[Role and related mechanisms of LiaSR two-component system in acid tolerance and biofilm formation of Streptococcus mutans].

Objective: To investigate the role and related mechanisms of the LiaSR two-component system in acid tolerance and biofilm formation abilities of Streptococcus mutans (Sm) 593. Methods: The growth curves of various Sm strains in pH=5.5 brian heart infusion (BHI) medium were analyzed. And colony forming unit (CFU) was also performed to evaluate the acid tolerance of Sm. Laurdan probe, H+-K+adenosine triphosphate (ATP)ase activity analysis kit, proton permeability assay and real-time fluorescence quantitative PCR (RT-qPCR) were conducted to detect the acid tolerant mechanisms of LiaSR two-component system in Sm. Crystal violet staining, CFU, SYTOX probe and anthrone-sulfuric method were used to analyze the properties and structures of the Sm biofilms. RT-qPCR was conducted to detect the expression levels of underlying regulated genes. Results: The growth of mutants in acidic BHI were inhibited (P<0.05). The acid tolerance of mutants significantly decreased compared to the wild-type strain (P<0.05). In mutants, the activity of H+-ATPase (917.06±59.53 and 469.53±47.65) were elevated by 7.22-folds and 3.70-folds compared to the wild-type strain (127.00±50.71) (P<0.001, P<0.001) and the encoded gene atpD (3.39±0.21 and 1.94±0.17) were also elevated by 3.39-folds and 1.94-folds compared to the wild-type strain (1.00±0.15) (P<0.001, P=0.001). The Laurdan generalized polarization of mutants (0.18±0.04 and 0.18±0.05) increased significantly compared to the wild-type strain (0.08±0.05) (P=0.006, P=0.003) and the expression levels of fabM gene were decreased in mutants (0.52±0.11 and 0.57±0.05) by 1/2 (P=0.014, P=0.022). In liaR deletion mutant, the reduced terminal pH (4.76±0.01) can also be observed (P<0.001). The total amount of the biofilms of three Sm didn't show significant differences (P>0.05). But the number of viable bacteria of mutants' biofilms were decreased [Sm 593: (12.00±2.80)×107 CFU/ml; Sm ΔliaS: (2.95±1.13)×107 CFU/ml; Sm ΔliaR: (7.25±1.60)×107 CFU/ml] (P=0.001, P=0.024). The extracellular DNA were increased by 18.00-folds and 6.50-folds in mutants' biofilms (128.73±15.65 and 46.38±5.52) compared to the wild-type strain (7.16±3.62) (P<0.001, P=0.003). Water-soluble exopolysaccharides could be found up-regulated in liaS deletion mutant [(138.73±10.12) µg/ml] (P=0.003) along with the expression level of gtfC gene (1.65±0.39) (P=0.014). The expression level of gtfD were elevated by 47.43-folds and 16.90-folds in mutants (P<0.001, P=0.010). Conclusions: The LiaSR two-component system can promote the expression of fabM gene and increase the fluidity of Sm which contributes to acid tolerance. The LiaR can also decrease the proton permeability and restrict the entrance of H+. The LiaSR two-component system can negatively regulate the production of the extracellular matrix in Sm biofilm.

PAD4 inhibitor promotes DNA damage and radiosensitivity of nasopharyngeal carcinoma cells.

Peptidylarginine deiminases 4 (PAD4), a kind of enzyme capable of converting protein arginine or mono-methylarginine into citrulline, has been identified to display a key role in diverse diseases. Radiotherapy is frequently used in nasopharyngeal carcinoma (NPC) treatment and induces DNA double strand breaks. In this study, whether PAD4 inhibitor YW3-56 affects the radiosensitivity of NPC cells was explored. RT-qPCR, immunofluorescence, western blot, clonogenic survival, and flow cytometry assays were used to assess the function of PAD4 and YW3-56 in NPC. We found the upregulation of PAD4 expression in NPC cells. PAD4 overexpression suppressed NPC cell apoptosis and promoted cell cycle, while PAD4 depletion had an opposite result. Moreover, the survival of NPC cells after irradiation was increased by overexpression of PAD4. PAD4 overexpression inhibited DNA damage and sensitivity of NPC cells to irradiation. Functional assays showed that YW3-56 treatment promoted DNA damage, apoptosis, and radiosensitivity of NPC cells. Importantly, YW3-56 treatment inhibited tumor growth in vivo. Overall, this study revealed the efficacy of PAD4 inhibitor YW3-56 in promoting sensitivity of NPC cells to irradiation.

