Photoinduced Generation of the π-Conjugated Zwitterionic State in the ESIPT Fluorophore of 2,4-Bisimidazolylphenol.

We demonstrate that 2,4-bis(4,5-diphenyl-1H-imidazol-2-yl)phenol (2,4-bImP) undergoes photoinduced conversion into the so-called "π-conjugated zwitterion" after causing an excited-state intramolecular proton transfer (ESIPT) reaction. The powder sample of 2,4-bImP exhibits largely Stokes-shifted fluorescence characteristics to ESIPT fluorophores. On the other hand, its originally colorless solutions become colored when exposed to UV light for several minutes, whose color depends on the type of solvent. In particular, the CHCl3 solution rapidly turns dark green with the absorption maximum around 700 nm, and the colored solution is nearly restored to original by alternating addition of acid and base. To explain such drastic and reversible color changes, we hypothesized that the occurrence of ESIPT (i.e., deprotonation of the phenol and protonation of the imidazolyl group at its 2-position) triggered the charge-separated structure between the negatively charged phenolate and the positively charged imidazoliumyl group at its 4-position, which allowed resonance with the neutral p-quinoid structure. The formation of this π-conjugated zwitterion was strongly supported by the results of 1H and 15N NMR and Raman measurements.

Low-Cost Soil Moisture Profile Probe Using Thin-Film Capacitors and a Capacitive Touch Sensor.

Soil moisture is an important property for agriculture, but currently commercialized soil moisture sensors are too expensive for many farmers. The objective of this study is to develop a low-cost soil moisture sensor using capacitors on a film substrate and a capacitive touch integrated circuit. The performance of the sensor was evaluated in two field experiments: a grape field and a mizuna greenhouse field. The developed sensor captured dynamic changes in soil moisture at 10, 20, and 30 cm depth, with a period of 10-14 days required after sensor installation for the contact between capacitors and soil to settle down. The measured soil moisture showed the influence of individual sensor differences, and the influence masked minor differences of less than 0.05 m³·m(-3) in the soil moisture at different locations. However, the developed sensor could detect large differences of more than 0.05 m³·m(-3), as well as the different magnitude of changes, in soil moisture. The price of the developed sensor was reduced to 300 U.S. dollars and can be reduced even more by further improvements suggested in this study and by mass production. Therefore, the developed sensor will be made more affordable to farmers as it requires low financial investment, and it can be utilized for decision-making in irrigation.

Reconstruction of a nonfunctional trabeculectomy bleb using an amniotic membrane-wrapped silicone sponge to treat refractory glaucoma.

BACKGROUND: This study was conducted to verify the usefulness of nonfunctional trabeculectomy bleb reconstruction using a silicone sponge wrapped with amniotic membrane. Its purpose was to allow aqueous humor to flow from the flap to the posterior orbital space. METHODS: Seven consecutive patients who had undergone two or more surgeries in one eye for refractory glaucoma followed by our operation were included in this study. Conjunctival adhesion to the sclera was detached with a limbus-based conjunctival incision, followed by reopening the former trabeculectomy flap. A 1.5 × 12 mm silicone sponge used for retinal detachment surgery was wrapped three to four times with amniotic membrane, placed longitudinally on the sclera, and fixed with 10-0 nylon sutures. The anterior end of the amniotic membrane was fixed underneath the scleral flap with sutures, and the conjunctival wound was closed. We periodically checked the intraocular pressure (IOP) and for complications. Follow-up periods ranged from 15 to 30 months (average 19.4 months). Surgical success was defined as a final IOP of ≤ 21 mmHg with or without additional treatment. We defined failure as an IOP of > 21 mmHg on the second of two consecutive visits after the first 4 weeks, or the need for additional glaucoma surgery. RESULTS: Surgery was successful in five of the seven eyes, although bleb needling was performed in two eyes and amniotic membrane patch covering for early aqueous leakage was needed in one eye. In four of the five successful eyes, IOP was well controlled for longer than the period between the previous and present surgeries. One of the unsuccessful eyes, with neovascular glaucoma, had high IOP with hyphema followed by phthisis of the eyeball. The other, with aqueous leakage via the conjunctival wound, required trabeculectomy in a different area. There were no other complications. CONCLUSIONS: Reconstruction of the nonfunctional trabeculectomy bleb using a silicone sponge wrapped with amniotic membrane can be a useful strategy for treating refractory glaucoma.

Synthesis of tertiary alkylphosphonate oligonucleotides through light-driven radical-polar crossover reactions.

