Intracellular signaling pathways involved in post-mitotic dopaminergic PC12 cell death induced by 6-hydroxydopamine

Oxidative stress has been shown to mediate neuron damage in Parkinson's disease (PD). In the present report, we intend to clarify the intracellular pathways mediating dopaminergic neuron death after oxidative stress production using post-mitotic PC12 cells treated with the neurotoxin 6-hydroxydopamine (6-OHDA). The use of post-mitotic cells is crucial, because one of the suggested intracellular pathways implicated in neuron death relates to the re-entry of neurons (post-mitotic cells) in the cell cycle. We find that 6-OHDA sequentially increases intracellular oxidants, functional cell damage and caspase-3 activation, leading to cell death after 12 h of incubation. Prevention of cell damage by different antioxidants supports the implication of oxidative stress in the observed neurotoxicity. Oxidative stress-dependent phosphorylation of the MAPK JNK and oxidative stress-independent PKB/Akt dephosphorylation are involved in 6-OHDA neurotoxicity. Decrease in p21(WAF1/CIP1) and cyclin-D1 expression, disappearance of the non-phosphorylated band of retinoblastoma protein (pRb), and expression of proliferating cell nuclear antigen, not present in PC12 post-mitotic cells, suggest a re-entry of differentiated cells into cell cycle. Our results indicate that such a re-entry is mediated by oxidative stress and is involved in 6-OHDA-induced cell death. We conclude that at least three intracellular pathways are involved in 6-OHDA-induced cell death in differentiated PC12 cells: JNK activation, cell cycle progression (both oxidative stress-dependent), and Akt dephosphorylation (not related to the increase of oxidants); the three pathways are necessary for the cells to die, since blocking one of them is sufficient to keep the cells alive.     

Multiple pathways cooperate to facilitate DNA replication fork progression through alkylated DNA

Eukaryotic genomes are especially vulnerable to DNA damage during the S phase of the cell cycle, when chromosomes must be duplicated. The stability of DNA replication forks is critical to achieve faithful chromosome replication and is severely compromised when forks encounter DNA lesions. To maintain genome integrity, replication forks need to be protected by the S-phase checkpoint and DNA insults must be repaired. Different pathways help to repair or tolerate the lesions in the DNA, but their contribution to the progression of replication forks through damaged DNA is not well known. Here we show in budding yeast that, when the DNA template is damaged with the alkylating agent methyl methanesulfonate (MMS), base excision repair, homologous recombination and DNA damage tolerance pathways, together with a functional S-phase checkpoint, are essential for the efficient progression of DNA replication forks and the maintenance of cell survival. In the absence of base excision repair, replication forks stall reversibly in cells exposed to MMS. This repair reaction is necessary to eliminate the lesions that impede fork progression and has to be coordinated with recombination and damage tolerance activities to avoid fork collapse and allow forks to resume and complete chromosome replication.     

Postnatal catch-up growth after fetal protein restriction programs proliferation of rat preadipocytes

We studied the in vitro proliferation and differentiation of rat preadipocytes to investigate whether catch-up growth after prenatal protein restriction may program adipose precursor cells leading to development of increased adipose tissue mass. Pregnant rat dams were fed either an isocaloric low-protein diet (LP-8%) or control diet (C-20%). During lactation, in order to induce catch-up growth, dams from LP group were fed with the C diet and litter size was reduced to four pups instead of eight. Preadipocytes were isolated from weanling male pups (28 days of age). Differentiation and proliferation were assessed across time. At late stages of preadipocyte differentiation, no difference was observed in lipid accumulation of C or LP cultures but the mRNA expression of leptin was enhanced in LP cells. At early stages of culture, a higher DNA and protein content accompanied by a higher rate of proliferation was measured in adipocytes from LP cultures. Moreover, the mRNA expression of cyclin D1 was increased in these cells whereas the expression of peroxisome proliferators-activated receptor gamma (PPARgamma) and steroyl regulatory element binding protein (SREBP-1c) was significantly reduced during early stages. The results suggest that prenatal exposure to a LP followed by rapid catch-up growth is associated with a higher rate for proliferation in preadipocytes.     

