Rapamycin Induces Apoptosis When Autophagy is Inhibited in T-47D Mammary Cells and Both Processes are Regulated by Phlda1

Autophagy is an evolutionarily conserved lysosomal degradation pathway and plays a critical role in the homeostatic process of recycling proteins and organelles. Functional relationships have been described between apoptosis and autophagy. Perturbations in the apoptotic machinery have been reported to induce autophagic cell deaths. Inhibition of autophagy in cancer cells has resulted in cell deaths that manifested hallmarks of apoptosis. However, the molecular relationships and the circumstances of which molecular pathways dictate the choice between apoptosis and autophagy are currently unknown. This study aims to identify specific gene expression of rapamycin-induced autophagy and the effects of rapamycin when the autophagy process is inhibited. In this study, we have demonstrated that rapamycin is capable of inducing autophagy in T-47D breast carcinoma cells. However, when the autophagy process was inhibited by 3-MA, the effects of rapamycin became apoptotic. The Phlda1 gene was found to be up-regulated in both autophagy and apoptosis and silencing this gene was found to reduce both activities, strongly suggests that Phlda1 mediates and positively regulates both autophagy and apoptosis pathways.     

Pulmonary haemorrhage as the earliest sign of severe leptospirosis in hamster model challenged with Leptospira interrogans strain HP358

BackgroundSevere leptospirosis is challenging as it could evolve rapidly and potentially fatal if appropriate management is not performed. An understanding of the progression and pathophysiology of Leptospira infection is important to determine the early changes that could be potentially used to predict the severe occurrence of leptospirosis. This study aimed to understand the kinetics pathogenesis of Leptospira interrogans strain HP358 in the hamster model and identify the early parameters that could be used as biomarkers to predict severe leptospirosis.Methodology/Principal findingsMale Syrian hamsters were infected with Leptospira interrogans strain HP358 and euthanized after 24 hours, 3, 4, 5, 6 and 7 days post-infection. Blood, lungs, liver and kidneys were collected for leptospiral detection, haematology, serum biochemistry and differential expression of pro- and anti-inflammatory markers. Macroscopic and microscopic organ damages were investigated. Leptospira interrogans strain HP358 was highly pathogenic and killed hamsters within 6–7 days post-infection. Pulmonary haemorrhage and blood vessel congestion in organs were noticed as the earliest pathological changes. The damages in organs and changes in biochemistry value were preceded by changes in haematology and immune gene expression.Conclusion/SignificanceThis study deciphered haemorrhage as the earliest manifestation of severe leptospirosis and high levels of IL-1β, CXCL10/IP-10, CCL3/MIP-α, neutrophils and low levels of lymphocytes and platelets serve as a cumulative panel of biomarkers in severe leptospirosis.     

Organotypic culture of human amnion cells in air-liquid interface as a potential substitute for skin regeneration

Background aimsThe aim of the present study was to evaluate the effects of air-liquid interface on the differentiation potential of human amnion epithelial cells (HAECs) to skin-like substitute in organotypic culture.MethodsHAECs at passage 1–2 were seeded onto a fibrin layer populated with human amnion mesenchymal cells to form the organotypic cultures. The organotypic HAECs were then cultured for 7, 14 and 21 d in two types of culture system: the submerged culture and the air-liquid interface culture. Cell morphogenesis was examined under the light and electron microscopes (transmission and scanning) and analyzed by immunohistochemistry.ResultsOrganotypic HAECs formed a single layer epithelium after 3 wk in submerged as well as air-liquid interface cultures. Ultrastructurally, desmosomes were observed in organotypic HAECs cultured in the air-liquid interface but not in the submerged culture. The presence of desmosomes marked the onset of early epidermal differentiation. Organotypic HAECs were positive against anti-CK18 and anti-CK14 in both the submerged and the air-liquid interface cultures. The co-expression of CK14 and CK18 suggested that differentiation of HAECs into skin may follow the process of embryonic skin development. However, weak expression of CK14 was observed after 2 and 3 wk of culture in air-liquid interface. CK10, involucrin, type IV collagen and laminin-5 expression was absent in organotypic HAECs. This observation reflects the initial process of embryonic epidermal differentiation and stratification.ConclusionsResults from the present study suggest that the air-liquid interface could stimulate early differentiation of organotypic HAECs to epidermal cells, with a potential use for skin regeneration.     

