Senescent cell removal via JAG1-NOTCH1 signalling in the epidermis.

Emerging evidence has pointed to the noxious effects of senescent cells in various tissues, and senescent cells in the epidermis are known to accumulate with age. We hypothesized that there is a mechanism by which senescent cells in the epidermis are preferentially removed and that the function of such removal mechanism declines as age increases. In this study, we investigated whether Notch signalling is involved in such senescent cell removal. We found that Notch1 receptor was expressed more highly in p16INK4a-positive senescent cells than in surrounding cells in human epidermis both in young and old subjects. On the other hand, the expression of its ligand JAG1 was decreased in the epidermis of aged subjects. When normal epidermal cells and UVB-irradiated senescent cells were mixed and three-dimensional reconstructed epidermis was developed in vitro, the senescent cells were preferentially removed from the basal layer and located in the upper layer. We also found that the depletion of senescent cells from the basal layer was suppressed by JAG1 knockdown in normal cells or using a Notch signalling inhibitor. From these results, Notch signalling may be involved in senescent cell removal in the epidermis and the age-related decrease of JAG1 expression in the basal layer may lead to accumulation of senescent cells owing to reduced activation of Notch signalling.

UVA causes dysfunction of ETBR and BMPR2 in vascular endothelial cells, resulting in structural abnormalities of the skin capillaries.

BACKGROUND: Capillary structural abnormalities cause skin disorders. Mottled redness, i.e., skin redness unevenness, may appear on the sun-exposed skin, suggesting capillary structural abnormalities, although its mechanism remains unclear. OBJECTIVE: To observe the capillary structures in the sun-exposed skin where skin redness unevenness is likely to occur, and clarify the mechanism of capillary structural abnormalities. METHODS: The tissue structures in the skin with skin redness unevenness were observed by LC-OCT. Subsequently, immunostaining of the sun-exposed skin where skin redness unevenness is often observed, was performed. Vascular endothelial cells were UV-irradiated to analyze the expression and functions of genes involved in the capillary structures and morphogenesis. RESULTS: The skin with skin redness unevenness exhibited scattering of dilated tubular tissue and disturbance of distribution uniformity. Immunostaining of the sun-exposed skin that were more likely to be exposed to UV rays also revealed similarly disorder of capillary structures. In addition, UVA-irradiated vascular endothelial cells exhibited increased expression of ETBR, involved in telangiectasia, decreased expression of BMPR2, involved in the morphogenesis and maintenance of the blood vessels, and reduced migration of the capillaries. CONCLUSION: UV rays alter ETBR and BMPR2 expression in the skin capillaries, and cause partial dilation and decreased migration, resulting in capillary structural abnormalities and causing skin redness unevenness.

Generation of Rat Monoclonal Antibody to Detect Hydrogen Sulfide and Polysulfides in Biological Samples.

Hydrogen sulfide (H2S) is endogenously produced by enzymes and via reactive persulfide/polysulfide degradation; it participates in a variety of biological processes under physiological and pathological conditions. H2S levels in biological fluids, such as plasma and serum, are correlated with the severity of various diseases. Therefore, development of a simple and selective H2S measurement method would be advantageous. This study aimed to generate antibodies specifically recognizing H2S derivatives and develop a colorimetric immunoassay for measuring H2S in biological samples. We used N-ethylmaleimide (NEM) as an H2S detection agent that forms a stable bis-S-adduct (NEM-S-NEM). We also prepared bis-S-heteroadduct with 3-maleimidopropionic acid, which, in conjugation with bovine serum albumin, was to immunize Japanese white rabbits and Wistar rats to enable generation of polyclonal and monoclonal antibodies, respectively. The generated antibodies were evaluated by competitive enzyme-linked immunosorbent assay. We could obtain two stable hybridoma cell lines producing monoclonal antibodies specific for NEM-S-NEM. By immunoassay with the monoclonal antibody, the H2S level in mouse plasma was determined as 0.2 µM, which was identical to the level detected by mass spectrometry. Taken together, these monoclonal antibodies can be a useful tool for a simple and highly selective immunoassay to detect H2S in biological samples.