Ultrasmall Molybdenum Disulfide Quantum Dots Cage Alzheimer's Amyloid Beta to Restore Membrane Fluidity.

Alzheimer's disease (AD) is a major cause of dementia characterized by the overexpression of transmembrane amyloid precursor protein and its neurotoxic byproduct amyloid beta (Aß). A small peptide of considerable hydrophobicity, Aß is aggregation prone catalyzed by the presence of cell membranes, among other environmental factors. Accordingly, current AD mitigation strategies often aim at breaking down the Aß-membrane communication, yet no data is available concerning the cohesive interplay of the three key entities of the cell membrane, Aß, and its inhibitor. Using a lipophilic Laurdan dye and confocal fluorescence microscopy, we observed cell membrane perturbation and actin reorganization induced by Aß oligomers but not by Aß monomers or amyloid fibrils. We further revealed recovery of membrane fluidity by ultrasmall MoS2 quantum dots, also shown in this study as a potent inhibitor of Aß amyloid aggregation. Using discrete molecular dynamics simulations, we uncovered the binding of MoS2 and Aß monomers as mediated by hydrophilic interactions between the quantum dots and the peptide N-terminus. In contrast, Aß oligomers and fibrils were surface-coated by the ultrasmall quantum dots in distinct testudo-like, reverse protein-corona formations to prevent their further association with the cell membrane and adverse effects downstream. This study offers a crucial new insight and a viable strategy for regulating the amyloid aggregation and membrane-axis of AD pathology with multifunctional nanomedicine.

ICT based water-soluble fluorescent probe for discriminating mono and dicarbonyl species and analysis in foods.

A unique and highly water-soluble ICT-based fluorescent probe is developed for efficient detection and discrimination of reactive monocarbonyl formaldehyde (FA) from dicarbonyl methylglyoxal (MGO)/glyoxal (GO) by modulating the ICT process, which was confirmed by photophysical and TD-DFT analysis. The probe is applied in cellular imaging and quantifying FA in preserved food and MGO in manuka honey.

Investigation of the Membrane Fluidity Regulation of Fatty Acid Intracellular Distribution by Fluorescence Lifetime Imaging of Novel Polarity Sensitive Fluorescent Derivatives.

Free fatty acids are essential structural components of the cell, and their intracellular distribution and effects on membrane organelles have crucial roles in regulating the metabolism, development, and cell cycle of most cell types. Here we engineered novel fluorescent, polarity-sensitive fatty acid derivatives, with the fatty acid aliphatic chain of increasing length (from 12 to 18 carbons). As in the laurdan probe, the lipophilic acyl tail is connected to the environmentally sensitive dimethylaminonaphthalene moiety. The fluorescence lifetime imaging analysis allowed us to monitor the intracellular distribution of the free fatty acids within the cell, and to simultaneously examine how the fluidity and the microviscosity of the membrane environment influence their localization. Each of these probes can thus be used to investigate the membrane fluidity regulation of the correspondent fatty acid intracellular distribution. We observed that, in PC-12 cells, fluorescent sensitive fatty acid derivatives with increased chain length compartmentalize more preferentially in the fluid regions, characterized by a low microviscosity. Moreover, fatty acid derivatives with the longest chain compartmentalize in lipid droplets and lysosomes with characteristic lifetimes, thus making these probes a promising tool for monitoring lipophagy and related events.

The Molecular Diversity of 1,8-diaminonaphthalene in Organic Chemistry.

