acts as an inhibitor of cyclooxygenases (COXs), which catalyze prostaglandins production. It is known that ASA serves as an apoptotic agent on cancer cells through the inhibition of the COX-2 enzyme. Here, we provide evidences that ASA also behaves as an agent inducing programmed cell death (PCD) in cell cultures of the model plant Arabidopsis thaliana, in a similar way than the well-established PCD-inducing agent H(2)O(2), although the induction of PCD by ASA requires much lower inducer concentrations. Moreover, ASA is herein shown to be a more efficient PCD-inducing agent than salicylic acid. ASA treatment of Arabidopsis cells induces typical PCD-linked morphological and biochemical changes, namely cell shrinkage, nuclear DNA degradation, loss of mitochondrial membrane potential, cytochrome c release from mitochondria and induction of caspase-like activity. However, the ASA effect can be partially reverted by jasmonic acid. Taking together, these results reveal the existence of common features in ASA-induced animal apoptosis and plant PCD, and also suggest that there are similarities between the pathways of synthesis and function of prostanoid-like lipid mediators in animal and plant organisms.     

Morphological aspects of fertilization in Phallusia (Ascidia) nigra (Ascidiacea,Tunicata)

The spermatozoa of Phallusia (Ascidia) nigra have an elongated head (approximately 5 m in length) in which a nucleus and a single mitochondrion are located side by side. There is no midpiece. The apex of the head is wedge-shaped. Acrosomal vesicles (approximately 55–65 nm in diameter) and moderately electron-dense material (MEDM) are present between the plasmalemma and the nuclear membranes in the anterior tip of the head. The MEDM occupies a central position and three or four acrosomal vesicles are seen in a line alongside it. The acrosomal vesicles disappear as the sperm makes contact with the surface of the chorion. Gamete fusion most likely occurs between a small process extending from the peripheral margin of the sperm apex and the egg surface, resulting in incorporation of the sperm into the egg from the anterior region of its head.     

Fine structure of the gastric epithelium of the ascidian Botryllus schlosseri. Mucous,endocrine and plicated cells

Summary The following five cell types have been recognized and defined on the basis of their fine structure in the gastric epithelium of B. schlosseri: vacuolated and zymogenic cells (described in a previous paper); ciliated mucous, endocrine and plicated cells. The ciliated mucous cells are distributed at the apex and the bottom of the gastric folds and along the dorsal groove. The mucus droplets appear to form from the Golgi complex as secretory granules of variable density and texture, which are released from the cell after fusion of their membranes with the apical plasma membrane. Holocrine or apocrine secretion has not been observed. The endocrine cells are scattered and are characterized by electron dense granules, especially numerous in the basal region of the cell. Finally, the plicated cells, present in the pyloric caecum, show rod-like microvilli, a well developed Golgi complex and abundant, deep infoldings of the basal plasma membrane, which are associated with numerous mitochondria. The possible role of the gastric cell types is discussed taking into account information concerning morphologically similar cells in other animals, as well as previously reported data on the biochemistry and physiology of digestion and excretion in ascidians.The authors are grateful to Mr. G. Tognon for technical help and to the Staff of the Stazione Idrobiologica di Chioggia for their assistance in collecting material. Work supported by a C.N.R. Grant from the Istituto di Biologia del Mare, Venezia, Contract n. 71.00396/04.115.542.     

Involution of the caudal musculature during metamorphosis in the ascidian,Botryllus schlosseri

Summary The caudal musculature of the free-swimming tadpole of the ascidian, B. schlosseri consists of cylindrical mononucleated cells connected in longitudinal rows flanking the axial notochord. During resorption of the larval tail, which is apparently induced by the contraction of the epidermis, muscle cells are dissociated and pushed into the body cavity where most of them are rapidly engulfed by phagocytes. In the initial stages of tail withdrawal muscle cells display surface alterations due to the disruption of intercellular junctions and disarrangement of myofibrils. Extensive degenerative changes, with shrinkage of mitochondria and disintegration of the contractile material are subsequently observed. Lysosomes and autophagic vacuoles are rarely seen and appear to play a secondary role in the degradation of the muscle cells, which occurs predominantly within the phagocytes. Myofilaments and myofibrils have never been observed within autophagic vacuoles. Clumps of muscle fragments and degenerated phagocytes undergo eventual dissolution in the blood lacunae, concomitantly with the differentiation of the young oozooid.This investigation was supported in part by a grant from the Muscular Dystrophy Associations of America and by CNR contract No. 7100396/04115542 from the Istituto di Biologia del Mare, Venice. We gratefully acknowledge the skillful assistance of Mr. G. Gallian, Mr. M. Fabbri and Mr. G. Tognon. We also thank the staff of the Stazione Idrobiologica at Chioggia for collecting the colonies.     