A liposomal formulation study of 2,7-dichlorodihydrofluorescein for detection of reactive oxygen species.

It is known that reactive oxygen species (ROS) are connected with various diseases, and many studies have examined redox conditions in the body. However, there is a problem with stability of ROS and selectivity of detection probe. In this study, we aimed to stabilize the detection probe, and have tried to encapsulate the probe in polyethyleneglycol (PEG)-modified liposomes. Dichlorodihydrofluorescein (DCDHF) is oxidized to dichlorofluorescein (DCF), a highly fluorescent product, by ROS. We tried liposomalization of DCDHF probes by the Bangham method. However, it was found that DCDHF was oxidized during preparation and converted to DCF. In contrast, when we performed to encapsulate DCDHF in the liposome after the preparation of empty liposome, the stabilized DCDHF encapsulated liposome was successfully proposed by the addition of DCDHF solution by freeze-drying and re-hydration. Furthermore, the encapsulated efficacy of PEG-modified liposomes was higher than unmodified liposomes. This DCDHF liposome was examined for reactivity with hydroxyl radical and peroxynitrite as highly ROS. It was confirmed that DCDHF liposome had a protective effect on the hydroxyl radical, though an effect of the liposomalization of DCDHF was not shown on reactivity of the peroxynitrite. Therefore, it is likely that the liposomalization of DCDHF has selectivity for certain radical species. It is hoped that these results can be applied to novel and simple diagnostics for redox detection of conditions in the body.

A reliable and sensitive immunoassay for the determination of crustacean protein in processed foods.

Among food allergens, crustacea such as shrimps, crabs, and lobsters are a frequent cause of adverse food reactions in allergic patients. The major allergen has been identified as a muscular protein, tropomyosin. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of crustacean protein in processed foods was developed using the sample dilution buffer that is added to porcine tropomyosin. The sandwich ELISA method was highly specific for the Decapoda group, apart from minor cross-reactivities to other crustacea and mollusks. The recovery ranged from 85 to 141%, while the intra- and interassay coefficients of variation were less than 2.8 and 8.4%, respectively.

Feasibility of simple chitosan sheet as drug delivery carrier.

Chitosan, a biodegradable and biocompatible polysaccharide, is a potentially useful material in various fields. We developed a simple chitosan sheet and examined the possibility of using an adriamycin-containing chitosan sheet as a drug carrier for controlled release. To prepare a carrier consisting only of chitosan, a chitosan suspension was subjected to acid-alkaline treatment, mixed with adriamycin, frozen and freeze-dried. The adriamycin-containing chitosan sheet was inserted into the peritoneal cavity of mice in order to investigate its biodegradation. The appearance of decomposition of chitosan was observed using scanning electron microscopy, and adriamycin in urine and liver was detected for 1 and 2 weeks, respectively. Adriamycin metabolites were detected in plasma for 2 weeks. Furthermore, adriamycin remained in the chitosan sheet without being metabolized after 2 months. These results suggested that the chitosan sheet prepared in this study might improve therapeutic efficacy in topical lesions as a carrier of sustained-release drugs.

Changes in the mutagenic and estrogenic activities of bisphenol A upon treatment with nitrite.

Bisphenol A (4,4'isopropylidenediphenol: BPA), an endocrine-disrupting chemical, is contained in food-packaging and can-coating agents as well as in dental sealants. Nitrite is present in vegetables, fish and tap water as an ingredient or contaminant, and also in human saliva. Here, we explored the possible generation of genotoxicity from the reactions of BPA and nitrite under acidic conditions, a situation simulating the stomach. We determined the changes in the mutagenic and estrogenic activities of BPA before and after nitrite treatment. Untreated BPA did not exhibit any mutagenicity. However, the mixture of BPA and sodium nitrite after incubation at pH 3.0 showed strong mutagenic activity toward Salmonella typhimurium strains TA 100 and TA 98 either with or without a metabolic activation system (S9 mix). The clastogenic properties of nitrite-treated and untreated BPA were analyzed by a micronucleus test with male ICR mice. A single gastric intubation of nitrite-treated BPA induced a significantly higher frequency of micronucleated reticulocytes (MNRETs) in mice. The results of analysis of electron spin resonance (ESR) suggest that the expression of the mutagenic activity of nitrite-treated BPA is related to the generation of radicals in the reaction mixture. By applying 1H and 13C NMR, AB-MS and APCI/LC/MS, we identified two compounds 3-nitrobisphenol A and 3,3'-dinitro-bisphenol A. These compounds were synthesized by the reaction of BPA with nitric acid. 3,3'-Dinitro-bisphenol induced a significantly greater frequency of MNRETs in male ICR mice. By applying a green fluorescent protein (GFP)-reporter expression system and an estrogen R(alpha) competitor screening kit, we found that nitrite-treated BPA and 3,3'-dinitro-bisphenol A showed weak estrogenic activity compared to that of untreated BPA.