Chemical modification of nucleotides can improve the metabolic stability and target specificity of oligonucleotide therapeutics, and alkylphosphonates have been employed as charge-neutral replacements for naturally-occurring phosphodiester backbones in these compounds. However, at present, the alkyl moieties that can be attached to phosphorus atoms in these compounds are limited to methyl groups or primary/secondary alkyls, and such alkylphosphonate moieties can degrade during oligonucleotide synthesis. The present work demonstrates the tertiary alkylation of the phosphorus atoms of phosphites bearing two 2'-deoxynuclosides. This process utilizes a carbocation generated via a light-driven radical-polar crossover mechanism. This protocol provides tertiary alkylphosphonate structures that are difficult to synthesize using existing methods. The conversion of these species to oligonucleotides having charge-neutral alkylphosphonate linkages through a phosphoramidite-based approach was also confirmed in this study.

[Epidemiological analysis of Streptococcus pneumoniae in Gifu prefecture and the northern district of Aichi prefecture--2009].

High pathogenicity and drug resistance of Streptococcus pneumoniae are serious problem in clinical practice. Since 1999, we have conducted epidemiologic analyses of S. pneumoniae in Chubu district. We report the results of the analysis conducted in 2009. Three hundred and eight (308) S. pneumoniae isolates with a gene coding for autolysin lyt-A, which had been isolated from patients at 21 medical institutions in Gifu prefecture and the northern part of Aichi prefecture in 2009, were enrolled in this study. The strains were classified according to their drug resistance based on the presence of the pbp mutation, and examined for the presence of the two macrolide-resistance genes, ermB and mefA. Moreover, they were serotyped using type-specific antisera. The mean age of the patients from whom these S. pneumoniae strains were isolated, was 23.4 +/- 30.1 years old, and children aged 15 years old or less accounted for 66% of all the patients. Genotype penicillin-susceptible S. pneumoniae (gPSSP), genotype penicillin-intermediate S. pneumoniae (gPISP) and genotype penicillin-resistant S. pneumoniae (gPRSP) were 22 (7.1%), 131 (42.5%) and 155 (50.3%), respectively. The strains with mefA positive and ermB negative, mefA negative and ermB positive, and mefA positive and ermB positive were 80 (26.0%), 153 (49.7%), and 47 (15.3%), respectively. The MIC90 values of tebipenem (TBPM) and faropenem were 0.06 microg/mL and 0.5 microg/mL, respectively. TBPM showed the high bactericidal activity against gPRSP. In carbapenems, panipenem and biapenem exhibited higher bactericidal activities. Quinolone-resistant S. pneumoniae (QRSP) were isolated from 10 (3.2%). QRSP dominated 5 (7.9%) and 3 (1.5%) among the elderly (over 65 years old) and children, respectively. (As for the serotype, serotypes 6, 19 and 23 were 60 (19.5%), 62 (20.1%), and 44 (14.3%), respectively. Further epidemiologic studies on S. pneumoniae might be required also in the future, including the relationship between the serotype and drug resistance.

Ets regulates peroxiredoxin1 and 5 expressions through their interaction with the high-mobility group protein B1.

Peroxiredoxins (Prdxs) are thiol-specific antioxidant proteins that are highly expressed in human cancer cells. Prdxs have been shown to be involved in tumor cell proliferation under conditions of microenvironmental stress such as hypoxia. We hypothesized that Prdxs could be categorized into two groups, stress-inducible and non-inducible ones. In this study, we analyzed the promoter activity and expression levels of five Prdx family members in human cancer cells. We found that both Prdx1 and Prdx5 are inducible after treatment with hydrogen peroxide or hypoxia, but that Prdx2, Prdx3, and Prdx4 are not or are only marginally inducible. We also found that Ets transcription factors are the key activators for stress-inducible expression. High-mobility group protein HMGB1 was shown to function as a coactivator through direct interactions with Ets transcription factors. The DNA binding of Ets transcription factors was significantly enhanced by HMGB1. Silencing of Ets1, Ets2, Prdx1, and Prdx5 expression sensitized cells to oxidative stress. These data indicate that transcription of Prdx genes mediated by Ets/HMG proteins might protect cells from oxidative stress.

p73gamma transactivates the p21 promoter through preferential interaction with the p300/CBP-associated factor in human prostate cancer cells.

