Following publication of the original article [1], the authors reported a change to one of the author names.
Following publication of the original article [1], the authors reported a change to one of the author names.
Background: The palmitate analogue 2-bromopalmitate (2-BP) is a non-selective membrane tethered cysteine alkylator of many membrane-associated enzymes that in the last years emerged as a general inhibitor of protein S-palmitoylation. Palmitoylation is a post-translational protein modification that adds palmitic acid to a cysteine residue through a thioester linkage, promoting membrane localization, protein stability, regulation of enzymatic activity, and the epigenetic regulation of gene expression. Little is known on such important process in the pathogenic protozoan Trypanosoma cruzi, the etiological agent of Chagas disease.
Results: The effect of 2-BP was analyzed on different developmental forms of Trypanosoma cruzi. The IC50/48 h value for culture epimastigotes was estimated as 130 μM. The IC50/24 h value for metacyclic trypomastigotes was 216 nM, while for intracellular amastigotes it was 242 μM and for cell derived trypomasigotes was 262 μM (IC50/24 h). Our data showed that 2-BP altered T. cruzi: 1) morphology, as assessed by bright field, scanning and transmission electron microscopy; 2) mitochondrial membrane potential, as shown by flow cytometry after incubation with rhodamine-123; 3) endocytosis, as seen after incubation with transferrin or albumin and analysis by flow cytometry/fluorescence microscopy; 4) in vitro metacyclogenesis; and 5) infectivity, as shown by host cell infection assays. On the other hand, lipid stress by incubation with palmitate did not alter epimastigote growth, metacyclic trypomastigotes viability or trypomastigote infectivity.
Conclusion: Our results indicate that 2-BP inhibits key cellular processes of T. cruzi that may be regulated by palmitoylation of vital proteins and suggest a metacyclic trypomastigote unique target dependency during the parasite development.
Background: Interactions of resident bacteria and/or their producing lipopolysaccharide (LPS) with sulcular epithelial keratinocytes may be regulated by autophagy in the gingival sulcus. In this study, we investigated an induction of bacterial autophagy in exfoliative sulcular keratinocytes of the gingival sulcus and cultured keratinocytes treated with Porphyromonas gingivalis-originated LPS (PgLPS).
Results: Exfoliative sulcular keratinocytes showed an induction of autophagy, in addition to increased expression of LPS-mediated factors including lipopolysaccharide-binding protein and toll-like receptors (TLRs), leading to co-localization of bacteria with autophagosomes. In contrast, exfoliative keratinocytes from the free gingiva did not show similar autophagy. Autophagy activity in human cultured keratinocyte cells (HaCaT) was induced by PgLPS, which was dependent partially on the AMP-activated protein kinase (AMPK) pathway via increased intracellular reactive oxygen species (ROS) and was in association with an activation of TLR4 signaling. After incubation of cultured keratinocytes with E.coli BioParticles following PgLPS stimulation, co-localization of bioparticles with autophagosomes was enhanced. Conversely, blockage of autophagy with 3-methyladenin and LPS-binding with polymyxin B led to significant reduction of co-localization of particles with autophagosomes.
Conclusion: These findings indicate that PgLPS-induced autophagy is at least partially responsible for interaction between bacteria and sulcular keratinocytes in the gingival sulcus.
Background: Elevated levels of environmental ionizing radiation can be a selective pressure for wildlife by producing reactive oxygen species and DNA damage. However, the underlying molecular mechanisms that are affected are not known.
Results: We isolated skin fibroblasts from bank voles (Myodes glareolus) inhabiting the Chernobyl nuclear power plant accident site where background radiation levels are about 100 times greater than in uncontaminated areas. After a 10 Gy dose of gamma radiation fibroblasts from Chernobyl animals recovered faster than fibroblasts isolated from bank voles living in uncontaminated control area. The Chernobyl fibroblasts were able to sustain significantly higher doses of an oxidant and they had, on average, a higher total antioxidant capacity than the control fibroblasts. Furthermore, the Chernobyl fibroblasts were also significantly more resistant than the control fibroblasts to continuous exposure to three DNA damaging drugs. After drug treatment transcription of p53-target gene pro-apoptotic Bax was higher in the control than in the Chernobyl fibroblasts.
Conclusion: Fibroblasts isolated from bank voles inhabiting Chernobyl nuclear power plant accident site show elevated antioxidant levels, lower sensitivity to apoptosis, and increased resistance against oxidative and DNA stresses. These cellular qualities may help bank voles inhabiting Chernobyl to cope with environmental radioactivity.