Polygonatum cyrtonema lectin, a potential antineoplastic drug targeting programmed cell death pathways

Polygonatum cyrtonema lectin (PCL), a mannose/sialic acid-binding plant lectin, has recently drawn a rising attention for cancer biologists because PCL bears remarkable anti-tumor activities and thus inducing programmed cell death (PCD) including apoptosis and autophagy in cancer cells. In this review, we focus on exploring the precise molecular mechanisms by which PCL induces cancer cell apoptotic death such as the caspase-dependent pathway, mitochondria-mediated ROS–p38–p53 pathway, Ras–Raf and PI3K–Akt pathways. In addition, we further elucidate that PCL induces cancer cell autophagic death via activating mitochondrial ROS–p38–p53 pathway, as well as via blocking Ras–Raf and PI3K–Akt pathways, suggesting an intricate relationship between autophagic and apoptotic death in PCL-induced cancer cells. In conclusion, these findings may provide a new perspective of Polygonatum cyrtonema lectin (PCL) as a potential anti-tumor drug targeting PCD pathways for future cancer therapeutics.     

On the development and reproduction of Botryllus schlossen (Tunicata) colonies from the eastern Mediterranean Sea: plasticity of life history traits

Summary

Analysis of developmental patterns of Botryllus schlosseri colonies from the eastern Mediterranean coast has been performed on 143 genets under different temperature regimens (15,20,27 °C) for up to 22 weeks. While the average maximal size as the length of the blastogenic cycle (BC) varied in respect to water temperature, ontogeny at all temperatures was characterized by 4 developmental stages: 1. the lag phase (the first 1–3 BCs, 1 bud/BC), 2. the exponential growth phase (5.5–7.8 BCs, up to 3 buds/BC), 3. the plateau phase, (7.4–8.2 BCs, 1 bud/BC), 4. the degenerative or the variable phase. Many (40.6%) colonies were not sexually reproductive, the others were male only (30.1%) or hermaphrodites. Colonies at the peak of reproduction develop 1.2 oocytes/zooid, and up to 57.7% produced >4 clutches. Analysing onset of reproduction with maximal colony size revealed 4 patterns, two for “male only” colonies and two for hermaphrodites that varied at different sea water temperatures. In two patterns, the onset of reproduction precedes colony maximal size, and in the others it starts at the peak size or thereafter. Zooids at the colony's periphery developed almost twice as many buds as did zooids at the center, but produced significantly lower numbers of eggs. Fragmentation was recorded in large colonies and was temperature dependent. Results are compared with the data available on populations from other localities indicating for dramatically different developmental modes characteristic to this cosmopolitan species.     

Mechanisms of programmed cell death during oogenesis in <Emphasis Type="Italic">Drosophila virilis</Emphasis>

We describe the features of programmed cell death occurring in the egg chambers of Drosophila virilis during mid-oogenesis and late oogenesis. During mid-oogenesis, the spontaneously degenerating egg chambers exhibit typical characteristics of apoptotic cell death. As revealed by propidium iodide, rhodamine-conjugated phalloidin staining, and the TUNEL assay, respectively, the nurse cells contain condensed chromatin, altered actin cytoskeleton, and fragmented DNA. In vitro caspase activity assays and immunostaining procedures demonstrate that the atretic egg chambers possess high levels of caspase activity. Features of autophagic cell death are also observed during D. virilis mid-oogenesis, as shown by monodansylcadaverine staining, together with an ultrastructural examination by transmission electron microscopy. During the late stages of oogenesis in D. virilis, once again, the two mechanisms, viz., nurse cell cluster apoptosis and autophagy, operate together, manifesting features of cell death similar to those detailed above. Moreover, an altered form of cytochrome c seems to be released from the mitochondria in the nurse cells proximal to the oocyte. We propose that apoptosis and autophagy function synergistically during oogenesis in D. virilis in order to achieve a more efficient elimination of the degenerated nurse cells and abnormal egg chambers. The present study was co-financed within Op. Education by the European Social Fund and by National Resources via a grant (HRAKLEITOS 70/3/7164) to Professor L.H. Margaritis.     