Several p73 variants have been reported with different carboxy-terminal structures and transcriptional activities. We showed that p73gamma had stronger transactivation activity than the other splicing variants such as alpha, beta and delta by analysing p21 promoter activity in human prostate cancer PC3 cells. The transactivation activity of p73gamma was similar to that of p53 and was enhanced by co-transfection with p300/CBP-associated factor (PCAF). In vitro pull-down assay, p73 variants were able to bind to PCAF with a similar extent. However, in vivo co-immunoprecipitation assays showed that p73gamma interacted preferentially with PCAF. Neither in vitro-translated nor in vivo-immunoprecipitated p73gamma were able to bind to oligonucleotides containing the p53 consensus binding site. However, p73gamma acetylated by PCAF restored DNA binding activity. Differential functions of p73 variants are supposed to be regulated by the structural differences of carboxy-terminal region. Our results revealed that p21 promoter activity was affected by differential interactions of p73 variants with PCAF and its acetylation.

An Obtuse-angled Corner Unit for Fluctuating Carbon Nanohoops.

The invention of corner units was the key factor that allowed the synthesis of cyclo-para-phenylenes with strained curved π-systems. Despite only a few scarce instances of the development of corner units to date, a variety of structural congeners have been synthesized. These preceding corner units commonly possessed directing angles of ≤90°, which enabled the macrocyclization of multiple units, up to six. In this study, we introduce an obtuse-angled corner unit for the synthesis of cyclo-para-phenylene congeners. The corner unit with oxanorbornadiene possessed a directing angle of 126° and thus allowed for the macrocyclization of larger structures with up to seven units. Reductive aromatization was applicable to complete the cyclo-para-phenylene structures and afforded the congeners with multiple anthracenylene panels. Structural studies with experimental and theoretical methods revealed a fluctuating structure with an intrinsic non-belt shape.

The effect of the leukotriene antagonist pranlukast on pediatric acute otitis media.

OBJECTIVE: Conventional treatment for acute otitis media mainly targets bacteria with antibiotics, neglecting to control for mediators of inflammation. Mediators of inflammation, such as leukotrienes, have been identified in patients with acute otitis media (AOM) or subsequent secretory otitis media (SOM). They can cause functional eustachian tube dysfunction or increase mucous in the middle ear, causing persistent SOM following AOM. The objective of the present study was to evaluate whether or not administration of pranlukast, a widely used leukotriene C4, D4, and E4 antagonist, together with antibiotics could inhibit the progression to SOM. METHODS: Children with AOM, who were from two to 12 years old, were randomly divided into two groups as follows: a control group in which 50 patients received antibiotic-based conventional treatment according to guidelines for treating AOM proposed by the Japan Otological Society (version 2006); and a pranlukast group, in which 52 patients were administered pranlukast for up to 28 days as well as given conventional treatment. Cases were regarded as persistent SOM when a tympanogram was type B or C2 four weeks after treatment was initiated. RESULTS: Two patients in the pranlukast group and 3 patients in the control group were excluded because they relapsed AOM within 28 days after initial treatment. Therefore, the analysis included 50 and 47 subjects in the pranlukast and control groups, respectively. The percentage of patients diagnosed with persistent SOM (22.0%) was significantly smaller in the pranlukast group compared with the control group (44.7%) (p = 0.018, chi-squared test). CONCLUSION: The results indicate that combined treatment of AOM with antibiotics and a leukotriene antagonist to control inflammation is useful for preventing progression to persistent SOM.

Transcription factors and drug resistance.

Intrinsic or acquired resistance to anticancer agents is a major obstacle to the success of chemotherapy. Anticancer agents are known to modulate signal transduction pathways and alter expression of genes that play an important role in drug resistance. Emerging evidence suggests that the complexity of genomic response against anticancer agents arise from elaborate gene expression by multiple transcription factors. Here, we briefly describe the development of solid tumours and the appearance of drug-resistant cells. We also review what is known of the transcription factors that are involved in resistance to drugs, particularly cisplatin.

Quercetin induces the expression of peroxiredoxins 3 and 5 via the Nrf2/NRF1 transcription pathway.

PURPOSE: The flavonoids have potent antioxidant and free-radical scavenging properties and are beneficial in the prevention and treatment of ocular diseases including glaucoma. The authors have previously reported that antiglaucoma agents could transcriptionally activate the antioxidant protein peroxiredoxin (PRDX)2. The purpose of this study was to investigate whether quercetin can activate transcription factors and induce the expression of the PRDX family. METHODS: To demonstrate whether quercetin can transcriptionally induce the expression of the PRDX family, trabecular meshwork cells were treated with quercetin, and PRDX expression and transcription factors were both investigated by Western blot analysis, reporter assays, and siRNA strategies. Subsequently, cellular sensitivity to oxidative stress was determined. RESULTS: Expression of the PRDX3 and PRDX5 genes was induced by quercetin in a time- and dose-dependent manner. NRF1 transactivates the promoter activity of both PRDX3 and PRDX5 but not PRDX2 and PRDX4. Quercetin can also induce the expression of Nrf2 and NRF1 but not of Ets1, Ets2, or Foxo3a. Knockdown of NRF1 expression significantly reduced the expression of both PRDX3 and PRDX5. Reporter assays showed that NRF1 transactivated the promoter activity of both PRDX3 and PRDX5 and that the downregulation of NRF1 with siRNA repressed the promoter activity of both PRDX3 and PRDX5. Furthermore, the downregulation of NRF1, PRDX3, and PRDX5 renders trabecular meshwork cells sensitive to hydrogen peroxide. Finally, NRF1 activation by quercetin was completely abolished by the knockdown of Nrf2. CONCLUSIONS: Quercetin upregulates the antioxidant peroxiredoxins through the activation of the Nrf2/NRF1 transcription pathway and protects against oxidative stress-induced ocular disease.

