Characterization of a UV-damage recognition factor in vitro that is associated with UV resistance in HeLa cells.

We have previously reported a cisplatin-selected HeLa cell line showing cross-resistance to ultraviolet (UV) radiation and overexpression of UV-damage recognition factors (Chao et al., Mol. Cell. Biol., 11, 2075-2080, 1991). Here, we further characterize a UV-damage recognition factor in vitro using a gel mobility shift assay. The results indicate that the damage-recognition factor is (i) localized mostly in the nucleus, (ii) protease-sensitive, (iii) RNA-independent, (iv) active in a wide range of ionic strengths (50-400 mM NaCl), (v) with a high affinity for UV-damaged DNA (50-fold molar excess competitor causes 50% recognition loss), and (vi) resistant to agents and that modify protein conformation (urea and NP-40), but slightly sensitive to CaCl2. The significance of the identified UV-damage recognition factor in the sensitivity or resistance of cells to UV is also discussed.     

INFLUENCE OF NATURAL ULTRAVIOLET RADIATION ON LOTIC PERIPHYTIC DIATOM COMMUNITY GROWTH,BIOMASS ACCRUAL,AND SPECIES COMPOSITION: SHORT-TERM VERSUS LONG-TERM EFFECTS1, 2

Growth rates, accumulation dynamics, and species succession of periphytic diatom communities were examined in the presence and absence of natural ultraviolet (UV) radiation using a series of outdoor, continuous-flow experimental flumes located on the South Thompson River, British Columbia. In a short-term experiment (2–3 wk), log-phase growth rates of naturally seeded diatom communities comprised of Tabellaria fenestrata (Lyngb.) Kütz., T. flocculosa (Roth) Kütz., Fragilaria crotonesis Kitton, and F. vaucheriae (Ehr.) Peter. exposed to 90% ambient photosynthetically active radiation (PAR) + UV were 30–40% lower than growth rates under 90% PAR alone. UV inhibition of growth rate was independent of the degree of P limitation within the range of relative specific growth rates (μ:μ_max-P) of 0.5–1.0. In a long-term trial, inhibition of attached diatom accumulation under 90% PAR + UV during the first 2–3 wk was corroborated. Reduction of full sunlight to 50% PAR + UV prevented the initial inhibition phase. The initial inihibitory effect of 90% PAR + UV on algal accumulation was reversed after 3–4 wk, and by 5 wk total diatom abundance (chlorophyll a, cell numbers and cell biovolumes) in communities exposed to PAR + UV were 2–4-old greater than in communities protected from UV. Under 90% PAR + UV and 50% PAR + UV, a succession to stalked diatom genera (Cymbella and Gomphoneis) occurred. Species succession under UV radiation doubled the mean cell size of the diatom communities. The shift from inhibition to a long-term increase in the autotrophic community under PAR + UV compared to PAR alone provides further evidence against the use of short-term incubation experiments to define the long-term implications of increases in UVB. These results suggest that the ecological effects of present-day levels of UVB and UVB:UVA ratios on autotrophic communities are not well understood and might be mediated through complex trophic level interactions.     

Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair

Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3′ side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV irradiation and cisplatin, is subjected to proteasome-mediated proteolytic degradation. Productive NER processing is required for XPG degradation as both UV and cisplatin treatment-induced XPG degradation is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In addition, the NER-related XPG degradation requires Cdt2, a component of an E3 ubiquitin ligase, CRL4^Cdt2. Micropore local UV irradiation and in situ Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-damage sites and interacts with XPG in the presence of PCNA. Importantly, Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of DNA polymerase δ and DNA repair synthesis. Collectively, our data support the idea of PCNA recruitment to damage sites which occurs in conjunction with XPG, recognition of the PCNA-bound XPG by CRL4^Cdt2 for specific ubiquitylation and finally the protein degradation. In essence, XPG elimination from DNA damage sites clears the chromatin space needed for the subsequent recruitment of DNA polymerase δ to the damage site and completion of gap-filling DNA synthesis during the final stage of NER.     

