Formaldehyde induces hyperphosphorylation and polymerization of Tau protein both in vitro and in vivo

Background

Chronic formaldehyde exposure leads to memory impairment and abnormal elevation of endogenous formaldehyde has been found in the brains of Alzheimer's disease (AD) patients. Hyperphosphorylated Tau protein with subsequent aggregates as neurofibrillary tangles (NFTs) is one of the typical pathological characteristics in AD brains. The mechanism underlying abnormally elevated concentrations of endogenous formaldehyde that induce Tau hyperphosphorylation is unknown.

Methods

N2a cells and mice were treated with formaldehyde for different time points, then Western blotting and immunocytochemistry were utilized to determine the phosphorylation and polymerization of Tau protein. HPLC was used to detect the concentration of formaldehyde in cell media.

Results

Under formaldehyde stress, Tau became hyperphosphorylated, not only in the cytoplasm, but also in the nucleus of neuroblastoma (N2a) cells, and mouse brains. Polymers of cellular phospho-Tau were also detected. Significant accumulation of glycogen synthase kinase-3β (GSK-3β) in the nucleus of N2a and mouse brain cells, and elevation of its phosphorylation at Y216, was observed under formaldehyde stress. Formaldehyde-induced Tau hyperphosphorylation was blocked in the presence of LiCl and CT99021, inhibitors of GSK-3β, and by RNAi interference.

Conclusions

Formaldehyde, which may cause age-related memory loss, can act as a factor triggering Tau hyperphosphorylation via GSK-3β catalysis and induces polymerization of Tau.

General significance

Investigation of formaldehyde-induced Tau hyperphosphorylation may provide novel insights into mechanisms underlying tauopathies.     

Different activities of the conserved lysine residues in the double-stranded RNA binding domains of RNA helicase A in vitro and in the cell

Background

RNA helicase A regulates a variety of RNA metabolism processes including HIV-1 replication and contains two double-stranded RNA binding domains (dsRBD1 and dsRBD2) at the N-terminus. Each dsRBD contains two invariant lysine residues critical for the binding of isolated dsRBDs to RNA. However, the role of these conserved lysine residues was not tested in the context of enzymatically active full-length RNA helicase A either in vitro or in the cells.

Methods

The conserved lysine residues in each or both of dsRBDs were substituted by alanine in the context of full-length RNA helicase A. The mutant RNA helicase A was purified from mammalian cells. The effects of these mutations were assessed either in vitro upon RNA binding and unwinding or in the cell during HIV-1 production upon RNA helicase A–RNA interaction and RNA helicase A-stimulated viral RNA processes.

Results

Unexpectedly, the substitution of the lysine residues by alanine in either or both of dsRBDs does not prevent purified full-length RNA helicase A from binding and unwinding duplex RNA in vitro. However, these mutations efficiently inhibit RNA helicase A-stimulated HIV-1 RNA metabolism including the accumulation of viral mRNA and tRNA^Lys3 annealing to viral RNA. Furthermore, these mutations do not prevent RNA helicase A from binding to HIV-1 RNA in vitro as well, but dramatically reduce RNA helicase A–HIV-1 RNA interaction in the cells.

Conclusions

The conserved lysine residues of dsRBDs play critical roles in the promotion of HIV-1 production by RNA helicase A.

General significance

The conserved lysine residues of dsRBDs are key to the interaction of RNA helicase A with substrate RNA in the cell, but not in vitro.     

Promoter methylation analysis of WNT/β-catenin pathway regulators and its association with expression of DNMT1 enzyme in colorectal cancer

Background

Aberrant DNA methylation as the most important reason making epigenetic silencing of genes is a main mechanism of gene inactivation in patients with colorectal cancer. In this study, we decided to identify promoter methylation status of ten genes encoding WNT negative regulators, and measure the expression of DNMT1 enzyme in colorectal cancer samples.