Background: TP53 gene mutations occur in more than 50% of human cancers and the vast majority of these mutations in human cancers are missense mutations, which broadly occur in DNA binding domain (DBD) (Amino acids 102-292) and mainly reside in six "hotspot" residues. TP53 G245C and R273H point mutations are two of the most frequent mutations in tumors and have been verified in several different cancers. In the previous study of the whole genome sequencing (WGS), we found some mutations of TP53 DBD in esophageal squamous cell carcinoma (ESCC) clinical samples. We focused on two high-frequent mutations TP53 p.G245C and TP53 p.R273H and investigated their oncogenic roles in ESCC cell lines, p53-defective cell lines H1299 and HCT116 p53-/-.
Results: MTS and colony formation assays showed that mutant TP53 G245C and R273H increased cell vitality and proliferation. Flow cytometry results revealed inhibition of ultraviolet radiation (UV)- and ionizing radiation (IR)- induced apoptosis and disruption of TP53-mediated cell cycle arrest after UV, IR and Nocodazole treatment. Transwell assays indicated that mutant TP53 G245C and R273H enhanced cell migration and invasion abilities. Moreover, western blot revealed that they were able to suppress the expression of TP53 downstream genes in the process of apoptosis and cell cycle arrest induced by UV, which suggests that these two mutations can influence apoptosis and growth arrest might be due, at least in part, to down-regulate the expression of P21, GADD45α and PARP.
Conclusions: These results indicate that mutant TP53 G245C and R273H can lead to more aggressive phenotypes and enhance cancer cell malignancy, which further uncover TP53 function in carcinogenesis and might be useful in clinical diagnosis and therapy of TP53 mutant cancers.
Background: Conditional reprogramming has enabled the development of long-lived, normal epithelial cell lines from mice and humans by in vitro culture with ROCK inhibitor on a feeder layer. We applied this technology to mouse small intestine to create 2D mouse intestinal epithelial monolayers (IEC monolayers) from genetic mouse models for functional analysis.
Results: IEC monolayers form epithelial colonies that proliferate on a feeder cell layer and are able to maintain their genotype over long-term passage. IEC monolayers form 3D spheroids in matrigel culture and monolayers on transwell inserts making them useful for functional analyses. IEC monolayers derived from the Cystic Fibrosis (CF) mouse model CFTR ∆F508 fail to respond to CFTR activator forskolin in 3D matrigel culture as measured by spheroid swelling and transwell monolayer culture via Ussing chamber electrophysiology. Tumor IEC monolayers generated from the ApcMin/+ mouse intestinal cancer model grow more quickly than wild-type (WT) IEC monolayers both on feeders and as spheroids in matrigel culture.
Conclusions: These results indicate that generation of IEC monolayers is a useful model system for growing large numbers of genotype-specific mouse intestinal epithelial cells that may be used in functional studies to examine molecular mechanisms of disease and to identify and assess novel therapeutic compounds.
Background: Tendinopathies are common and difficult to resolve due to the formation of scar tissue that reduces the mechanical integrity of the tissue, leading to frequent reinjury. Tenocytes respond to both excessive loading and unloading by producing pro-inflammatory mediators, suggesting that these cells are actively involved in the development of tendon degeneration. The transcription factor scleraxis (Scx) is required for the development of force-transmitting tendon during development and for mechanically stimulated tenogenesis of stem cells, but its function in adult tenocytes is less well-defined. The aim of this study was to further define the role of Scx in mediating the adult tenocyte mechanoresponse.
Results: Equine tenocytes exposed to siRNA targeting Scx or a control siRNA were maintained under cyclic mechanical strain before being submitted for RNA-seq analysis. Focal adhesions and extracellular matrix-receptor interaction were among the top gene networks downregulated in Scx knockdown tenocytes. Correspondingly, tenocytes exposed to Scx siRNA were significantly softer, with longer vinculin-containing focal adhesions, and an impaired ability to migrate on soft surfaces. Other pathways affected by Scx knockdown included increased oxidative phosphorylation and diseases caused by endoplasmic reticular stress, pointing to a larger role for Scx in maintaining tenocyte homeostasis.
Conclusions: Our study identifies several novel roles for Scx in adult tenocytes, which suggest that Scx facilitates mechanosensing by regulating the expression of several mechanosensitive focal adhesion proteins. Furthermore, we identified a number of other pathways and targets affected by Scx knockdown that have the potential to elucidate the role that tenocytes may play in the development of degenerative tendinopathy.