Reappraisal of early Miocene rails (Aves,Rallidae) from central France: diversity and character evolution

In Europe, Miocene rails (Aves, Rallidae) are quite abundant, but their phylogenetic placement in the context of recent forms has remained elusive. Rails from the early Miocene of the Saint‐Gérand‐le‐Puy area in central France were first described in the 19th century, and currently, only two species are recognized, namely Palaeoaramides christyi and Paraortygometra porzanoides. Our examination of the material however suggests the presence of four, likely coeval, species of rail from these deposits. Palaeoaramides eximius, previously synonymized with Palaeoaramides christyi, is here shown to probably be a distinct species, and a previously unrecognized rail, Baselrallus intermedius gen. et sp. nov., is described. To find out how these fossil rails are related to modern Rallidae, we compared them with an extensive sample of extant rails and identified plesiomorphic and derived features for crown group Rallidae. Our assessment does not support a particularly close relationship of either Palaeoaramides to Aramides or Paraortygometra to Crex (Ortygometra), and overall, these fossil rails are more primitive than previously assumed. Based on our observations of the morphology of the previously undescribed humerus of Palaeoaramides, we show this taxon to be outside crown group Rallidae, and perhaps closely related to the early Oligocene taxon Belgirallus. On the other hand, Paraortygometra porzanoides bears a resemblance to recent flufftails (Sarothrura spp.) in some elements, but whether it can be included in a clade together with flufftails is uncertain.     

Src kinases relax adherens junctions between the neighbors of apoptotic cells to permit apical extrusion

Epithelia can eliminate apoptotic cells by apical extrusion. This is a complex morphogenetic event where expulsion of the apoptotic cell is accompanied by rearrangement of its immediate neighbors to form a rosette. A key mechanism for extrusion is constriction of an actomyosin network that neighbor cells form at their interface with the apoptotic cell. Here we report a complementary process of cytoskeletal relaxation that occurs when cortical contractility is down-regulated at the junctions between those neighbor cells themselves. This reflects a mechanosensitive Src family kinase (SFK) signaling pathway that is activated in neighbor cells when the apoptotic cell relaxes shortly after injury. Inhibiting SFK signaling blocks both the expulsion of apoptotic cells and the rosette formation among their neighbor cells. This reveals the complex pattern of spatially distinct contraction and relaxation that must be established in the neighboring epithelium for apoptotic cells to be extruded.     

Differential Migration and Activation Profile of Monocytes after Trophoblast Interaction

Macrophages at the maternal-placental interface coordinate opposite demands under the control of trophoblast cells such as the response against pathogens on one hand, and apoptotic cell clearance and wound healing with the production of suppressor cytokines. Here, we investigated whether trophoblast cells induce maternal monocyte activation towards an alternative activated macrophage profile and whether bacterial or viral stimuli modulate their migratory properties. We used an in vitro model of the maternal-placental interface represented by co-cultures of CD14+ cells isolated from fertile women with first trimester trophoblast cell line (Swan-71 cells) in the presence or absence of pathogen associated molecular pattern (PAMP) stimuli lipopolysaccharide (LPS), peptidoglycan (PGN) or poly [I:C]). Maternal CD14+ cells showed increased CD16 and CD39 expression, both markers associated to an alternative activation profile, with no changes in CD80 expression after trophoblast cell interaction. These changes were accompanied by increased IL-10 and decreased IL-12 production by CD14+ cells. After stimulation with LPS, PGN or poly [I:C], monocytes co-cultured with trophoblast cells had lower production of TNF-α and IL-1β compared with non co-cultured monocytes. Interestingly, monocyte migration towards trophoblast cells was prevented in the presence of LPS or PGN but not after 24h of stimulation with poly [I:C]. LPS or PGN also decreased CCR5, CXCL-8 and CCL5 expression. Finally, trophoblast cells co-cultured with monocytes in the presence of pathological stimuli failed to increase chemokine expression, indicating a bidirectional effect. In conclusion, trophoblast might ‘instruct’ maternal monocytes to express an alternative activation profile and restrain their early recruitment under pathological threats as one of the first strategies to avoid potential tissue damage at the maternal-placental interface.     