Results

Aberrant methylation of APC gene was statistically significant associated with age over 50 (p = 0.017), DDK3 with male (p < 0.0001), SFRP4, WIF1, and WNT5a with increasing tumor stage (p = 0.004, p = 0.029, and p = 0.004), SFRP4 and WIF1 with tumor differentiation (p = 0.009 and p = 0.031) and SFRP2 and SFRP5 with histological type (p = 0.001 and p = 0.025). The increasing number of methylated genes correlated with the expression levels of the DNMT1 mRNA.

Conclusions

The rate of gene promoter methylation of WNT pathway regulators is high in colorectal cancer cells. Hyper-methylation is associated with increased expression of the DNMT1 enzyme.     

Deinococcus radiodurans' SRA-HNH domain containing protein Shp (Dr1533) is involved in faithful genome inheritance maintenance following DNA damage

Background

Deinococcus radiodurans R1 (DR) survives conditions of extreme desiccation, irradiation and exposure to genotoxic chemicals, due to efficient DNA breaks repair, also through Mn²⁺ protection of DNA repair enzymes.

Molecular mechanism of Arabidopsis thaliana profilins as antifungal proteins

Background

It remains an open question whether plant phloem sap proteins are functionally involved in plant defense mechanisms.

Excision of the oxidatively formed 5-hydroxyhydantoin and 5-hydroxy-5-methylhydantoin pyrimidine lesions by Escherichia coli and Saccharomyces cerevisiae DNA N-glycosylases

Background

(5R?) and (5S?) diastereomers of 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd) are major oxidation products of 2′-deoxycytidine and thymidine respectively. If not repaired, when present in cellular DNA, these base lesions may be processed by DNA polymerases that induce mutagenic and cell lethality processes.

Methods

Synthetic oligonucleotides that contained a unique 5-hydroxyhydantoin (5-OH-Hyd) or 5-hydroxy-5-methylhydantoin (5-OH-5-Me-Hyd) nucleobase were used as probes for repair studies involving several E. coli, yeast and human purified DNA N-glycosylases. Enzymatic reaction mixtures were analyzed by denaturing polyacrylamide gel electrophoresis after radiolabeling of DNA oligomers or by MALDI-TOF mass spectrometry measurements.

Results

In vitro DNA excision experiments carried out with endo III, endo VIII, Fpg, Ntg1 and Ntg2, show that both base lesions are substrates for these DNA N-glycosylases. The yeast and human Ogg1 proteins (yOgg1 and hOgg1 respectively) and E. coli AlkA were unable to cleave the N-glycosidic bond of the 5-OH-Hyd and 5-OH-5-Me-Hyd lesions. Comparison of the k_cat/K_m ratio reveals that 8-oxo-7,8-dihydroguanine is only a slightly better substrate than 5-OH-Hyd and 5-OH-5-Me-Hyd. The kinetic results obtained with endo III indicate that 5-OH-Hyd and 5-OH-5-Me-Hyd are much better substrates than 5-hydroxycytosine, a well known oxidized pyrimidine substrate for this DNA N-glycosylase.

Conclusions

The present study supports a biological relevance of the base excision repair processes toward the hydantoin lesions, while the removal by the Fpg and endo III proteins are effected at better or comparable rates to that of the removal of 8-oxoGua and 5-OH-Cyt, two established cellular substrates.

General significance

The study provides new insights into the substrate specificity of DNA N-glycosylases involved in the base excision repair of oxidized bases, together with complementary information on the biological role of hydantoin type lesions.     

Antibacterial performance of nanoscaled visible-light responsive platinum-containing titania photocatalyst in vitro and in vivo

Background

Traditional antibacterial photocatalysts are primarily induced by ultraviolet light to elicit antibacterial reactive oxygen species. New generation visible-light responsive photocatalysts were discovered, offering greater opportunity to use photocatalysts as disinfectants in our living environment. Recently, we found that visible-light responsive platinum-containing titania (TiO₂–Pt) exerted high performance antibacterial property against soil-borne pathogens even in soil highly contaminated water. However, its physical and photocatalytic properties, and the application in vivo have not been well-characterized.