Protective effect of spin trap agent, N-tert-butyl-alpha-phenylnitrone on hyperoxia-induced oxidative stress and its potential as a nitric oxide donor.

We have previously suggested that the spin trap agent, N-tert-butyl-alpha-phenylnitrone (PBN) can function not only as an antioxidant but also as a nitric oxide (NO) donor. To characterize the pharmacological activities of PBN against oxidative damage, we examined the effect of PBN on NO generation under hyperoxic conditions. The formation of NO in mice exposed to 95% oxygen was determined using a NOx analyzer and electron spin resonance (ESR). Levels of NOx, an oxidative product of NO, increased in the blood of mice under these conditions. However, the increase was returned to a normal level by the NOS (nitric oxide synthase) inhibitor, L-NMMA, indicating that the NO was formed via a biosynthetic pathway. In addition, ESR spectra of the liver and brain of control and experimental mice that were measured using Fe(DETC)2 as an NO trap reagent showed strong ESR signals from NO complexes in the livers of mice exposed to 95% oxygen. When examining the effect of PBN in mice, PBN reduced the NOx formation in the blood under the same hyperoxic conditions. In addition, the ESR intensity of the NO complex was weaker in the PBN-treated mice than in the non-treated mice, showing that PBN possess anti-inflammatory properties. However, under a normal atmosphere, NOx and ESR analyses showed that NO levels increased in PBN-treated mice but not in control mice. These findings suggested that PBN functions as an NO donor under specific physiological conditions. PBN appears to protect against hyperoxia-induced NO toxicity by anti-inflammatory action rather than by serving as an NO donor.

Spin probe study on the interaction of chitosan-derived polymer surfactants with lipid membrane.

Chitosan-derived polymer surfactants, sulfated N-acyl-chitosan (S-Cn-Chitosan), were synthesized and compared with commonly used low-molecular-weight surfactants, sodium dodecyl sulfate (SDS), dodecyltrimethyl ammonium chloride (DTAC), and octaethyleneglycol mono n-dodecyl ether (BL8SY), in their interaction with a lipid membrane using a spin probe method. A suspension of dipalmitoylphosphatidyl-choline (DPPC) spin-labeled strongly (10%) with a spin probe, 1-palmitoyl-2-(12-doxyl) stearoyl-phosphatidylcholine, was mixed with the surfactant solutions. The dissolution time of the DPPC membrane was estimated from the peak height change vs time, which was caused by the decrease in spin-exchange interaction. The times were 2, 4, and 70 s for BL8SY, SDS, and DTAC and 1.2 and 8.8 h for S-C10-Chitosan and S-C14-Chitosan, respectively, showing that the dissolution of the lipid membrane with polymer surfactants was far slower than that with low-molecular-weight surfactants. In addition, the time depended on the length of the alkyl chains of the polymer surfactants. Simulations of the ESR spectra of the DPPC-surfactant systems containing small amounts of surfactants were carried out in order to examine how the membrane structure was changed by the incorporation of surfactant molecules. By this analysis, it was revealed that the rigidity of the membrane was decreased by the addition of low-molecular-weight surfactants in the order of DTAC>SDS>BL8SY, inverse to the order of dissolution times. S-Cn-Chitosan, in contrast, increased the rigidity of the membrane, suggesting that polymer surfactants adhered to the lipid membrane and tightly enfolded the riposome anchoring their alkyl chains.