Transcriptional regulation of activating transcription factor 4 under oxidative stress in retinal pigment epithelial ARPE-19/HPV-16 cells.

PURPOSE: Oxidative stress plays an important role in the pathogenesis of various ocular diseases such as retinopathy, glaucoma, and age-related macular degeneration. Activating transcription factor 4 (ATF4) is induced by various stressors, including endoplasmic reticulum (ER) and oxidative stress, and ATF4 expression is regulated translationally through the PERK pathway of eIF2α phosphorylation. Transcriptional regulation of the ATF4 gene under oxidative stress was investigated in human papillomavirus 16 (HPV-16)-transformed retinal pigment epithelial ARPE-19/HPV-16 cells. METHODS: Retinal pigment epithelial cells, trabecular meshwork cells, and corneal endothelial cells were treated with anoxia and thapsigargin (TG). Gene expression of ATF4 and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and transcription factors was investigated by Western blot analysis, reporter assays, chromatin immunoprecipitation (ChIP) assays, and small interfering (si)RNA strategies. Cellular sensitivity to oxidative stress was determined. RESULTS: The expression of two transcriptional factors, ATF4 and Nrf2, was significantly induced by anoxia and TG. The Nrf2 regulator Keap1 was downregulated by anoxia. Downregulation of Nrf2 abolished ATF4 expression. On the other hand, downregulation of Keap1 enhanced the expression of both Nrf2 and ATF4. The promoter activity of ATF4 was transactivated by the co-transfection of Nrf2 expression plasmids and reduced by the transfection of Nrf2-specific siRNA. The ChIP assays demonstrated that Nrf2 bound to the promoter of the ATF4 gene. Nrf2 downregulation nearly abolished the ATF4 induction by anoxia and TG. Consistent with these findings, the promoter activity of ATF4 was augmented by treatment with TG, HCA, H(2)O(2), and anoxia. However, stress induction of ATF4 promoter activity was observed, even when a mutation was introduced into the antioxidant-responsive elements site. Furthermore, stress induction of the ATF4 promoter was completely abolished when the 5' untranslated region of the ATF4 gene was deleted. Downregulation of ATF4 rendered ARPE-19/HPV-16 cells sensitive to oxidative stress. CONCLUSIONS: These results suggest that the stress induction of ATF4 is significantly regulated transcriptionally through a Nrf2-dependent mechanism and may be a double-edged sword in the pathogenesis of various retinopathies.

Nipradilol and timolol induce Foxo3a and peroxiredoxin 2 expression and protect trabecular meshwork cells from oxidative stress.

PURPOSE: Oxidative stress plays an important role in pathogenesis of glaucoma. The purpose of this study is to investigate the novel effect of antiglaucoma drugs on the expression of antioxidant peroxiredoxins of trabecular meshwork (TM) cells. METHODS: The expression of the peroxiredoxin family was investigated using immortalized TM cell lines. Cells were treated with antiglaucoma drugs and analyzed for the expression of peroxiredoxin, and cellular sensitivity to oxidative stress. Furthermore, the effect of antiglaucoma drugs on the molecular regulation of the expression of peroxiredoxin was examined using a reporter assay and siRNA strategy. RESULTS: Glaucomatous TM cells highly express peroxiredoxin 2 when compared with normal TM cells. Nipradilol and timolol, but not latanoprost, induce the expression of peroxiredoxin 2 through the activation of the Foxo3a transcription factor. TM cells showed reduced sensitivity to H(2)O(2) when cells were treated with either nipradilol or timolol, but not with latanoprost. In addition, both Foxo3a and PRDX2 expression were enhanced by drug-induced signal transduction through its receptor. CONCLUSIONS: These results indicate that both nipradilol and timolol possess a novel mechanism of action and function as potent protective agents against oxidative stress.