Methods

Transmission electron microscopy, energy dispersive spectroscopy, X-ray photoelectron spectroscopy, X-ray diffraction, ultraviolet–visible absorption spectrum and the removal rate of nitrogen oxides were therefore analyzed. The antibacterial performance under in vitro and in vivo conditions was evaluated.

Results

The apparent quantum efficiency for visible light illuminated TiO₂–Pt is relatively higher than several other titania photocatalysts. The killing effect achieved approximately 2 log reductions of pathogenic bacteria in vitro. Illumination of injected TiO₂–Pt successfully ameliorated the subcutaneous infection in mice.

Conclusions

This is the first demonstration of in vivo antibacterial use of TiO₂–Pt nanoparticles. When compared to nanoparticles of some other visible-light responsive photocatalysts, TiO₂–Pt nanoparticles induced less adverse effects such as exacerbated platelet clearance and hepatic cytotoxicity in vivo.

General significance

These findings suggest that the TiO₂–Pt may have potential application on the development of an antibacterial material in both in vitro and in vivo settings.     

Entamoeba histolytica thioredoxin reductase: Molecular and functional characterization of its atypical properties

Background

Entamoeba histolytica, an intestinal protozoan that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen and nitrogen species during tissue invasion. Thioredoxin reductase catalyzes the reversible transfer of reducing equivalents between NADPH and thioredoxin, a small protein that plays key metabolic functions in maintaining the intracellular redox balance.

Methods

The present work deals with in vitro steady state kinetic studies aimed to reach a better understanding of the kinetic and structural properties of thioredoxin reductase from E. histolytica (EhTRXR).

Results

Our results support that native EhTRXR is a homodimeric covalent protein that is able to catalyze the NAD(P)H-dependent reduction of amoebic thioredoxins and S‐nitrosothiols. In addition, the enzyme exhibited NAD(P)H dependent oxidase activity, which generates hydrogen peroxide from molecular oxygen. The enzyme can reduce compounds like methylene blue, quinones, ferricyanide or nitro-derivatives; all alternative substrates displaying a relative high capacity to inhibit disulfide reductase activity of EhTRXR.

Conclusions and general significance

Interestingly, EhTRXR exhibited kinetic and structural properties that differ from other low molecular weight TRXR. The TRX system could play an important role in the parasite defense against reactive species. The latter should be critical during the extra intestinal phase of the amoebic infection. So far we know, this is the first in depth characterization of EhTRXR activity and functionality.     

Rapid uptake of lipophilic triphenylphosphonium cations by mitochondria in vivo following intravenous injection: Implications for mitochondria-specific therapies and probes

Background

Mitochondrial dysfunction contributes to a range of pathologies, consequently there is a need to monitor mitochondrial function and to intervene pharmacologically to prevent mitochondrial damage. One approach to this is to deliver antioxidants, probes and pharmacophores to mitochondria by conjugation to the lipophilic triphenylphosphonium (TPP) cation that is taken up selectively by mitochondria driven by the membrane potential.

Conclusions

Oral administration of TPP-conjugated antioxidants protects against mitochondrial damage in vivo. However, there is also a need to deliver molecules rapidly to mitochondria to respond quickly to pathologies and for the real-time assessment of mitochondrial function.

Methods

To see if this was possible we investigated how rapidly TPP cations were taken up by mitochondria in vivo following intravenous (iv) administration.

Results

AlkylTPP cations were accumulated selectively by mitochondria within mice within 5 min of iv injection. The extent of uptake was enhanced 10–30-fold relative to simple alkylTPP cations by attaching functional groups to the TPP cation via long, hydrophobic alkyl chains. Conclusions: Mitochondria-targeted antioxidants, probes and pharmacophores can be delivered into mitochondria within minutes of iv administration.

General significance

These findings greatly extend the utility of mitochondria-targeted lipophilic cations as therapies and probes.     

