Formation Of Double Membranes Research Articles

Nicotinamide Increases Intracellular NAD+ Content to Enhance Autophagy-Mediated Group A Streptococcal Clearance in Endothelial Cells.

Group A streptococcus (GAS) is a versatile pathogen that causes a wide spectrum of diseases in humans. Invading host cells is a known strategy for GAS to avoid antibiotic killing and immune recognition. However, the underlying mechanisms of GAS resistance to intracellular killing need to be explored. Endothelial HMEC-1 cells were infected with GAS, methicillin-resistant Staphylococcus aureus (MRSA) and Salmonella Typhimurium under nicotinamide (NAM)-supplemented conditions. The intracellular NAD+ level and cell viability were respectively measured by NAD+ quantification kit and protease-based cytotoxicity assay. Moreover, the intracellular bacteria were analyzed by colony-forming assay, transmission electron microscopy, and confocal microscopy. We found that supplementation with exogenous nicotinamide during infection significantly inhibited the growth of intracellular GAS in endothelial cells. Moreover, the NAD+ content and NAD+/NADH ratio of GAS-infected endothelial cells were dramatically increased, whereas the cell cytotoxicity was decreased by exogenous nicotinamide treatment. After knockdown of the autophagy-related ATG9A, the intracellular bacterial load was increased in nicotinamide-treated endothelial cells. The results of Western blot and transmission electron microscopy also revealed that cells treated with nicotinamide can increase autophagy-associated LC3 conversion and double-membrane formation during GAS infection. Confocal microscopy images further showed that more GAS-containing vacuoles were colocalized with lysosome under nicotinamide-supplemented conditions than without nicotinamide treatment. In contrast to GAS, supplementation with exogenous nicotinamide did not effectively inhibit the growth of MRSA or S. Typhimurium in endothelial cells. These results indicate that intracellular NAD+ homeostasis is crucial for controlling intracellular GAS infection in endothelial cells. In addition, nicotinamide may be a potential new therapeutic agent to overcome persistent infections of GAS.

Double membrane formation in heterogeneous vesicles.

Lipids are capable of forming a variety of structures, including multi-lamellar vesicles. Layered lipid membranes are found in cell organelles, such as autophagosomes and mitochondria. Here, we present a mechanism for the formation of a double-walled vesicle (i.e., two lipid bilayers) from a unilamellar vesicle through the partitioning and phase separation of a small molecule. Using molecular dynamics simulations, we show that double membrane formation proceeds via a nucleation and growth process - i.e., after a critical concentration of the small molecules, a patch of double membrane nucleates and grows to cover the entire vesicle. We discuss the implications of this mechanism and theoretical approaches for understanding the evolution and formation of double membranes.

Oxidative stress-elicited autophagosome accumulation contributes to human neuroblastoma SH-SY5Y cell death induced by PBDE-47

Polybrominated diphenyl ethers, a ubiquitous persistent organic pollutant used as brominated flame retardants, is known to damage nervous system, however the underlying mechanism is still elusive. In this study, we used human neuroblastoma SH-SY5Y cells to explore the effects of PBDE-47 on autophagy and investigate the role of autophagy in PBDE-47-induced cell death. Results showed PBDE-47 could increase autophagic level (performation of cell ultrastructure with double membrane formation, MDC-positive cells raised, autophagy-related proteins LC3-II, Beclin1 and P62 increased) after cells exposed to PBDE-47. Then cells were exposed to PBDE-47 (1, 5, 10μmol/L) respectively for 1, 3, 6, 9, 12, 18, 24h, and the results showed that PBDE-47 increased the levels of LC3-II, Beclin1 and P62 in 5, 10μmol/L (9, 12, 18, 24h) PBDE-47 exposed groups. Furthermore, ROS scavenger N-Acetyl-l-cysteine (NAC), autophagic inhibitor 3-methyladenine (3-MA) and 5μmol/L PBDE-47 treated for 9h and 24h were chosen for the follow-up research. Moreover, 3-MA significantly improved cell viability when cells exposed to 5 and 10μmol/L PBDE-47, indicating that PBDE-47-induced autophagic cell death. Importantly, NAC could decrease PBDE-47-induced LC3-II, Beclin1 and P62 expression. We concluded that autophagosome accumulation mediated by oxidative stress may contribute to SH-SY5Y cell death induced by PBDE-47.