紫外照射对DNA修复基因RAD24转录的影响

以酿酒酵母HY684为实验对象,用波长254nm紫外线分别在0,37,50和70J/m~2等强度下照射不同时间,提取总RNA,应用North-ern杂交方法检测RAD24基因转录水平的变化。结果显示37J/m~2、50J/m~2照射20分钟到120分钟明显提高RAD24基因转录水平,70J/m~2照射时,则恢复至正常水平,这说明低剂量紫外线照射可提高RAD24基因转录水平,该基因的表达具有损伤诱导性。     

Rapid modulation of ultraviolet shielding in plants is influenced by solar ultraviolet radiation and linked to alterations in flavonoids

The accumulation of ultraviolet (UV)‐absorbing compounds (flavonoids and related phenylpropanoids) and the resultant decrease in epidermal UV transmittance (T_UV) are primary protective mechanisms employed by plants against potentially damaging solar UV radiation and are critical components of the overall acclimation response of plants to changing solar UV environments. Whether plants can adjust this UV sunscreen protection in response to rapid changes in UV, as occurs on a diurnal basis, is largely unexplored. Here, we use a combination of approaches to demonstrate that plants can modulate their UV‐screening properties within minutes to hours, and these changes are driven, in part, by UV radiation. For the cultivated species Abelmoschus esculentus, large (30–50%) and reversible changes in T_UV occurred on a diurnal basis, and these adjustments were associated with changes in the concentrations of whole‐leaf UV‐absorbing compounds and several quercetin glycosides. Similar results were found for two other species (Vicia faba and Solanum lycopersicum), but no such changes were detected in Zea mays. These findings reveal a much more dynamic UV‐protection mechanism than previously recognized, raise important questions concerning the costs and benefits of UV‐protection strategies in plants and have practical implications for employing UV to enhance crop vigor and quality in controlled environments.     

Recent developments in photochemical and chemical AOPs in water treatment: a mini-review

Photochemical and chemical oxidation methods, belonging to a wide group of advanced oxidation processes (AOPs), are extensively studied but still under constant development. The aim of this study was to review the recent developments mainly in UV/H₂O₂ process and Fenton’s reactions. Ultraviolet (UV) radiation is an essential factor in photochemical oxidation. In the development of UV radiation sources the energy efficiency and the use of toxicant free materials have become major objectives. Also the emitted wavelength plays a significant role. Besides, there are alternatives for UV radiation sources, such as radio frequency (RF) plasmas, which can operate in water without additional chemicals. A remarkable development in the field of chemical oxidation has been novel catalyst materials for Fenton’s reaction enabling the efficient reaction in neutral or almost neutral conditions, instead of pH below 3. In addition, varieties of method integrations, e.g., combinations of AOPs or AOPs integrated to biological processes, and other enhancements such as combining ultrasound (US) with AOP have led to improved water treatment efficiencies.     

Endothelin‐1 and α‐melanocortin have redundant effects on global genome repair in UV‐irradiated human melanocytes despite distinct signaling pathways

Human melanocyte homeostasis is sustained by paracrine factors that reduce the genotoxic effects of ultraviolet radiation (UV), the major etiological factor for melanoma. The keratinocyte‐derived endothelin‐1 (End‐1) and α‐melanocyte‐stimulating hormone (α‐MSH) regulate human melanocyte function, proliferation and survival, and enhance repair of UV‐induced DNA photoproducts by binding to the G_q‐ and G_i‐protein‐coupled endothelin B receptor (EDNRB), and the G_s‐protein‐coupled melanocortin 1 receptor (MC1R), respectively. We hereby report that End‐1 and α‐MSH regulate common effectors of the DNA damage response to UV, despite distinct signaling pathways. Both factors activate the two DNA damage sensors ataxia telangiectasia and Rad3‐related and ataxia telangiectasia mutated, enhance DNA damage recognition by reducing soluble nuclear and chromatin‐bound DNA damage binding protein 2, and increase total and chromatin‐bound xeroderma pigmentosum (XP) C. Additionally, α‐MSH and End‐1 increase total levels and chromatin localization of the damage verification protein XPA, and the levels of γH2AX, which facilitates recruitment of DNA repair proteins to DNA lesions. Activation of EDNRB compensates for MC1R loss of function, thereby reducing the risk of malignant transformation of these vulnerable melanocytes. Therefore, MC1R and EDNRB signaling pathways represent redundant mechanisms that inhibit the genotoxic effects of UV and melanomagenesis.     