Downregulation of MEG3 and upregulation of EZH2 cooperatively promote neuroblastoma progression

Neuroblastoma (NB), an embryonic tumour originating from sympathetic crest cells, is the most common extracranial solid tumour type in children with poor overall prognosis. Accumulating evidence has demonstrated the involvement of long non‐coding RNA (lncRNA) in numerous biological processes and their associations with embryonic development and multiple diseases. Ectopic lncRNA expression is linked to malignant tumours. Previous studies by our team indicate that MEG3 attenuates NB autophagy through inhibition of FOXO1 and epithelial‐mesenchymal transition via the mTOR pathway in vitro. Moreover, MEG3 and EZH2 negatively regulate each other. In present study, we first collected 60 NB tissues and 20 adjacent tissues for Quantitative real‐time polymerase chain reaction (Q‐PCR) experiments and performed clinical correlation analysis of the results. At the same time, nude mice were used for subcutaneous tumour formation to detect the effect of MEG3 in vivo. Two NB cell lines, SK‐N‐AS and SK‐N‐BE(2)C, were overexpressed MEG3 and rescued with EZH2 and then were subjected to proliferation, migration, invasion, apoptosis and autophagy experiments. RNA‐binding protein immunoprecipitation (RIP) and Co‐Immunoprecipitation (Co‐IP) experiments were performed to explore the molecular mechanism of MEG3 and EZH2 interaction. Q‐PCR revealed that MEG3 expression was negatively correlated with INSS stage and risk grade of NB. Moreover, MEG3 overexpression was associated with inhibition of NB growth in vivo. MEG3 exerted an anti‐cancer effect via stimulatory effects on EZH2 ubiquitination leading to its degradation. Conversely, EZH2 interacted with DNMT1 and HDAC1 to induce silencing of MEG3. The EZH2 inhibitor, DZNep, and HDAC inhibitor, SAHA, displayed synergistic activity against NB. Combined treatment with DZNep and SAHA inhibited proliferation, migration and invasion of NB through suppression of the PI3K/AKT/mTOR/FOXO1 pathway. In conclusion, downregulation of MEG3 and upregulation of EZH2 forms a feedback loop that concertedly promotes the development of NB. Combined blockage of EZH2 and HDAC1 with the appropriate inhibitors may therefore present an effective treatment strategy for NB cases with low MEG3 and high EZH2 expression.     

Dual blockade of VEGFR1 and VEGFR2 by a novel peptide abrogates VEGF-driven angiogenesis,tumor growth,and metastasis through PI3K/AKT and MAPK/ERK1/2 pathway

Background

Neutralization of vascular endothelial growth factor receptor 1 (VEGFR1) and/or VEGFR2 is a widely used means of inhibiting tumor angiogenesis.

Decursin attenuates hepatic fibrogenesis through interrupting TGF-beta-mediated NAD(P)H oxidase activation and Smad signaling in vivo and in vitro

Aims

We studied that a potent antifibrotic effect of decursin on in vivo liver damage model and the mechanism in inhibiting which transforming growth factor (TGF)-β1-induced human hepatic stellate cells (HSCs) activation.

Main methods

Liver injury was induced in vivo by intraperitoneal injection of carbon tetrachloride (CCl₄) with or without decursin for 4 weeks in mice. Human hepatic stellate cell line, an immortalized human HSC line, was used in in vitro assay system. The effects of decursin on HSC activation were measured by analyzing the expression of α-smooth muscle actin (α-SMA) and collagen I in liver tissue and human HSCs.

Key findings

Decursin treatment significantly reduced the ratio of liver/body weight, α-SMA activation, and type I collagen overexpression in CCl₄ treated mice liver. The elevated serum levels, including ALT, AST, and ALP, were also decreased by decursin treatment. Treatment of decursin markedly proved the generation of reactive oxygen species, NAD(P)H oxidase (NOX) protein (1, 2, and 4) upregulation, NOX activity, and superoxide anion production in HSCs by TGF-β1. It also significantly reduced TGF-β1-induced Smad 2/3 phosphorylation, nuclear translocation of Smad 4, and association of Smad 2/3–Smad 4 complex. Consistent with in vitro results, decursin treatment effectively blocked the levels of NOX protein, and Smad 2/3 phosphorylation in injured mice liver.