Single GUV method reveals interaction of tea catechin (-)-epigallocatechin gallate with lipid membranes.

Tea catechins, which are flavonoids and the main components of green tea extracts, are thought to have antibacterial and antioxidant activity. Several studies indicate that lipid membranes are one of the targets of the antibacterial activity of catechins. Studies using a suspension of large unilamellar vesicles (LUVs) indicate that catechin causes gradual leakage of internal contents from LUVs. However, the detailed characteristics of the interaction of catechins with lipid membranes remain unclear. In this study, we investigated the interaction of (-)-epigallocatechin gallate (EGCg), a major catechin in tea extract, with single giant unilamellar vesicles (GUVs) of egg phosphatidylcholine (egg PC) using phase-contrast fluorescence microscopy and the single GUV method. We prepared GUVs of lipid membranes of egg PC in a physiological ion concentration ( approximately 150 mM NaCl) using the polyethylene glycol-lipid method. Low concentrations of EGCg at and above 30 muM induced rapid leakage of a fluorescent probe, calcein, from the inside of single egg PC-GUVs; after the leakage, the GUVs changed into small lumps of lipid membranes. On the other hand, phase-contrast microscopic images revealed the detailed process of the EGCg-induced burst of GUVs, the decrease in their diameter, and their transformation into small lumps. The dependence of the fraction of burst GUVs on EGCg concentration was almost the same as that of the fraction of leaked GUV. This correlation strongly indicates that the leakage of calcein from the inside to the outside of the GUV occurred as a result of the burst of the GUV. The fraction of completely leaked GUV and the fraction of the burst GUV increased with time and also increased with increasing EGCg concentration. We compared the EGCg-induced leakage from single GUVs with EGCg-induced leakage from a LUV suspension. The analysis of the EGCg-induced shape changes shows that the binding of EGCg to the external monolayer of the GUV increases its membrane area, inducing an increase in its surface pressure. Small angle x-ray scattering experiments indicate that the intermembrane distance of multilamellar vesicles of PC membrane greatly decreased at EGCg concentrations above the threshold, suggesting that neighboring membranes came in close contact with each other. On the basis of these results, we discuss the mechanism of the EGCg-induced bursting of vesicles.

Interaction of (+)-catechin with a lipid bilayer studied by the spin probe method.

The interaction of (+)-catechin with a lipid bilayer was examined by the spin probe method. The spin probe, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), was dissolved in an aqueous dipalmitoylphosphatidylcholine (DPPC) dispersion containing (+)-catechin. The temperature dependence of the TEMPO parameter was measured. The increase of this parameter due to pretransition was eliminated by the addition of (+)-catechin, suggesting that it was adsorbed to the lipid membrane surface in the gel state, which hindered the change of the membrane from a flat to wavy structure. In the temperature region of the main transition, the TEMPO parameter increased rapidly, then gradually with increasing temperature, which could be explained by the eutectic phase diagram. The rotational correlation time of a spin probe 16-doxylstearic acid and the order parameter of 5-doxylstearic acid in the aqueous dispersion system of egg yolk phosphatidylcholine revealed that the motion of the alkyl chain in the liquid crystal state was hindered in the center of the membrane as well as near the surface by the adsorption of (+)-catechin.

Stability of membrane potential in heart mitochondria: single mitochondrion imaging.

Mitochondrial membrane potential (delta psi(m)) plays an important role in cellular activity. Although delta psi(m) of intracellular mitochondria are relatively stable, the recent experiments with isolated mitochondria demonstrate that individual mitochondria show frequent fluctuations of delta psi(m). The current study is performed to investigate the factors that stabilize delta psi(m) in cells by observing delta psi(m) of individual isolated mitochondria with fluorescence microscopy. Here, we report that (1) the transient depolarizations are also induced for mitochondria in plasma membrane permeabilized cells, (2) almost all mitochondria isolated from porcine hearts show the transient depolarizations that is enhanced with the net efflux of protons from the matrix to the intermembrane space, and (3) ATP and ADP significantly inhibit the transient depolarizations by plural mechanisms. These results suggest that the suppression of acute alkalinization of the matrix together with the presence of ATP and ADP contributes to the stabilization of delta psi(m) in cells.