Study on photosynthetic responses and chlorophyll fluorescence in Rhizophora mucronata seedlings under shade regimes

The photosynthetic performance of mangrove Rhizophora mucronata seedlings grown under seasonally full light (HL), 50 % shade (ML), and 80 % shade (LL) conditions was characterized by gas exchange, and chlorophyll fluorescence. The carboxylation efficiency significantly affected the seasonal change of the photosynthetic capacity. Temperature and light might have synergic effect on the carboxylation efficiency. The photosynthetic rate (PN) of R. mucronata seedlings under shade regimes, however, could not be attributed to variability in chlorophyll, C _i , ΦPSII, ETR or qP values but more to differences in carboxylation efficiency, g _max, and E _max. HL and ML plants had higher PN, g _s and E than the LL ones. Nevertheless, LL leaves exhibited low photoinhibition susceptibility. The high non-photochemical quenching in HL leaves may show that applied light intensity probably exceeded the photosynthetic capability. The findings indicate that ML treatments provided the best condition to obtain such carbon fixation capacity.     

Light- and sodium azide-induced death of RGC-5 cells in culture occurs via different mechanisms

Previous studies have shown that light impinging on the retina in situ has the capacity to kill neuronal and non-neuronal cells in vitro by interacting directly with mitochondrial constituents. A number of fluorophores are associated with mitochondria which can potentially absorb different wave-lengths of light, including cytochrome oxidase. The aim of the present study was to compare the death mechanism of a light insult to RGC-5 cells in culture with that of sodium azide. Sodium azide’s main toxic action is in inhibiting the function of cytochrome oxidase in the mitochondrial electron transport chain. Our studies showed that light and sodium azide kill RGC-5 cells via different mechanisms although some similarities do occur. Both inducers of cell death caused the generation of reactive oxygen species (ROS), the expression of phosphatidylserine, the breakdown of DNA and the activation of p38 MAPK, resulting in its translocation from the nucleus to the cytoplasm. However, light-induced cell death occurs via necroptosis, in that it was inhibited by necrostatin-1 and was caspase-independent. This was not the case for sodium azide, where the death process was caspase-dependent, occurred via apoptosis and was unaffected by necrostatin-1. Moreover, light caused an activation of the apoptosis inducing factor (AIF), c-Jun, JNK and HO-1, but it did not affect alpha fodrin or caspase-3. In contrast, sodium azide caused the activation of alpha fodrin and the stimulation of caspase-3 content without influencing AIF, c-Jun, JNK or HO-1. Therefore we conclude that light does not have a specific action on cytochrome oxidase in mitochondria to cause cell death.     

Differential regulation of macrophage CCAAT-enhancer binding protein isoforms by lipopolysaccharide and cytokines

The regulation of the C/EBP family in macrophages by LPS and cytokines is of potentially crucial importance in several pathophysiological conditions. The action of LPS and three cytokines on the expression of C/EBP mRNA, protein and functional DNA binding activity in the murine J774.2 cell line was therefore studied. Exposure of the cells to LPS, IL-1, IFN-gamma and TNF-alpha produced a reduction of C/EBP alpha mRNA levels and a corresponding increase in the expression of C/EBP beta and C/EBP delta. EMSA showed time-dependent changes in the DNA binding activity of individual C/EBP isoforms and demonstrated the participation of heterodimers between the different members in DNA-protein interactions. Additionally, mediator-specific changes in the kinetics and magnitude of C/EBP mRNA expression pattern and profile of DNA-protein interactions were observed. These studies provide novel insights into the potential mechanisms that may be responsible for the mediator-specific regulation of macrophage gene expression through the C/EBP family.     

Development of anti-acanthamoebic approaches

International Microbiology - Acanthamoeba keratitis is a sight-endangering eye infection, and causative organism Acanthamoeba presents a significant concern to public health, given escalation of...