Programmed cell death protein 4 down-regulates Y-box binding protein-1 expression via a direct interaction with Twist1 to suppress cancer cell growth.

Programmed cell death protein 4 (PDCD4) has recently been shown to be involved in both transcription and translation, and to regulate cell growth. However, the mechanisms underlying PDCD4 function are not well understood. In this study, we show that PDCD4 interacts directly with the transcription factor Twist1 and leads to reduced cell growth through the down-regulation of the Twist1 target gene Y-box binding protein-1 (YB-1). PDCD4 interacts with the DNA binding domain of Twist1, inhibiting its DNA binding ability and YB-1 expression. Immunohistochemical analysis showed that an inverse correlation between nuclear PDCD4 and YB-1 expression levels was observed in 37 clinical prostate cancer specimens. Growth suppression by PDCD4 expression was completely recovered by either Twist1 or YB-1 expression. Moreover, PDCD4-overexpressing cells are sensitive to cisplatin and paclitaxel but not to etoposide or 5-fluorouracil. In summary, PDCD4 negatively regulates YB-1 expression via its interaction with Twist1 and is involved in cancer cell growth and chemoresistance.

Twist promotes tumor cell growth through YB-1 expression.

YB-1 controls gene expression through both transcriptional and translational mechanisms and is involved in various biological activities such as brain development, chemoresistance, and tumor progression. We have previously shown that YB-1 is overexpressed in cisplatin-resistant cells and is involved in resistance against DNA-damaging agents. Structural analysis of the YB-1 promoter reveals that several E-boxes may participate in the regulation of YB-1 expression. Here, we show that the E-box-binding transcription factor Twist is overexpressed in cisplatin-resistant cells and that YB-1 is a target gene of Twist. Silencing of either Twist or YB-1 expression induces G(1) phase cell cycle arrest of tumor cell growth. Significantly, reexpression of YB-1 led to increase colony formation when Twist expression was down-regulated by small interfering RNA. However, cotransfection of Twist expression plasmid could not increase colony formation when YB-1 expression was down-regulated. Collectively, these data suggest that YB-1 is a major downstream target of Twist. Both YB-1 and Twist expression could induce tumor progression, promoting cell growth and driving oncogenesis in various cancers. Thus, both YB-1 and Twist may represent promising molecular targets for cancer therapy.

Tip60 is regulated by circadian transcription factor clock and is involved in cisplatin resistance.

Histone modification is important for maintaining chromatin structure and function. Recently, histone acetylation has been shown to have a critical regulatory role in both transcription and DNA repair. We report here that expression of histone acetyltransferase (HAT) genes is associated with cisplatin resistance. We found that Tip60 is overexpressed in cisplatin-resistant cells. The expression of two other HAT genes, HAT1 and MYST1, did not differ between drug-sensitive and -resistant cells. Knockdown of Tip60 expression rendered cells sensitive to cisplatin but not to oxaliplatin, vincristine, and etoposide. Tip60 expression is significantly correlated with cisplatin sensitivity in human lung cancer cell lines. Interestingly, the promoter region of the Tip60 gene contains several E boxes, and its expression was regulated by the E-box binding circadian transcription factor Clock but not by other E-box binding transcription factors such as c-Myc, Twist, and USF1. Hyperacetylation of H3K14 and H4K16 was found in cisplatin-resistant cells. The microarray study reveals that several genes for DNA repair are down-regulated by the knockdown of Tip60 expression. Our data show that HAT gene expression is required for cisplatin resistance and suggest that Clock and Tip60 regulate not only transcription, but also DNA repair, through periodic histone acetylation.

DNA topoisomerase inhibitor, etoposide, enhances GC-box-dependent promoter activity via Sp1 phosphorylation.

Modification of transcription factors by anticancer agents plays an important role in both apoptotic and survival signaling. Here we report that both DNA topoisomerase I and II inhibitors such as SN-38 and etoposide, but not cisplatin, 5-fluorouracil or actinomycin D, can induce phosphorylation of the transcription factor Sp1. Furthermore, DNA topoisomerase inhibitors were shown to transactivate GC-box-dependent promoters such as the SV40 and vascular endothelial growth factor promoters. The phosphorylated form of Sp1 was detectable within 30 min of etoposide treatment and was greatly diminished by the presence of the PI3K inhibitor wortmannin and by DNA-dependent protein kinase (DNA-PK) knockdown. We also confirmed that the phosphorylated form of DNA-PK was increased by treatment with both etoposide and SN-38. Taken together, these findings demonstrate a novel genomic response to anticancer agents that induce Sp1 phosphorylation, and might contribute to tumor progression and drug resistance.