Mitochondrial alterations related to programmed cell death in tobacco cells under aluminium stress

The present investigation was undertaken to verify whether mitochondria play a significant role in aluminium (Al) toxicity, using the mitochondria isolated from tobacco cells (Nicotiana tabacum, non-chlorophyllic cell line SL) under Al stress. An inhibition of respiration was observed in terms of state-III, state-IV, succinate-dependent, alternative oxidase (AOX)-pathway capacity and cytochrome (CYT)-pathway capacity, respectively, in the mitochondria isolated from tobacco cells subjected to Al stress for 18 h. In accordance with the respiratory inhibition, the mitochondrial ATP content showed a significant decrease under Al treatment. An enhancement of reactive oxygen species (ROS) production under state-III respiration was observed in the mitochondria isolated from Al-treated cells, which would create an oxidative stress situation. The opening of mitochondrial permeability transition pore (MPTP) was seen more extensively in mitochondria isolated from Al-treated cells than in those isolated from control cells. This was Ca(2+) dependent and well modulated by dithioerythritol (DTE) and Pi, but insensitive to cyclosporine A (CsA). The collapse of inner mitochondrial membrane potential (DeltaPsi(m)) was also observed with a release of cytochrome c from mitochondria. A great decrease in the ATP content was also seen under Al stress. Transmission electron microscopy analysis of Al-treated cells also corroborated our biochemical data with distortion in membrane architecture in mitochondria. TUNEL-positive nuclei in Al-treated cells strongly indicated the occurrence of nuclear fragmentation. From the above study, it was concluded that Al toxicity affects severely the mitochondrial respiratory functions and alters the redox status studied in vitro and also the internal structure, which seems to cause finally cell death in tobacco cells.     

Phosphite accelerates programmed cell death in phosphate-starved oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) suspension cell cultures

Phosphite (H₂PO₃^–, Phi) prevents the acclimation of plants and yeast to orthophosphate (Pi, HPO₄^2–) deprivation by specifically obstructing the derepression of genes encoding proteins characteristic of their Pi-starvation response. In this study, we report that prolonged (i.e., 3–4 weeks) culture of Brassica napus L. suspension cells in Pi-deficient (–Pi) media leads to programmed cell death (PCD). However, when the B. napus cells were subcultured into –Pi media containing 2 mM Phi, they initiated PCD within 5 days, with 95% cell death observed by day 9. Dying cells exhibited several morphological and biochemical features characteristic of PCD, including protoplast shrinkage, chromatin condensation, and fragmentation of nuclear DNA. Immunoblotting indicated that B. napus cells undergoing PCD upregulated a 30-kDa cysteine endoprotease that is induced during PCD in the inner integument cells of developing B. napus seeds. It is concluded that PCD in B. napus suspension cells is triggered by extended Pi starvation, and that Phi treatment greatly accelerates this process. Our results also infer that the adaptive value of acclimating at the molecular level to Pi-stress is to extend the viability of –Pi B. napus cell cultures by about 3 weeks.Abbreviations APase acid phosphatase (EC 3.1.3.2) - BnCysP B. napus cysteine proteinase - DAPI 4,6-diamidino-2-phenylindole - FDA fluorescein diacetate - PCD programmed cell death - Phi phosphite - +Pi and –Pi Pi-sufficient and -deficient, respectively - PI propidium iodide - PSI Pi-starvation inducible     

动物细胞培养过程中的细胞自然凋亡

细胞培养过程中的细胞自然凋亡是细胞受环境压力的影响而发生的现象。随着细胞自然凋亡的分子生物学和生物化学研究的深入,对以动物细胞产品生产为目的的细胞培养产业将产生极有价值的影响。采用DNA重组技术把预防细胞自然凋亡的基因导入细胞和在培基中加入具有抗细胞自然凋亡的化合物等手段已用于预防或减缓细胞培养过程中的细胞自然凋亡。这些技术将大大延长细胞达到饱和密度后的培养时间,从而使细胞培养系统的生产效率得以显著提高。     