Production,HPLC analysis,and in situ apoptotic activities of swainsonine toward lepidopteran,Sf‐21 cell line

Swainsonine, a secondary metabolite from Metarhizium anisopliae has been extensively studied in the complementary areas of therapeutics and toxicology. This work aims to develop a simple UV‐HPLC method of analyses for swainsonine in Metarhizium fermentation broth and to explore its in situ entomotoxic activities. The partially purified broth was quantitatively analyzed using middle UV (205 nm)‐reverse phase HPLC method with different mobile phases and gradient programmes. Swainsonine was eluted as single peak at (t_e) 6.0–6.9 min with average concentration of 4.04 ± 0.52 μg/mL using optimal mobile phase (0.1% trifluoroacetic acid in water and acetonitrile). The mass spectrometry analysis further indicated the characteristic MS1 species for swainsonine, [M+H]⁺ 174.30 in corresponding HPLC peaks. The antiproliferative effects of swainsonine on lepidopteran, Sf‐21 cells were determined through 3‐(4, 5‐dimethylthia‐zol‐2‐yl)?2, 5‐diphenyl tetrazolium bromide (IC_{50 standard} = 3.90 μM and IC_{50 purified} = 5.27 μM) and trypan blue dye exclusion (IC_{50 standard} = 6.91 μM and IC_{50 purified} = 8.67 μM) assays. The fluorescence activated cell sorting evaluation of Sf‐21 cells showed nearly 35% and 42% of population in various apoptotic stages at 36 h, when treated with standard and purified swainsonine, respectively. The morphodimensional field emission scanning electron and atomic force microscopic analyses further confirmed the characteristic apoptotic features like membrane blebbings, ruptures and volume shrinkage in the lepidopteran cells after 24–36 h of post‐treatment incubation. The study describes the potential entomotoxic activities of swainsonine and its role in the virulence of Metarhizium spp. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1196–1205, 2014     

Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair

Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3′ side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV irradiation and cisplatin, is subjected to proteasome-mediated proteolytic degradation. Productive NER processing is required for XPG degradation as both UV and cisplatin treatment-induced XPG degradation is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In addition, the NER-related XPG degradation requires Cdt2, a component of an E3 ubiquitin ligase, CRL4^Cdt2. Micropore local UV irradiation and in situ Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-damage sites and interacts with XPG in the presence of PCNA. Importantly, Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of DNA polymerase δ and DNA repair synthesis. Collectively, our data support the idea of PCNA recruitment to damage sites which occurs in conjunction with XPG, recognition of the PCNA-bound XPG by CRL4^Cdt2 for specific ubiquitylation and finally the protein degradation. In essence, XPG elimination from DNA damage sites clears the chromatin space needed for the subsequent recruitment of DNA polymerase δ to the damage site and completion of gap-filling DNA synthesis during the final stage of NER.     