Significance

Decursin blocked CCl₄-induced liver fibrosis and inhibited TGF-β1-mediated HSC activation in vitro. These data demonstrated that decursin exhibited hepatoprotective effects on experimental fibrosis, potentially by inhibiting the TGF-β1 induced NOX activation and Smad signaling.     

Potential role for Ext1-dependent heparan sulfate in regulating P311 gene expression in A549 carcinoma cells

Background

Exostosin-1 (EXT1), a member of the EXT protein family, is indispensable for synthesis of heparan sulfate (HS) chains that bind to and modulate the signaling efficiency of numerous growth factor activities. We have previously shown that Ext1 mutated mouse embryonic fibroblasts produce short sulfated HS chains which dramatically influence tumor cell behavior in a 3-dimensional (3D) heterospheroid system composed of tumor cells and fibroblasts.

Plasminogen activator inhibitor-1 and tenascin-C secreted by equine mesenchymal stromal cells stimulate dermal fibroblast migration in vitro and contribute to wound healing in vivo

Background

Impaired cutaneous wound healing is common in humans, and treatments are often ineffective. Based on the significant emotional and economic burden of impaired wound healing, innovative therapies are needed. The potential of mesenchymal stromal cell (MSC)–secreted factors to treat cutaneous wounds is an active area of research that is in need of refinement before effective clinical trials can be initiated. The aims of the present study were to (i) study which MSC-secreted factors stimulate dermal fibroblast (DF) migration in vitro and (ii) evaluate the potential of these factors to promote wound healing in vivo.

Endoglin based in vivo near-infrared fluorescence imaging of tumor models in mice using activatable liposomes

Background

Endoglin (CD105) is overexpressed on tumor cells and tumor vasculatures, making it a potential target for diagnostic imaging and therapy of different neoplasms. Therefore, studies on nanocarrier systems designed for endoglin-directed diagnostic and drug delivery purposes would expose the feasibility of targeting endoglin with therapeutics.

Demonstration of the hepatocyte growth factor signaling pathway in the in vitro neuritogenic activity of chondroitin sulfate from ray fish cartilage

Background

Chondroitin sulfate (CS) is a ubiquitous component of the cell surface and extracellular matrix and its sugar backbone consists of repeating disaccharide units: D-glucuronic acid (GlcUA)β1-3N-acetyl-D-galactosamine (GalNAc). Although CS participates in diverse biological processes such as growth factor signaling and the nervous system's development, the mechanism underlying the functions is not well understood.

Methods

CS was isolated from ray fish cartilage, an industrial waste, and its structure and neurite outgrowth-promoting (NOP) activity were analyzed to investigate a potential application to nerve regeneration.

Results

The major disaccharide unit in the CS preparation was GlcUA-GalNAc(6-O-sulfate) (61.9%). Minor proportions of GlcUA-GalNAc(4-O-sulfate) (27.0%), GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate) (8.5%), and GlcUA-GalNAc (2.7%) were also detected. The preparation showed NOP activity in vitro, and this activity was suppressed by antibodies against hepatocyte growth factor (HGF) and its receptor c-Met, suggesting the involvement of the HGF signaling pathway in the expression of the in vitro NOP activity of the CS preparation. The specific binding of HGF to the CS preparation was also demonstrated by surface plasmon resonance spectroscopy.

Conclusions and general significance

The NOP activity of CS from ray cartilage was demonstrated to be expressed through the HGF signaling pathway, suggesting that ray cartilage CS may be useful for studying the cooperative function of CS and HGF.     

Deciphering the role of dimer interface in intrinsic dynamics and allosteric pathways underlying the functional transformation of DNMT3A

Background

DNMT3A, as de novo DNA methyltransferase, is essential for regulating gene expression through cellular development and differentiation. The functions of DNMT3A rely on its oligomeric states and allosteric regulations between its catalytic domain and binding partners. Despite recent resolution of autoinhibitory and active DNMT3A/3L crystal structures, the mechanism of their functional motions and interdomain allostery in regulating the activity remains to be established.