Background: Copines are calcium-dependent phospholipid-binding proteins found in many eukaryotic organisms and are thought to be involved in signaling pathways that regulate a wide variety of cellular processes. Copines are characterized by having two C2 domains at the N-terminus accompanied by an A domain at the C-terminus. Six copine genes have been identified in the Dictyostelium genome, cpnA - cpnF.
Results: Independent cell lines expressing CpnA, CpnB, CpnC, CpnE, or CpnF tagged with green fluorescent protein (GFP) were created as tools to study copine protein membrane-binding and localization. In general, the GFP-tagged copine proteins appeared to localize to the cytoplasm in live cells. GFP-tagged CpnB, CpnC, and CpnF were also found in the nucleus. When cells were fixed or when live cells were treated with calcium ionophore, the GFP-tagged copine proteins were found associated with the plasma membrane and vesicular organelles. When starved Dictyostelium cells were stimulated with cAMP, which causes a transitory increase in calcium concentration, all of the copines translocated to the plasma membrane, but with varying magnitudes and on and off times, suggesting each of the copines has distinct calcium-sensitivities and/or membrane-binding properties. In vitro membrane binding assays showed that all of the GFP-tagged copines pelleted with cellular membranes in the presence of calcium; yet, each copine displayed distinct calcium-independent membrane-binding in the absence of calcium. A lipid overlay assay with purified GFP-tagged copine proteins was used to screen for specific phospholipid-binding targets. Similar to other proteins that contain C2 domains, GFP-tagged copines bound to a variety of acidic phospholipids. CpnA, CpnB, and CpnE bound strongly to PS, PI(4)P, and PI(4,5)P2, while CpnC and CpnF bound strongly to PI(4)P.
Conclusions: Our studies show that the Dictyostelium copines are soluble cytoplasmic and nuclear proteins that have the ability to bind intracellular membranes. Moreover, copines display different membrane-binding properties suggesting they play distinct roles in the cell. The transient translocation of copines to the plasma membrane in response to cAMP suggests copines may play a specific role in chemotaxis signaling.
Background: Adipocyte differentiation of human mesenchymal stem cells (hMSCs) is dependent on mitochondrial metabolism and reactive oxygen species (ROS) to initiate adipocyte differentiation. Although anethole has been known as an anti-oxidant and lipid peroxidation inhibitor, there is little investigated about its role in adipogenic differentiation.
Methods: The effects on cytotoxicity and proliferation of anethole in hMSCs were measured by the MTT assay. The anti-adipogenic effect of anethole on hMSCs was analyzed by Oil Red O staining and western blot analysis. The anti-oxidant activity of anethole on hMSC was assessed by flowcytometry and fluorescence staining using 2',7' -dichlorofluorescin diacetate (DCFDA). The western blotting was used to detect of phospho-Akt, phospho-mTOR, phospho-p70S6K, PPARγ, and phsopho-AMP-activated kinase (AMPK).
Results: Anethole suppressed the adipogenic differentiation of hMSCs through down-regulation of Akt-mTOR-p70S6K-PPARγ and up-regulation of AMPK. Anethole affected oxidative conditions through ROS generation. Anethole also rescued AMPK activity and reduced activation of mTOR-p70S6K-PPARγ under oxidative conditions in presence of exogenous hydrogen peroxide.
Conclusion: ROS and mTOR regulation is a crucial factor in adipogenic differentiation, anethole has an important role in regulating activities of mTOR/PPARγ and ROS control in adipogenic differentiation of hMSCs.
Background: Apoptosis, the most well-known type of programmed cell death, can induce in a paracrine manner a proliferative response in neighboring surviving cells called apoptosis-induced proliferation (AiP). While having obvious benefits when triggered in developmental processes, AiP is a serious obstacle in cancer therapy, where apoptosis is frequently induced by chemotherapy. Therefore, in this study, we evaluated the capacity of an alternative type of cell death, called caspase-independent cell death, to promote proliferation.
Results: Using a novel in vitro isogenic cellular model to trigger either apoptosis or caspase-independent cell death, we found that the later has no obvious compensatory proliferation effects on neighboring cells.
Conclusions: This study enforces the idea that alternative types of cell death such as caspase-independent cell death could be considered to replace apoptosis in the context of cancer treatment.
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