Ursolic acid promotes cancer cell death by inducing Atg5‐dependent autophagy

Ursolic acid (UA) has been reported to possess anticancer activities. Although some of the anticancer activities of UA have been explained by its apoptosis-inducing properties, the mechanisms underlying its anticancer actions are largely unknown. We have found that UA-activated autophagy induced cytotoxicity and reduced tumor growth of cervical cancer cells TC-1 in a concentration-dependent manner. UA did not induce apoptosis of TC-1 cells in vitro as determined by annexin V/propidium iodide staining, DNA fragmentation, and Western blot analysis of the apoptosis-related proteins. We found that UA increased punctate staining of light chain 3 (LC3), which is an autophagy marker. LC3II, the processed form of LC3I which is formed during the formation of double membranes, was induced by UA treatment. These results were further confirmed by transmission electron microscopy. Wortmannin, an inhibitor of autophagy, and a small interfering RNA (siRNA) for autophagy-related genes (Atg5) reduced LC3II and simultaneously increased the survival of TC-1 cells treated with UA. We also found that LC3II was significantly reduced and that survival was increased in Atg5-/- mouse embryonic fibroblast (MEF) cells compared to Atg5+/+ MEF cells under UA treatment. However, silencing BECN1 by siRNA affected neither the expression of LC3II nor the survival of TC-1 cells under UA treatment. These results suggest that autophagy is a major mechanism by which UA kills TC-1 cells. It is Atg5 rather than BECN1 that plays a crucial role in UA-induced autophagic cell death in TC-1 cells. The activation of autophagy by UA may become a potential cancer therapeutic strategy complementing the apoptosis-based therapies. Furthermore, regulation of Atg5 may improve the efficacy of UA in cancer treatment.

Ultrastructure of the Cochlear Hair Cells in the Guinea Pig after Injection of Nitrogen Mustard-N-Oxide

Nitrogen Mustard-N-Oxide was given to six guinea pigs intraperitoneally, and morphological changes of the outer hair cells in the cochlea were investigated with electron microscopy. The results obtained were as follows:1. Swelling of the cytoplasma of the cuticle free area at the top of the cell was found in various degrees. In some of them, cuticular plate was covered and the auditory hairs were partially surrounded by the swelled cytoplasma.2. Formation of double membranes was found. The double membranes were parallel to the cytoplasmic membrane, or whorl of the membranes was made in the cytoplasma.3. A rod-shaped structure was presented in a large number of the cells.4. No vacuolization was observed in any case.5. With the increase of degeneration, the outer hair cell became atrophic, then it was destroyed and disappeared, while the supporting cells were remarkably swollen.

Some Degenerative Phenomena in Drosophila Ovaries

ABSTRACT A description is given of degenerative phenomena in the Drosophila ovary. Three types of degeneration are dealt with: (1) degeneration of the follicle cells; (2) degeneration occurring in the unfertilized egg, a major feature of which is the segregation of the different constituents of the egg into comparatively large masses; and (3) degenerative phenomena in the nurse cells and oocytes of abnormal eggs produced in ovaries homozygous for deep-orange. In all these instances it is noteworthy that a considerable degree of structural organization (formation of double membranes, or sets of parallel or concentric membranes, &c.) occurs in cytoplasm which is clearly degenerating. It is suggested that such organization does not always represent the formation of functioning organelles, but may be a mere consequence of the inherent physicochemical properties of the materials involved. In degenerating deep-orange ovaries masses of fragmented porous membranes are found. These seem to be derived from the nuclear membranes of the nurse cells, which appear to persist even when the rest of the cell is rather completely destroyed. Fragments of nuclear envelopes tend to lie close together and parallel to one another, and it is suggested that this indicates that there is some force of attraction between them, which may be comparable to the forces involved in the formation of stacks of annulate lamellae, or of concentric or parallel double-membrane structures in ergastoplasm, and in chromosome pairing in healthy functional cells.