Anisomycin Selectively Desensitizes Signalling Components Involved in Stress Kinase Activation and fos and jun Induction

Anisomycin, a translational inhibitor secreted by Streptomyces spp., strongly activates the stress-activated mitogen-activated protein (MAP) kinases JNK/SAPK (c-Jun NH₂-terminal kinase/stress-activated protein kinase) and p38/RK in mammalian cells, resulting in rapid induction of immediate-early (IE) genes in the nucleus. Here, we have characterized this response further with respect to homologous and heterologous desensitization of IE gene induction and stress kinase activation. We show that anisomycin acts exactly like a signalling agonist in eliciting highly specific and virtually complete homologous desensitization. Anisomycin desensitization of a panel of IE genes (c-fos, fosB, c-jun, junB, and junD), using epidermal growth factor (EGF), basic fibroblast growth factor, (bFGF), tumor necrosis factor alpha (TNF-α), anisomycin, tetradecanoyl phorbol acetate (TPA), and UV radiation as secondary stimuli, was found to be extremely specific both with respect to the secondary stimuli and at the level of individual genes. Further, we show that anisomycin-induced homologous desensitization is caused by the fact that anisomycin no longer activates the JNK/SAPK and p38/RK MAP kinase cascades in desensitized cells. In anisomycin-desensitized cells, activation of JNK/SAPKs by UV radiation and hyperosmolarity is almost completely lost, and that of the p38/RK cascade is reduced to about 50% of the normal response. However, all other stimuli produced normal or augmented activation of these two kinase cascades in anisomycin-desensitized cells. These data show that anisomycin behaves like a true signalling agonist and suggest that the anisomycin-desensitized signalling component(s) is not involved in JNK/SAPK or p38/RK activation by EGF, bFGF, TNF-α, or TPA but may play a significant role in UV- and hyperosmolarity-stimulated responses.     

Photosynthetic performance,DNA damage and repair in gametes of the endemic Antarctic brown alga Ascoseira mirabilis exposed to ultraviolet radiation

Abstract Stress physiology on the reproductive cells of Antarctic macroalgae remained unstudied. Ascoseira mirabilis is endemic to the Antarctic region, an isolated ecosystem exposed to extreme environmental conditions. Moreover, stratospheric ozone depletion leads to increasing ultraviolet radiation (280–400 nm) at the earth's surface, thus it is necessary to investigate the capacity of reproductive cells to cope with different UV irradiances. This study is aimed to investigate the impact of exposure to different spectral irradiance on the photosynthetic performance, DNA damage and gamete morphology of the A. mirabilis. Gametangia, gametes and zygotes of the upper sublittoral brown alga A. mirabilis were exposed to photosynthetically active radiation (PAR = P; 400–700 nm), P + UV‐A radiation (UV‐A, 320–400 nm) and P + UV‐A + UV‐B radiation (UV‐B, 280–320 nm). Rapid photosynthesis versus irradiance curves of freshly released propagules were measured. Photosynthetic efficiencies and DNA damage (in terms of cyclobutane pyrimidine dimers) were determined after 1, 2, 4 and 8 h exposure as well as after 2 days of recovery in dim white light. Saturation irradiance (I_k) in freshly released propagules was 52 μmol photons m⁻² s⁻¹. Exposure for 1 h under 22 μmol photons m⁻² s⁻¹ of PAR significantly reduced the optimum quantum yield (F_v/F_m), suggesting that propagules are low light adapted. Furthermore, UVR significantly contributed to the photoinhibition of photosynthesis. Increasing dose as a function of exposure time additionally exacerbated the effects of different light treatments. The amount of DNA damage increased with the UV‐B dose but an efficient repair mechanism was observed in gametes pre‐exposed to a dose lower than 5.8 × 10³ J m⁻² of UV‐B. The results of this study demonstrate the negative impact of UV‐B radiation. However, gametes of A. mirabilis are capable of photosynthetic recovery and DNA repair when the stress factor is removed. This capacity was observed to be dependent on the fitness of the parental sporophyte.     