BACKGROUND: 1,8-diaminonaphthalen (1,8-DAN) with special organic structure was applied in organic synthesis to provide efficient complex scaffolds, through the two or fourcomponent fashion. This review highlights its recent application in organic reactions under different conditions and heterogynous catalysts to produce various molecules, which were used as medicines, sensors, and dyes. OBJECTIVE: 1,8-diaminonaphthalene (1,8-DAN) is a bicyclic compound with two amino groups which has received much attention in organic chemistry due to their medicinal activities such as antifungal, antimicrobial, antiulcer, antitumor, oxidation dyestuff, hair color, fluorescent, and chemo-sensors. In continuation of our studies, herin, recent application of 1,8-DAN in organic synthesis was reviewed. CONCLUSION: In conclusion, the application of 1,8-DAN 1 in different reactions was reviewd through the various conditions to yield the target compounds with high medicinal activities, and potential for sensitive detection of different ions. The target compounds including 1,8-DAN 1 with various structures were provided such as perimidines, perimidinones, aminonaphthalenes which were produced through different methods as brought for further study in this review.

Highly selective chemosensor for reactive carbonyl species based on simple 1,8-diaminonaphthalene.

Reactive carbonyl species (RCSs) including one carbon formaldehyde (FA) and dicarbonyl compounds such as methylglyoxal (MGO) and glyoxal (GO) are produced during demethylase reactions and various glucose metabolic pathways respectively. Elevation of the RCSs concentrations in cells is due to abnormal DNA damage, glycation adducts with macromolecules that lead to various neurotoxic diseases. Hence, regular monitoring of these RCSs with an easy tool is of utmost interest. However, conventional methods such as chromatography and mass spectrometry for the detection of these species are not so economically viable. These issues were well addressed by the non-invasive reactivity-based fluorescence techniques. However, tedious synthesis, only specific to either mono aldehyde is limited to detect multiple RCSs in physiologies by synthesized fluorophores. An alternative, simple small molecules are widely applied as commercial biomarkers such as terephthalate and 2,3-diaminonaphthalene (NAP) for hydroxy radical (OH·) and nitric oxide (NO) respectively. Herein, we report an analogue of NAP, 1,8-diamino naphthalene (DAN) is an efficient chemosensor for highly sensitive detection of FA, MGO and GO with minimum detection limits of 0.95-3.97 µM. Surprisingly, DAN shows a "turn on" response towards RCSs but remaining silent towards NO which are exactly opposite to commercial probe NAP. Exogenous RCSs imaging in vitro cancerous cells shows the efficacy of the probe and its potential application for RCSs monitoring in cancer cells, generation of toxic byproducts.

Melittin Induces Local Order Changes in Artificial and Biological Membranes as Revealed by Spectral Analysis of Laurdan Fluorescence.

Antimicrobial peptides (AMPs) are a class of molecules widely used in applications on eukaryotic and prokaryotic cells. Independent of the peptide target, all of them need to first pass or interact with the plasma membrane of the cells. In order to have a better image of the peptide action mechanism with respect to the particular features of the membrane it is necessary to better understand the changes induced by AMPs in the membranes. Laurdan, a lipid membrane probe sensitive to polarity changes in the environment, is used in this study for assessing changes induced by melittin, a well-known peptide, both in model and natural lipid membranes. More importantly, we showed that generalized polarization (GP) values are not always efficient or sufficient to properly characterize the changes in the membrane. We proved that a better method to investigate these changes is to use the previously described log-normal deconvolution allowing us to infer other parameters: the difference between the relative areas of elementary peak (ΔSr), and the ratio of elementary peaks areas (Rs). Melittin induced a slight decrease in local membrane fluidity in homogeneous lipid membranes. The addition of cholesterol stabilizes the membrane more in the presence of melittin. An opposite response was observed in the case of heterogeneous lipid membranes in cells, the local order of lipids being diminished. RS proved to be the most sensitive parameter characterizing the local membrane order, allowing us to distinguish among the responses to melittin of both classes of membrane we investigated (liposomes and cellular membranes). Molecular simulation of the melittin pore in homogeneous lipid bilayer suggests that lipids are more closely packed in the proximity of the melittin pore (a smaller area per lipid), supporting the experimental observation.

TAT-Modified Gold Nanoparticles Enhance the Antitumor Activity of PAD4 Inhibitors.