Functional Assessment of Four Types of Disintegrants and their Effect on the Spironolactone Release Properties

Spironolactone is a drug derived from sterols that exhibits an incomplete oral absorption due to its low water solubility and slow dissolution rate. In this study, formulations of spironolactone with four disintegrants named as croscarmellose sodium, crospovidone, sodium starch glycolate and microcrystalline cellulose II (MCCII) were conducted. The effect of those disintegrants on the tensile strength, disintegration time and dissolution rate of spironolactone-based compacts was evaluated using a factorial design with three categorical factors (filler, lubricant, and disintegrant). The swelling values, water uptake and water sorption studies of these disintegrants all suggested that MCCII compacts disintegrate by a wicking mechanism similar to that of crospovidone, whereas a swelling mechanism was dominant for sodium starch glycolate and croscarmellose sodium. The disintegration time of MCCII and sodium starch glycolate remained unchanged with magnesium stearate. However, this lubricant delayed the disintegration time of crospovidone and croscarmellose sodium. MCCII presented the fastest disintegration time independent of the medium and lubricant employed. The water sorption ratio and swelling values determined sodium starch glycolate followed by croscarmellose sodium as the largest swelling materials, whereas crospovidone and MCCII where the least swelling disintegrants. The swelling property of sodium starch glycolate and croscarmellose sodium was strongly affected by the medium pH. The disintegration time of spironolactone compacts was faster when starch was used as a filler due to the formation of soft compacts. In this case, the type of filler employed rather than the disintegrant had a major effect on the disintegration and dissolution times of spironolactone.     

Lysophosphatidic acid‐triggered pathways promote the acquisition of trophoblast endovascular phenotype in vitro

Successful implantation and placentation requires that extravillous cytotrophoblast acquires an endovascular phenotype and remodels uterine spiral arteries. Defects in this mechanism correlate with severe obstetric complications as implantation failure and preeclampsia. Lysophosphatidic acid (LPA) participates in embryo implantation and contributes to vascular physiology in different biological systems. However, the role of LPA on trophoblast endovascular transformation has not been studied. Due to difficulties in studying human pregnancy in vivo, we adopted a pharmacological approach in vitro to investigate LPA action in various aspects of trophoblast endovascular response, such as the formation of endothelial capillary‐like structures, migration, and proliferation. The HTR‐8/SVneo cell line established from human first trimester cytotrophoblast was used to model the acquisition of the endovascular phenotype by the invading trophoblast. LPA increased HTR‐8/SVneo tube formation, migration (wound healing assay and phalloidin staining) and proliferation (MTT assay). LPA G protein‐coupled receptors, LPA₁ and LPA₃, were expressed in HTR‐8/SVneo. By using selective antagonists, we showed that enhanced tubulogenesis was mediated by LPA₃. In addition, cyclooxygenase‐2 and inducible nitric oxide synthase pathways participated in LPA‐stimulated tubulogenesis. Inducible nitric oxide synthase was activated downstream cyclooxygenase‐2. Furthermore, prostaglandin E2 and a nitric oxide donor (SNAP) increased trophoblast tube formation in a concentration‐dependent manner. Finally, we observed that cyclooxygenase‐2 and inducible nitric oxide synthase were localized in the nucleus, and LPA did not modify their cellular distribution. Our results show that LPA‐triggered regulatory pathways promote trophoblast endovascular response in vitro, suggesting a new role for LPA during spiral artery remodeling at the maternal‐fetal interface.