Analysis of the interacting partners eIF4F and 3′‐CITE required for Melon necrotic spot virus cap‐independent translation

We have shown previously that the translation of Melon necrotic spot virus (MNSV, family Tombusviridae, genus Carmovirus) RNAs is controlled by a 3′‐cap‐independent translation enhancer (CITE), which is genetically and functionally dependent on the eukaryotic translation initiation factor (eIF) 4E. Here, we describe structural and functional analyses of the MNSV‐Mα5 3′‐CITE and its translation initiation factor partner. We first mapped the minimal 3′‐CITE (Ma5TE) to a 45‐nucleotide sequence, which consists of a stem‐loop structure with two internal loops, similar to other I‐shaped 3′‐CITEs. UV crosslinking, followed by gel retardation assays, indicated that Ma5TE interacts in vitro with the complex formed by eIF4E + eIF4G_980–1159 (eIF4F_p20), but not with each subunit alone or with eIF4E + eIF4G_1003–1092, suggesting binding either through interaction with eIF4E following a conformational change induced by its binding to eIF4G_980–1159, or through a double interaction with eIF4E and eIF4G_980–1159. Critical residues for this interaction reside in an internal bulge of Ma5TE, so that their mutation abolished binding to eIF4E + eIF4G_1003–1092 and cap‐independent translation. We also developed an in vivo system to test the effect of mutations in eIF4E in Ma5TE‐driven cap‐independent translation, showing that conserved amino acids in a positively charged RNA‐binding motif around amino acid position 228, implicated in eIF4E–eIF4G binding or belonging to the cap‐recognition pocket, are essential for cap‐independent translation controlled by Ma5TE, and thus for the multiplication of MNSV.     

Recombinant Bacillus thuringiensis strain shows high insecticidal activity against Plutella xylostella and Leptinotarsa decemlineata without affecting nontarget species in the field

Aims: To construct a recombinant Bacillus thuringiensis (Bt) strain with broad insecticidal spectrum and investigate its impact on nontarget organisms in field. Method and Results: The cry-type gene of wild Bt strain UV17 was identified and a novel cry1Ba gene was cloned. The cry3Aa7 gene, which was highly toxic to coleopteran pests, was introduced into UV17, and a recombinant strain designated as UV173A was obtained. Bioassay results showed that UV173A was not only highly toxic against Plutella xylostella (50% lethal concentration [LC₅₀] = 18·03 μg ml^–1), but also against coleopteran Leptinotarsa decernlineata (LC₅₀ = 0·19 mg ml^–1). The recombinant strain was then tested in field trials to monitor its spatial variation of population and to investigate the impact on nontarget invertebrates. Conclusions: A recombinant Bt stain UV173A with broad insecticidal spectrum was obtained, and it did not cause adverse effects on the population of nontarget organisms. Significance and Impact of the Study: The results obtained here indicated that cry1Ba3 gene may be useful for the resistance management of P. xylostella, and the recombinant stain UV173A was potential for field application against some crucifer vegetable pests as well as L. decemlineata.     

Responses of entomopathogenic fungi to the mutagen 4-nitroquinoline 1-oxide

Survival of entomopathogenic fungi under solar ultraviolet (UV) radiation is paramount to the success of biological control of insect pests and disease vectors. The mutagenic compound 4-nitroquinoline 1-oxide (4-NQO) is often used to mimic the biological effects of UV radiation on organisms. Therefore, we asked whether tolerance to 4-NQO could predict tolerance to UV radiation in thirty isolates of entomopathogenic fungi and one isolate of a xerophilic fungus. A dendrogram obtained from cluster analyses based on the 50 and 90 % inhibitory concentrations (IC₅₀ and IC₉₀, respectively) divided the fungal isolates into six clusters numbered consecutively based on their tolerance to 4-NQO. Cluster 6 contained species with highest tolerance to 4-NQO (IC₅₀ > 4.7 μM), including Mariannaea pruinosa, Lecanicillium aphanocladii, and Torrubiella homopterorum. Cluster 1 contained species least tolerant to 4-NQO (IC₅₀ < 0.2 μM), such as Metarhizium acridum (ARSEF 324), Tolypocladium geodes, and Metarhizium brunneum (ARSEF 7711). With few exceptions, the majority of Metarhizium species showed moderate to low tolerances (IC₅₀ between 0.4 and 0.9 μM) and were placed in cluster 2. Cluster 3 included species with moderate tolerance (IC₅₀ between 1.0 and 1.2 μM). In cluster 4 were species with moderate to high tolerance (IC₅₀ between 1.3 and 1.6 μM). Cluster 5 contained the species with high tolerance (IC₅₀ between 1.9 and 4.0 μM). The most UV tolerant isolate of M. acridum, ARSEF 324, was the least tolerant to 4-NQO. Also, L. aphanocladii, which is very susceptible to UV radiation, showed high tolerance to 4-NQO. Our results indicate that tolerance to 4-NQO does not correlate with tolerance to UV radiation. Therefore this chemical compound is not a predictor of UV tolerance in entomopathogenic fungi.     