PURPOSE: Histone citrullination by peptidylarginine deiminases 4 (PAD4) regulates the gene expression of tumor suppressor. In our previously study, YW3-56 (356) was developed as a potent PAD4 inhibitor for cancer therapy with novel function in the autophagy pathway. To enhance the antitumor activity, the PAD4 inhibitor 356 was modified by the well-established cationic penetrating peptide RKKRRQRRR (peptide TAT) and gold nanoparticles to obtain 356-TAT-AuNPs which could enhance the permeability of chemical drug in solid tumor. METHODS: 356-TAT-AuNPs were prepared, and their morphology were characterized. The antitumor activity of 356-TAT-AuNPs was evaluated in vitro and in vivo. RESULTS: 356-TAT-AuNPs exhibited higher anticancer activity against HCT-116, MCF-7 and A549 cells than 356 and 356-AuNPs. Compared with 356 and 356-AuNPs, 356-TAT-AuNPs entered the cytoplasm and nuclear, exhibited stronger anticancer activity by increasing apoptosis, inducing autophagy and inhibiting of histone H3 citrullination, and in HCT-116 xenograft mouse model, 356-TAT-AuNPs could improve the antitumor activity. CONCLUSION: The modified AuNPs with peptide TAT as drug delivery system are potent in delaying tumor growth and could be a powerful vehicle for profitable anticancer drug development. We believe that peptide TAT modification strategy may provide a simple and valuable method for improving antitumor activity of PAD4 inhibitors for clinical use.

An experimental system for real-time fluorescence recordings of cell membrane changes induced by electroporation.

The electroporation of cells is nowadays used for a large variety of purposes, from basic research to cancer therapy and food processing. Understanding molecular mechanisms of the main processes involved in electroporation is thus of significant interest. In the present work, we propose an experimental system to record in real time the evolution of any cell parameter which can be evaluated by fluorescence (before, during and after application of the electroporation pulses to cells in suspension). The system is based on the design of adequate electroporation electrodes, compatible with a standard spectrofluorometer cuvette housing. The electric field intensity generated when pulses are applied was carefully characterized for different geometries of the electrodes, to choose a construction ensuring the greatest homogeneity of the field in combination with the best possible illumination of the sample. As an example of the method's application, we present here generalized polarization kinetics for a varying number of electroporation pulses applied to a cell suspension; the general polarization parameter is strongly correlated to water presence in the hydrophobic membrane core. The system may be used for many other fluorescence measurements useful for the characterization of the electroporation process.

A Fluorescent Kinase Inhibitor that Exhibits Diagnostic Changes in Emission upon Binding.

The development of a fluorescent LCK inhibitor that exhibits favourable solvatochromic properties upon binding the kinase is described. Fluorescent properties were realised through the inclusion of a prodan-derived fluorophore into the pharmacophore of an ATP-competitive kinase inhibitor. Fluorescence titration experiments demonstrate the solvatochromic properties of the inhibitor, in which dramatic increase in emission intensity and hypsochromic shift in emission maxima are clearly observed upon binding LCK. Microscopy experiments in cellular contexts together with flow cytometry show that the fluorescence intensity of the inhibitor correlates with the LCK concentration. Furthermore, multiphoton microscopy experiments demonstrate both the rapid cellular uptake of the inhibitor and that the two-photon cross section of the inhibitor is amenable for excitation at 700 nm.

Solvation properties of raft-like model membranes.

Dimethyl sulfoxide (DMSO) is a universal water-soluble solvent widely used in many biotechnological and medical applications, such as cells cryopreservation, and for the treatment of different human diseases (e.g. amyloidosis). Despite the great number of reported studies, the effects of DMSO on the physico-chemical properties of biological membranes are poorly understood. Often, these studies are limited to model membranes composed of phosphatidylcholines (PCs) and cholesterol (Chol). In this work, we explored the effect of DMSO on liposomes composed of the natural egg sphingomyelin (ESM) and Chol as raft-like model membranes. With a multi-technique approach we probe the structure and the thermal stability of ESM/Chol bilayer at different Chol mole fractions. In particular, we investigate the ESM-solvent interactions to clarify the role of DMSO in perturbing the solvating conditions of lipid vesicles and show that the addition of DMSO increases the thermal stability of vesicles. An increase of transition temperature, a decrease of both enthalpy and entropy as well as a decrease of the cooperativity of the gel to liquid phase transition are observed at 0.1 DMSO mole fraction. Fluorescence experiments with the probe Laurdan and FTIR spectra strongly indicate that DMSO exerts a dehydration effect on the membrane. Besides, FTIR measurements with tungsten hexacarbonyl, in combination with fluorescence data of the probe NBD-PE, indicate that DMSO promotes the formation of a highly packed membrane by reducing the thickness of the membrane.