Changes in the mutagenic and estrogenic activities of 17beta-estradiol after treatment with nitrite.

We determined the changes in the mutagenic and estrogenic activities of 17beta-estradiol after a nitrite treatment. Nitrite-treated 17beta-estradiol showed mutagenic activities toward Salmonella typhimurium strains TA 100 and TA 98. We confirmed that nitrite-treated 17beta-estradiol generated radicals from the results of an analysis of electron spin resonance. By applying an instrumental analysis, we identified 2-nitro-17beta-estradiol to have been formed in the reaction mixture. 2-Nitro-17beta-estradiol did not exhibit mutagenic activities toward Salmonella typhimurium strains, suggesting that other mutagens might have been formed in the reaction mixture. The clastogenic properties of nitrite-treated 17beta-estradiol and 2-nitro-17beta-estradiol were analyzed by a micronucleus test with male ICR mice. Nitrite-treated 17beta-estradiol and 2-nitro-17beta-estradiol induced a significantly higher frequency of micronucleated reticulocytes in mice. The estrogenic activity of 2-nitro-17beta-estradiol was found to be lower than that of 17beta-estradiol. These data suggest that a daily oral intake of 17beta-estradiol and nitrite might induce the formation of mutagenic compounds in our body.

Synthesis of lipophilic poly-lauroyl-(+)-catechins and radical-scavenging activity.

Lipophilic catechins were synthesized to improve absorption into living bodies and obtain new antioxidants effective in lipid bilayers. The hydroxyl (OH) groups of (+)-catechin was acylated randomly using lauroyl chloride. The mixture was separated by preparative HPLC, and 3-lauroyl-, 3',4'-dilauroyl- and 3,3',4'-trilauroyl-catechins (3-LC, 3',4'-LC, and 3,3',4'-LC) were obtained, their structures being determined by (1)H NMR. Their radical scavenging activity was measured in a ethanol solution using the 1,1-diphenyl-2-picrylhydrazyl radical, and was compared with that of (+)-catechin. The activity of 3-LC was almost same as that of (+)-catechin, but those of 3',4'-LC and 3,3',4'-LC were small, showing that the blocking of phenolic OH groups in the B ring lowered the activity. The scavenging activity on lipophilic radicals in a liposome system was also measured, and the activities were in the order of 3-LC > 3',4'-LC = (+)-catechin. These results suggested that radical scavenging activity in the lipid membrane depended not only on the number and the relative positions of phenolic OH groups of catechins but also on affinity to the membrane.

Nitric oxide and effect of a radical scavenger N-tert-butyl-alpha-phenylnitrone on stroke in a rat model.

To characterize the role of nitric oxide (NO) in stroke, NO was measured using an in vivo microdialysis technique and electron spin resonance spectrometry in malignant stroke-prone spontaneously hypertensive rats (M-SHRSP), stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY). The brain dialysate NO level was higher in SHRSP than in WKY. NO was not detected in M-SHRSP hippocampus microdialysate after stroke except after the administration of N-tert-butyl-alpha-phenylnitrone (PBN). In addition, very little NO was generated in M-SHRSP brain tissue with hemorrhage. These data demonstrate an association between NO and stroke in M-SHRSP. Further, PBN administration results in maintenance of NO levels after stroke in M-SHRSP.

Repetitive transient depolarizations of the inner mitochondrial membrane induced by proton pumping.

Single mitochondria show the spontaneous fluctuations of DeltaPsim. In this study, to examine the mechanism of the fluctuations, we observed DeltaPsim in single isolated heart mitochondria using time-resolved fluorescence microscopy. Addition of malate, succinate, or ascorbate plus TMPD to mitochondria induced polarization of the inner membrane followed by repeated cycles of rapid depolarizations and immediate repolarizations. ADP significantly decreased the frequency of the rapid depolarizations, but the ADP effect was counteracted by oligomycin. On the other hand, the rapid depolarizations did not occur when mitochondria were polarized by the efflux of K(+) from the matrix. The rapid depolarizations became frequent with the increase in the substrate concentration or pH of the buffer. These results suggest that the rapid depolarizations depend on the net translocation of protons from the matrix. The frequency of the rapid depolarizations was not affected by ROS scavengers, Ca(2+), CsA, or BA. In addition, the obvious increase in the permeability of the inner membrane to calcein (MW 623) that was entrapped in the matrix was not observed upon the transient depolarization. The mechanisms of the spontaneous oscillations of DeltaPsim are discussed in relation to the matrix pH and the permeability transitions.