Spodoptera frugiperda FKBP‐46 is a consensus p53 motif binding protein

p53 protein, the central molecule of the apoptosis pathway, is mutated in 50% of the human cancers. Of late, p53 homologues have been identified from different invertebrates including Drosophila melanogaster, Caenorhabditis elegans, Squid, and Clams. We report the identification of a p53‐like protein in Spodoptera frugiperda (Sf9) insect cells, which is activated during oxidative stress, caused by exposure to UV‐B or H₂O₂, and binds to p53 consensus DNA binding motifs as well as other p53 cognate motifs. Sf9 p53 motif‐binding protein is similar to murine and Drosophila p53 in terms of molecular size, which is around 50–60 kDa, as evident from UV cross‐linking, and displays DNA binding characteristics similar to both insect and vertebrate p53 as seen from electrophoretic mobility shift assays. The N‐terminal sequencing of the purified Sf9 p53 motif‐binding protein reveals extensive homology to the pro‐apoptotic FK‐506 binding protein (FKBP‐46), earlier identified in Sf9 cells as a factor which interacts with murine casein kinase. FKBP, an evolutionarily conserved protein of mammalian origin functions as a pro‐apoptotic factor. Identification of FKBP‐46 as a novel p53 motif‐binding protein in insect cells adds a new facet to our understanding of the mechanisms of apoptosis under oxidative stress in the absence of a typical p53 homologue. J. Cell. Biochem. 114: 899–907, 2013. © 2012 Wiley Periodicals, Inc.     

Microstructural tuning and band gap engineering of calcium hydroxides: a novel approach by pH variation

Here we report a simple, inexpensive, energy benign, yet novel pH-driven chemical precipitation technique to achieve microstructural and band gap engineering of calcium hydroxide nanoparticles (CHNPs). The chemical precipitation route involved the use of 0.4–1.6 M Ca(NO₃)₂.4H₂O solutions as the precursor and 1 M NaOH solution as the precipitator. The simple variation in precursor molarity induces a pH change from about 12.4 to 11.3 in the reactant solution. The CHNPs characterized by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), and ultraviolet–visible (UV–Vis) spectroscopy techniques confirm a jump of nanocrystallite size from ~50–70 nm with a concomitant reduction of direct optical band gap energy from ~5.38–5.26 eV. The possible mechanisms that could be operative behind obtaining microstructurally tuned (MT)-CHNPS and band gap engineering (BGE) are discussed from both theoretical and physical process perspectives. Furthermore, the implications of these novel results for possible futuristic applications are briefly hinted upon.     

Endothelium-dependent vasorelaxation in rat thoracic aorta by Mansoa hirsuta D.C.