Effects of artepillin C on model membranes displaying liquid immiscibility

It has been hypothesized that the therapeutic effects of artepillin C, a natural compound derived from Brazilian green propolis, are likely related to its partition in the lipid bilayer component of biological membranes. To test this hypothesis, we investigated the effects of the major compound of green propolis, artepillin C, on model membranes (small and giant unilamelar vesicles) composed of ternary lipid mixtures containing cholesterol, which display liquid-ordered (lo) and liquid-disordered (ld) phase coexistence. Specifically, we explored potential changes in relevant membrane parameters upon addition of artepillin C presenting both neutral and deprotonated states by means of small angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and confocal and multiphoton excitation fluorescence microscopy. Thermotropic analysis obtained from DSC experiments indicated a loss in the lipid cooperativity of lo phase at equilibrium conditions, while at similar conditions spontaneous formation of unilamellar vesicles from SAXS experiments showed that deprotonated artepillin C preferentially located at the surface of the membrane. Time-resolved experiments using fluorescence microscopy showed that at doses above 100 µM, artepillin C in its neutral state interacted with both liquid-ordered and liquid-disordered phases, inducing curvature stress and promoting dehydration at the membrane interface.

Phospholipid components of the synthetic pulmonary surfactant CHF5633 probed by fluorescence spectroscopy.

CHF5633 (Chiesi Farmaceutici, Italy) is a synthetic pulmonary surfactant currently under clinical development for the treatment of Respiratory Distress Syndrome in premature infants. The product is composed of phospholipids in liposomal organization, together with two peptide analogues of human surfactant proteins B and C. Phospholipids in liposomes can undergo oxidation of unsaturated lipids and hydrolysis, with formation of fatty acids and lysolipids, both affecting the physico-chemical properties of the formulation. We exploited two fluorescence probes, Prodan and ADIFAB, to evaluate the stability of the phospholipid components of CHF5633. While Prodan enters the phospholipid bilayer and probes the polarity of this environment, ADIFAB binds free fatty acids in the aqueous phase, allowing to determine their concentration. Changes of Prodan fluorescence emission indicated an increase in the polarity of the phospholipid bilayer as a function of time. This behavior is coupled with an increase in fatty acids concentration in the aqueous phase, as determined by ADIFAB, and an increase in lysolipids concentration, as determined by HPLC-MS. Prodan and ADIFAB resulted efficient probes to monitor phospholipids hydrolysis in liposomes, reporting an increased stability of CHF5633 at pH values higher than 6.5.

Gel-Phase-like Ordered Membrane Properties Observed in Dispersed Oleic Acid/1-Oleoylglycerol Self-Assemblies: Systematic Characterization Using Raman Spectroscopy and a Laurdan Fluorescent Probe.

Aqueous dispersions of oleic acid (OA) and those modified with 1-oleoylglycerol (monoolein, MO) form various kinds of self-assembled structures: micelles, vesicles, oil-in-water (O/W) emulsions, hexagonal phases, and dispersed cubic phases. Conventionally, these self-assembled structures have been characterized using cryogenic transmission electron microscopy or X-ray diffraction spectroscopy. However, these methodologies require specialized treatment before they can be used, which may lead to the self-assemblies not adopting their true equilibrium state. Herein, we systematically characterized the self-assemblies composed of OA and MO in aqueous solution using Raman spectroscopy and fluorescent probe 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan). The OA/MO dispersions at pH 5.0 showed increased chain packing in comparison to the OA micelle at pH 11 or OA vesicle at pH 9.0, which were characterized by the intensity ratio of the Raman peaks at 2850 and 2890 cm-1, R = I2890/I2850. In the Laurdan fluorescence measurements, the obtained spectra were deconvoluted to two peak fractions (A1: λem= 490 nm; A2: λem = 440 nm), and the peak area ratio, A1/(A1 + A2), was defined as the membrane hydrophilicity Øm. The Øm value of the OA/MO dispersion at pH 5.0 was similar to that of the OA O/W emulsion, indicating that the membrane surfaces of these self-assemblies were relatively dehydrated compared to the OA micelle or OA vesicle. To categorize the type of self-assembly dispersion, a Cartesian diagram plot was systematically drawn: R on the x axis and Øm on the y axis, with the cross point at x = 1, y = 0.5. By comparing the membrane properties of the OA-based micelles, O/W emulsions, and dispersed cubic phases, we determined that the OA/MO dispersion at pH 5.0 possessed higher chain packing (R > 1) and a dehydrated membrane surface (Øm < 0.5), which is similar to that of the ordered membranes in gel phases. This characterization method can be useful in evaluating the ordered membrane properties in dispersed self-assemblies in aqueous media.