ESR characterization of a novel spin-trapping agent, 15N-labeled N-tert-butyl-alpha-phenylnitrone, as a nitric oxide donor.

We previously found that one of the pharmacological effects of N-tert-butyl-alpha-phenylnitrone (PBN) is the release of nitric oxide (NO) under oxidative conditions. However, to confirm this hypothesis in vivo, NO released from PBN must be distinguished from NO produced in biological systems, and therefore we undertook the synthesis of PBN using labeled 15N to identify its corresponding 15NO in vivo. The properties were examined with an ESR spectrometer. To synthesize 15N-PBN, the starting material, ammonium-15N chloride, was converted to 2-amino-15N-2-methylpropane, oxidized to 2-methyl-2-nitropropane-15N, and finally reacted with benzaldehyde to give 15N-PBN. The final product was purified by repeated sublimation. With ferrous sulfate-methyl glucamine dithiocarbamate complex, Fe (MGD)2, as a trapping agent to measure the NO levels of 15N-PBN or 14N-PBN in vitro, the peak intensity of 15NO[Fe(MGD)2] was over 50% stronger than that of 14NO[Fe(MGD)2], and that 15NO and 14NO had the corresponding two-and three line hyperfine structures due to their nuclear spin quantum numbers. Subsequently, the ESR spectrum of 15NO derived from 15N-PBN was significantly different than that of lipopolysaccharide (LPS)-induced NO, which was derived from biological cells, and therefore we have demonstrated the possibility to distinguish 15NO from PBN and 14NO generated from cells. These results suggested that 15N-PBN is a useful molecule, not only as a spin-trapping agent, but also as an NO donor to explore the pharmacological mechanisms of PBN in vivo.

Detection of 2-deoxy-D-ribose radicals generated by the reaction with the hydroxyl radical using a rapid flow-ESR method.

ESR spectrum of the short-lived radicals derived from 2-deoxy-D-ribose by the reaction with the hydroxyl radical (HO*) was measured using a rapid flow method. A dielectric mixing resonator was used for the measurement, which made it possible to measure the highly sensitive ESR spectra of the radicals with a lifetime of the order of milliseconds. A complex spectrum was obtained and the spectral simulation was done to show that it was the superposition of the signals due to five radicals (I-V). Three of them were those formed by the dehydrogenation with the HO* at C-1 (I), C-3 (II), and C-4 (III) positions of the 2-deoxy-D-ribose molecule. The other two (IV and V) were carbonyl-conjugated radicals formed by the elimination of a water molecule from III and II. The results showed that dehydrogenation occurred randomly at the positions where hydroxyl groups are attached, but the most preferred position was C-3 and the radical position moved from C-3 to C-4 by the elimination of water molecule.

Kinetic analysis of the effect of (-)-epigallocatechin gallate on the DNA scission induced by Fe(II).

The DNA strand scission induced by Fe(II) in a citrate buffer solution and the effect of (-)-epigallocatechin gallate (EGCg) were kinetically analyzed. The rate of consumption of dissolved oxygen by Fe(II) in each of these solutions was measured and paralleled that DNA scission. Coordinated EGCg accelerated these reactions. Curves of the time-course characteristics of DNA scission were simulated by using the rate constant of oxygen consumption and by assuming that scission with the hydroxyl radical (OH), which was formed from the dissolved oxygen, proceeded competitively with the scavenging of OH by citrate, Cl- ions and EGCg added. Free EGCg acted as a DNA scission inhibitor to scavenge OH, in contrast to the case of the coordinated one. This analysis is useful for estimating the rate constant of the reaction between an antioxidant and OH, and might provide a new method for measuring the OH-scavenging activity.