Cytoplasmic inclusions containing α-synuclein (α-Syn) fibrils, referred to as Lewy bodies (LBs), are the signature neuropathological hallmarks of Parkinson's disease (PD). Although α-Syn fibrils can be generated from recombinant α-Syn protein in vitro, the production of fibrillar α-Syn inclusions similar to authentic LBs in cultured cells has not been achieved. We show here that intracellular α-Syn aggregation can be triggered by the introduction of exogenously produced recombinant α-Syn fibrils into cultured cells engineered to overexpress α-Syn. Unlike unassembled α-Syn, these α-Syn fibrils "seeded" recruitment of endogenous soluble α-Syn protein and their conversion into insoluble, hyperphosphorylated, and ubiquitinated pathological species. Thus, this cell model recapitulates key features of LBs in human PD brains. Also, these findings support the concept that intracellular α-Syn aggregation is normally limited by the number of active nucleation sites present in the cytoplasm and that small quantities of α-Syn fibrils can alter this balance by acting as seeds for aggregation.
Modification of cellular functions by overexpression of genes is increasingly practised for research of signalling pathways, but restricted by limitations of low efficiency. We investigated whether the novel technique of magnetofection (MF) could enhance gene transfer to cultured primary endothelial cells. MF of human umbilical vein endothelial cells (HUVEC) increased transfection efficiency of a luciferase reporter gene up to 360-fold compared to various conventional transfection systems. In contrast, there was only an up to 1.6-fold increase in toxicity caused by MF suggesting that the advantages of MF outbalanced the increase in toxicity. MF efficiently increased transfection efficiency using several commercially available cationic lipid transfection reagents and polyethyleneimine (PEI). Using PEI, even confluent HUVEC could be efficiently transfected to express luciferase activity. Using a green fluorescent protein vector maximum percentages of transfected cells amounted up to 38.7% while PEI without MF resulted in only 1.3% transfected cells. Likewise, in porcine aortic endothelial cells MF increased expression of a luciferase or a β-galactosidase reporter, reaching an efficiency of 37.5% of cells. MF is an effective tool for pDNA transfection of endothelial cells allowing high efficiencies. It may be of great use for investigating protein function in cell culture experiments.
Accumulating evidences indicate that pulmonary exposure to carbon nanotubes (CNTs) is associated with increased risk of lung diseases, whereas the effect on the vascular system is less studied. We investigated vascular effects of 2 types of multiwalled CNTs (MWCNTs) in apolipoprotein E-/ mice, wild-type mice, and cultured cells. The ApoE(/) mice had accelerated plaque progression in aorta after 5 intracheal instillations of MWCNT (25.6 g/mouse weekly for 5 weeks). The exposure was associated with pulmonary inflammation, lipid peroxidation, and increased expression of inflammatory, oxidative stress, DNA repair, and vascular activation response genes. The level of oxidatively damaged DNA in lung tissue was unaltered, probably due to increased DNA repair capacities. Despite upregulation of inflammatory genes in the liver, effects on systemic cytokines and lipid peroxidation were minimal. The exposure to MWCNTs in cultured human endothelial cells increased the expression of cell adhesion molecules (ICAM1 and VCAM1). In cocultures, there was increased adhesion of monocytes to endothelial cells after exposure to MWCNT. The exposure to both types of MWCNT was also associated with increased lipid accumulation in monocytic-derived foam cells, which was dependent on concomitant oxidative stress because the antioxidant N-acetylcysteine inhibited the lipid accumulation. Collectively, our results indicate that exposure to MWCNT is associated with accelerated progression of atherosclerosis, which could be related to both increased adherence of monocytes onto the endothelium and oxidative stress-mediated transformation of monocytes to foam cells.
► Kisspeptin immunoreactive substance is expressed in chicken ovarian granulosa cells. ► Kisspeptin-10 (Kp-10) stimulates progesterone secretion in these cells from preovulatory (F –F ) follicles. ► Stimulated progesterone secretion parallels upregulation of StAR, P450scc and 3β-HSD mRNA expression. The effect of kisspeptin-10 (Kp-10) on the secretion of progesterone (P ) was investigated in cultured granulosa cells from F to F follicles of hens. The results showed that granulosa cells were stained with clear signals for kisspeptin using immunocytochemistry with the specific antibody against Kp-10. Among 10, 100 and 1000 nM concentrations tested, 100 nM Kp-10 treated for 24 h significantly increased P secretion in granulosa cells from F to F follicles. After 24 h and 48 h of treatment, 100 nM Kp-10 showed a significant increase in P secretion, while after 72 h of treatment P secretion was markedly decreased by Kp-10 compared to the control group ( 0.05). These results indicate that Kp-10 stimulates P secretion in cultured chicken granulosa cells, which was associated with an up-regulation in StAR, P450scc and 3β-HSD gene transcription.
Publicly available sequence databases of the small subunit ribosomal RNA gene, also known as 16S rRNA in bacteria and archaea, are growing rapidly, and the number of entries currently exceeds 4 million. However, a unified classification and nomenclature framework for all bacteria and archaea does not yet exist. In this Analysis article, we propose rational taxonomic boundaries for high taxa of bacteria and archaea on the basis of 16S rRNA gene sequence identities and suggest a rationale for the circumscription of uncultured taxa that is compatible with the taxonomy of cultured bacteria and archaea. Our analyses show that only nearly complete 16S rRNA sequences give accurate measures of taxonomic diversity. In addition, our analyses suggest that most of the 16S rRNA sequences of the high taxa will be discovered in environmental surveys by the end of the current decade.