细菌感染与Gasdermin家族介导的宿主细胞程序性死亡的研究进展

侵入宿主后,细菌生长、繁殖并与宿主相互作用,引发机体不同程度的病理变化。为抑制细菌致病过程,宿主免疫系统产生抗感染免疫应答,感染的发生和发展取决于细菌对机体的致病性与机体抗细菌免疫的相互抗争。在细菌所致感染性疾病的发生、发展过程中,细菌与宿主细胞的拮抗往往涉及程序性细胞死亡(programmed cell death, PCD)这一过程。新近发现Gasdermin家族成员Gasdermin D和Gasdermin E参与PCD过程,并在其中发挥重要作用,跟踪其研究进展将有助于应对细菌感染造成的威胁。     

A house divided: Ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death

Programmed cell death is an important physiological response to many forms of cellular stress. The signaling cascades that result in programmed cell death are as elaborate as those that promote cell survival, and it is clear that coordination of both protein- and lipid-mediated signals is crucial for proper cell execution. Sphingolipids are a large class of lipids whose diverse members share the common feature of a long-chain sphingoid base, e.g., sphingosine. Many sphingolipids have been shown to play essential roles in both death signaling and survival. Ceramide, an N-acylsphingosine, has been implicated in cell death following a myriad of cellular stresses. Sphingosine itself can induce cell death but via pathways both similar and dissimilar to those of ceramide. Sphingosine-1-phosphate, on the other hand, is an anti-apoptotic molecule that mediates a host of cellular effects antagonistic to those of its pro-apoptotic sphingolipid siblings. Extraordinarily, these lipid mediators are metabolically juxtaposed, suggesting that the regulation of their metabolism is of the utmost importance in determining cell fate. In this review, we briefly examine the role of ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death and highlight the potential roles that these lipids play in the pathway to apoptosis.     

Evidence for programmed cell death and activation of specific caspase-like enzymes in the tomato fruit heat stress response

The tomato (Lycopersicon esculentum) fruit is the best available model to study the stress response of fleshy fruit. Programmed cell death (PCD) plays an important role in stress responses in mammals and plants. In this study, we provide evidence that PCD is triggered in the tomato fruit heat stress response by detection of the sequential diagnostic PCD events, including release of cytochrome c, activation of caspase-like proteases and the presence of TUNEL-positive nuclei. Investigating the time course of these events for 12 h after heat treatment indicated that cytochrome c release and caspase-like protease activation occurred rapidly and were consistent with the onset of DNA fragmentation. In addition, LEHDase and DEVDase enzymes were specifically activated in tomato fruit pericarp during the heat treatment and recovery time. There was no significant activation of YVADase or IETDase proteases. Preincubation of pericarp discs with the broad-spectrum, cell-permeable caspase inhibitor Z-VAD-FMK, suppressed heat-induced cell death measured by trypan blue, accompanied by a decrease in LEHDase and DEVDase activities. Gui-Qin Qu and Xiang Liu contributed equally to this work.     

Clinical and pathological significance of programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) expression in high grade serous ovarian cancer

We investigated programmed cell death 1 (PD-1) / programmed cell death ligand 1 (PD-L1) expression in high grade serous ovarian cancer (HGSOC) and its relationship to tumor infiltrating lymphocytes (TIL) and prognosis. Formalin fixed paraffin embedded (FFPE) samples of 94 HGSOC cases were included in the study. Immunohistochemical analysis (CD3, CD4, CD8, PD-1 and PD-L1) was performed. Samples were analyzed for expression of immune proteins in the peritumoral stromal and intratumoral areas, scored, and expression was correlated with overall survival, stage, and age. PD-L1 staining ratio with a score greater than 0 was found to have lower survival. There were two positive staining patterns, patchy/diffuse and patchy/focal patterns, in 24 (25.5%) cases. Considering the threshold value ≥5%, we demonstrated that the PD-L1 positive cancer cell membrane immunoreactivity rate and patchy/diffuse PD-L1 expression were 9.6% (n = 9). There was statistically significant relationship between high PD-1 scores and PD-L1 cases of ≥ 5%. A statistically significant difference was found between PD-L1 staining and survival in patients with a threshold ≥ 5%. However an appropriate rate for treatment was determined in 9.6% cases. There was a statistically significant correlation between PD-1 positive TIL score and intratumoral CD3, peritumoral stromal CD3, intratumoral CD4 and intratumoral CD8 positive cells. Survival was lower in cases with higher PD-L1 positive stromal TIL score.