The vasodilator effect of the ethanolic extract of Mansoa hirsuta leaves (EEF) was assayed in rat aortic rings. EEF produced a concentration-dependent vasodilatation (pIC₅₀=5.1±0.2), which was absent in endothelium-denuded vessels. The vasodilator effect of EEF was similar to a standardized ethanolic extract of Hancornia speciosa Gomes (pIC₅₀=5.1±0.1). The endothelium-dependent vasodilatation induced by EEF was abolished by L-NAME (100 μM), a nitric oxide (NO) synthase inhibitor, but not by indomethacin (10 μM; pIC₅₀=4.9±0.2), a cyclooxygenase inhibitor. The concentration–response curve of EEF was not modified by the addition of superoxide dismutase (SOD; 300 U/ml). In addition, EEF (50 μg/ml) displaced the 3-morpholino-sidnonimine (SIN-1; p<0.05) concentration–effect curve to the left, as well as SOD (300 U/ml). These findings lead us to conclude that EEF induces a NO- and endothelium-dependent vasodilatation in rat aortic preparations, and that this effect is, at least in some extent, due to an increase in the NO bioavailability as consequence of its antioxidant activity. The HPLC-DAD profile recorded for EEF indicates the presence of four major peaks with close retention times, exhibiting similar UV spectra with wavelength maxima compatible with heterogeneous proanthocyanidins.     

Inhibitors of histone deacetylases in class I and class II suppress human osteoclasts in vitro

Histone deacetylase inhibitors (HDACi) suppress cancer cell growth, inflammation, and bone resorption. The aim of this study was to determine the effect of inhibitors of different HDAC classes on human osteoclast activity in vitro. Human osteoclasts generated from blood mononuclear cells stimulated with receptor activator of nuclear factor kappa B (RANK) ligand were treated with a novel compound targeting classes I and II HDACs (1179.4b), MS‐275 (targets class I HDACs), 2664.12 (targets class II HDACs), or suberoylanilide hydroxamic acid (SAHA; targets classes I and II HDACs). Osteoclast differentiation was assessed by expression of tartrate resistant acid phosphatase and resorption of dentine. Expression of mRNA encoding for osteoclast genes including RANK, calcitonin receptor (CTR), c‐Fos, tumur necrosis factor (TNF) receptor associated factor (TRAF)6, nuclear factor of activated T cells (NFATc1), interferon‐β, TNF‐like weak inducer of apoptosis (TWEAK), and osteoclast‐associated receptor (OSCAR) were assessed. Expression of HDACs 1–10 during osteoclast development was also assessed. 1179.4b significantly reduced osteoclast activity (IC₅₀ < 0.16 nM). MS‐275 (IC₅₀ 54.4 nM) and 2664.12 (IC₅₀ > 100 nM) were markedly less effective. A combination of MS‐275 and 2664.12 inhibited osteoclast activity similar to 1179.4b (IC₅₀ 0.35 nM). SAHA was shown to suppress osteoclast activity (IC₅₀ 12 nM). 1179.4b significantly (P < 0.05) reduced NFATc1, CTR, and OSCAR expression during the later stages of osteoclast development. Class I HDAC 8 and Class II HDAC5 were both elevated (P < 0.05) during osteoclast development. Results suggest that inhibition of both classes I and II HDACs may be required to suppress human osteoclastic bone resorption in vitro. J. Cell. Physiol. 226: 3233–3241, 2011. © 2011 Wiley Periodicals, Inc.     

A fast and simple approach to the quantitative evaluation of fibrinogen coagulation

Fibrinogen is essential in the intrinsic and extrinsic blood coagulation process. Inhibition of fibrinogen aggregation could lead to anticoagulation effects. The availability of methods for easy quantitative evaluation of the coagulation process is critical to studying coagulation and its inhibition. A commonly used method is UV–Vis absorbance (405 nm) detection by a micro-plate reader. However, because of the heterogeneous nature of the resulting mixture in a coagulation process, transmission-based optical measurements give large variations. Herein, a very simple and easy method is developed for the quantitative measurements of the coagulation process. The method was validated using three known thrombin inhibitors: 4-(2-aminoethyl) benzenesulfonyl fluoride (IC₅₀: 0.01 mM), p-amidinophenyl methanesulfonyl fluoride (IC₅₀: 0.18 mM) and PMSF (IC₅₀: 0.23 mM).