N-Phenyl-2-naphthylamine as a Novel MALDI Matrix for Analysis and in Situ Imaging of Small Molecules.

Due to its strong ultraviolet absorption, low background interference in the small molecular range, and salt tolerance capacity, N-phenyl-2-naphthylamine (PNA) was developed as a novel matrix in the present study for analysis and imaging of small molecules by matrix-assisted laser desorption/ionization mass spectrometry time-of-fight (MALDI-TOF MS). The newly developed matrix displayed good performance in analysis of a wide range of small-molecule metabolites including free fatty acids, amino acids, peptides, antioxidants, and phospholipids. In addition, PNA-assisted LDI MS imaging of small molecules in brain tissue of rats subjected to middle cerebral artery occlusion (MCAO) revealed unique distributions and changes of 89 small-molecule metabolites including amino acids, antioxidants, free fatty acids, phospholipids, and sphingolipids in brain tissue 24 h postsurgery. Fifty-nine of the altered metabolites were identified, and all the changed metabolites were subject to relative quantitation and statistical analysis. The newly developed matrix has great potential application in the field of biomedical research.

Quantification of N-phenyl-2-naphthylamine by gas chromatography and isotope-dilution mass spectrometry and its percutaneous absorption ex vivo under workplace conditions.

N-Phenyl-2-naphthylamine (P2NA) is an antioxidant used to protect rubbers from flex-cracking. P2NA can be converted in vivo to 2NA, one of the most potent bladder carcinogens. Here, we report the specific and ultra-sensitive quantification of P2NA in the receptor fluid of Franz diffusion cells by gas chromatography and isotope-dilution tandem-mass spectroscopy (GC-MS/MS). The experimental conditions were optimized to minimize losses of P2NA due to surface absorption on glass, plastic, and rubber material, and subsequently validated. Static and dynamic diffusion cell conditions were used to study the percutaneous penetration of P2NA into freshly prepared porcine skin. The experimental settings closely resembled those of the printing industry in the 1960s/1970s in Germany where P2NA-containing solutions in dichloromethane have been used. P2NA penetrated the skin at very low levels (0.02 ± 0.01 µg/cm2/h) with a cumulative penetrated amount of 0.80 ± 0.26 µg/cm2, a lag time of 6.33 ± 2.21 h and under dynamic conditions. Compared to the receptor fluid, 10-40-fold higher concentrations were found in the skin, predominantly in the dermis and the stratum corneum. Dichloromethane acted as a penetration enhancer by increasing the cumulative penetrated amounts and the recovery of P2NA in both the receptor fluid and the skin, while shortening its lag time. However, the flux remained unaffected. Due to its accumulation in subcutaneous layers, we finally proved that P2NA is continuously released into the receptor fluid despite exposure cessation up to 160 h. Overall, the results show that close attention has to be paid to dermal absorption of P2NA in exposed workers.

Suppressive effects of RXR agonist PA024 on adrenal CYP11B2 expression, aldosterone secretion and blood pressure.