Benzophenone-3 (BP-3; oxybenzone) is an ingredient in sunscreen lotions and personal-care products that protects against the damaging effects of ultraviolet light. Oxybenzone is an emerging contaminant of concern in marine environments—produced by swimmers and municipal, residential, and boat/ship wastewater discharges. We examined the effects of oxybenzone on the larval form (planula) of the coral Stylophora pistillata, as well as its toxicity in vitro to coral cells from this and six other coral species. Oxybenzone is a photo-toxicant; adverse effects are exacerbated in the light. Whether in darkness or light, oxybenzone transformed planulae from a motile state to a deformed, sessile condition. Planulae exhibited an increasing rate of coral bleaching in response to increasing concentrations of oxybenzone. Oxybenzone is a genotoxicant to corals, exhibiting a positive relationship between DNA-AP lesions and increasing oxybenzone concentrations. Oxybenzone is a skeletal endocrine disruptor; it induced ossification of the planula, encasing the entire planula in its own skeleton. The LC50 of planulae exposed to oxybenzone in the light for an 8- and 24-h exposure was 3.1 mg/L and 139 µg/L, respectively. The LC50s for oxybenzone in darkness for the same time points were 16.8 mg/L and 779 µg/L. Deformity EC20 levels (24 h) of planulae exposed to oxybenzone were 6.5 µg/L in the light and 10 µg/L in darkness. Coral cell LC50s (4 h, in the light) for 7 different coral species ranges from 8 to 340 µg/L, whereas LC20s (4 h, in the light) for the same species ranges from 0.062 to 8 µg/L. Coral reef contamination of oxybenzone in the U.S. Virgin Islands ranged from 75 µg/L to 1.4 mg/L, whereas Hawaiian sites were contaminated between 0.8 and 19.2 µg/L. Oxybenzone poses a hazard to coral reef conservation and threatens the resiliency of coral reefs to climate change.
Danggui Buxue Tang (DBT), a Chinese herbal decoction used to treat ailments in women, contains Radix Astragali (Huangqi; RA) and Radix Angelicae Sinensis (Danggui; RAS). The weight ratio of RA to RAS used in DBT must be 5:1 as stipulated as early as AD 1247; however, DBT’s mechanism of action has never been described. Here, the estrogenic effects of DBT were investigated by determining the phosphorylations of estrogen receptor α (ERα) and extracellular signal-regulated kinase 1/2 (Erk1/2) in cultured MCF-7 cells. The application of DBT triggered the phosphorylation of ERα and Erk1/2 in a time-dependent manner. In contrast to the effect of estrogen, DBT triggered ERα phosphorylation at both S118 and S167. This DBT-specific phosphorylation was not triggered by an extract of one of the individual herbs, or by mixing the extracts of RA and RAS. DBT-induced downstream signals are described here. These signals suggest the uniqueness of this Chinese herbal decoction that requires a well-defined formulation.
Background Pyridoxine (VB6), which acts as a coenzyme in the biosynthesis of niacin, is formulated in pharmaceuticals to treat skin roughness. However, the mechanism of action of VB6 is not known precisely. Objective This study was conducted to clarify the influence of highly oxidative conditions on the expression of skin moisture-related mRNAs and to evaluate the preventive effects of VB6 focusing on antioxidant behaviour. Methods Intracellular levels of reactive oxygen species (ROS) in normal human epidermal keratinocytes (NHEKs) were determined using the 2 ',7 '-dichlorofluorescein diacetate assay. Real-time PCR was employed to investigate the influence of higher oxidative conditions on the expression of mRNAs encoding serine palmitoyl transferase (SPT) and filaggrin, and to characterize the mechanism of the antioxidant effect of VB6. Intracellular glutathione was quantified using an assay based on the glutathione recycling system with 5,5 '-dithiobis (2-nitrobenzoic acid) reagent and glutathione reductase. Carbonylated proteins (CPs) were semi-quantified by detecting aldehyde residues. Results Treatment of NHEKs with BSO increased the level of intracellular CPs by interfering with intracellular glutathione synthesis. Further, treatment with BSO down-regulated the expression level of SPT mRNA, but VB6 restored SPT mRNA expression in BSO-treated NHEKs. VB6 decreased the level of intracellular CPs with or without BSO treatment in a dose-dependent manner. In addition, VB6 increased levels of intracellular NADH/NADPH and glutathione through the activation of nuclear factor E2-related factor 2 (Nrf2) signalling. Conclusion These results suggest that highly oxidative conditions cause an impaired skin barrier function due to the down-regulation of SPT that results in skin roughness. VB6 improved the down-regulation of SPT mRNA expression initiated by highly oxidative conditions by enhancing the intracellular antioxidant system.
Dermal papillae ( DP ) play key roles in hair growth and regeneration by regulating follicular cell activity. Owing to the established roles of exosomes (Exos) in the regulation of cell functions, we investigated whether DP ‐derived Exos, especially those from three‐dimensional (3D)‐cultured DP cells, affect hair growth, cycling and regeneration. Exos derived from 3D DP (3D DP ‐Exos) promoted the proliferation of DP cells and outer root sheath ( ORS ) cells and increased the expression of growth factors ( IGF ‐1, KGF and HGF ) in DP cells. 3D DP ‐Exo treatment also increased hair shaft elongation in cultured human hair follicles. In addition, local injections of 3D DP ‐Exos induced anagen from telogen and also prolonged anagen in mice. Moreover, Exo treatment in human DP spheres augmented hair follicle neogenesis when the DP spheres were implanted with mouse epidermal cells. Similar results were obtained using Exos derived from 2D‐cultured DP cells (2D DP ‐Exo). Collectively, our data strongly suggest that Exos derived from DP cells promote hair growth and hair regeneration by regulating the activity of follicular dermal and epidermal cells; accordingly, these findings have implications for the development of therapeutic strategies for hair loss.