The effects of retinoids on adrenal aldosterone synthase gene (CYP11B2) expression and aldosterone secretion are still unknown. We therefore examined the effects of nuclear retinoid X receptor (RXR) pan-agonist PA024 on CYP11B2 expression, aldosterone secretion and blood pressure, to elucidate its potential as a novel anti-hypertensive drug. We demonstrated that PA024 significantly suppressed angiotensin II (Ang II)-induced CYP11B2 mRNA expression, promoter activity and aldosterone secretion in human adrenocortical H295R cells. Human CYP11B2 promoter functional analyses using its deletion and point mutants indicated that the suppression of CYP11B2 promoter activity by PA024 was in the region from -1521 (full length) to -106 including the NBRE-1 and the Ad5 elements, and the Ad5 element may be mainly involved in the PA024-mediated suppression. PA024 also significantly suppressed the Ang II-induced mRNA expression of transcription factors NURR1 and NGFIB that bind to and activate the Ad5 element. NURR1 overexpression demonstrated that the decrease of NURR1 expression may contribute to the PA024-mediated suppression of CYP11B2 transcription. PA024 also suppressed the Ang II-induced mRNA expression of StAR, HSD3ß2 and CYP21A2, a steroidogenic enzyme group involved in aldosterone biosynthesis. Additionally, the PA024-mediated CYP11B2 transcription suppression was shown to be exerted via RXRα. Moreover, the combination of PPARγ agonist pioglitazone and PA024 caused synergistic suppressive effects on CYP11B2 mRNA expression. Finally, PA024 treatment significantly lowered both the systolic and diastolic blood pressure in Tsukuba hypertensive mice (hRN8-12 x hAG2-5). Thus, RXR pan-agonist PA024 may be a candidate anti-hypertensive drugs that acts via the suppression of aldosterone synthesis and secretion.

Dermal penetration and resorption of beta-naphthylamine and N-phenyl-beta-naphthylamine from lubricants in an ex vivo human skin model.

Dermal Penetration of aromatic amines (AA's), often suspected or known to be carcinogenic, can play an important role in the overall human exposure. However, information on penetration of certain AA's is poor and inconsistent. Penetration of the former lubricant additive N-phenyl-beta-naphthylamine (PBNA) and its contaminant beta-naphthylamine (BNA) a known carcinogen was investigated and the influence of formulation and co-application characterized. Percutaneous penetration of BNA and PBNA through freshly excised human skin (n = 8; 48 h) was investigated using an ex vivo diffusion cell model. Both AA's were applied in a technical-conform lubricant or dissolved in hexane. The amount of BNA and PBNA applied to skin was 0.52 and 259 µg/0.64 cm2. The analytical determination of AA's was performed by GC-MS. Both, BNA and PBNA penetrated through human skin (38 vs. 5% of applied dose). In contrast to BNA, the percutaneous penetration of PBNA continued beyond the end of exposure. Co-exposure of both AA's increased the intradermal uptake of BNA and PBNA (p < 0.05). Exposure in lubricant showed the least overall penetration (2.9 and 1.9% of applied dose). The results clearly reveal that dermal penetration of both AA's depends strongly on the mode of application. Co-application and formulation alters the penetration of the AA's.

Effect of retinoic acid on midkine gene expression in rat anterior pituitary cells.

Retinoic acid (RA) is converted from retinal by retinaldehyde dehydrogenases (RALDHs) and is an essential signaling molecule in embryonic and adult tissue. We previously reported that RALDH1 was produced in the rat anterior pituitary gland and hypothesized that RA was generated in the gland. Midkine (MK) is an RA-inducible growth factor, and MK production in the rat anterior pituitary gland was recently reported. However, the mechanism that regulates gene expression of MK in the pituitary gland has not been determined. To investigate regulation of MK production in the anterior pituitary gland, we analyzed changes in MK mRNA in cultured rat anterior pituitary cells. We identified MK-expressing cells by double-staining with in situ hybridization and immunohistochemical techniques for RALDH1. MK mRNA was expressed in RALDH1-producing cells in the anterior pituitary gland. Using isolated anterior pituitary cells of rats, we examined the effect of RA on gene expression of MK. Quantitative real-time PCR revealed that 72 h exposure to a concentration of 10-6 M of retinal and all-trans retinoic acid increased MK mRNA levels by about 2-fold. Moreover, the stimulatory effect of all-trans retinoic acid was mimicked by the RA receptor agonist Am80. This is the first report to show that RA is important in regulating MK expression in rat anterior pituitary gland.