Selective recognition of thymidine homopolymer (poly T) oligonucleotide with cobalt(II)–4‐[(5‐chloro‐2‐pyridyl)azo]‐1,3‐diaminobenzene complex

The interactions of cobalt(II)–4‐[(5‐chloro‐2‐pyridyl)azo]‐1,3‐diaminobenzene (5‐Cl‐PADAB) complex with different kinds of homopolymer oligonucleotides in basic medium were investigated based on the measurements of resonance light scattering, UV–vis, circular dichroism spectra and dark field light‐scattering imaging. Experiments showed that only thymidine homopolymer (poly T) oligonucleotides with the length in the range of poly T₆ to poly T₁₈ could interact with the Co(II)–5‐Cl‐PADAB complex in alkaline conditions and cause evident color and spectral change. Thus, the binary complex of Co(II)–5‐Cl‐PADAB could be employed as a visual probe for selectively recognizing the poly T oligonucleotides. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis of some novel quinone diimine derivatives of benzo‐15‐crown‐5 for application in Hg2+ recognition

A series of novel fluoroionophore bearing derivatives of benzo‐15‐crown‐5 were synthesized by the amination of benzo‐15‐crown‐5 followed by condensation with different quinones in the presence of titanium tetrachloride (TiCl₄) and 1,4‐diazabicyclo‐[2.2.2]octane. The compounds were characterized by infrared, ¹H and ¹³C nuclear magnetic resonance, mass spectroscopy and elemental analysis. Absorption and fluorescence spectral characteristics of these compounds were studied. It was observed that the anthraquinone derivative was acting as an Hg²⁺ ion sensor. Copyright © 2013 John Wiley & Sons, Ltd.     

Validated spectrofluorimetric method for the determination of carbamazepine in pharmaceutical dosage forms after reaction with 4‐chloro‐7‐‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐Cl)

A sensitive and simple spectrofluorimetric method has been developed and validated for the determination of the anti‐epileptic drug carbamazepine (CBZ) in its dosage forms. The method was based on a nucleophilic substitution reaction of CBZ with 4‐chloro‐7‐nitrobenzo‐2‐ oxa‐1,3‐diazole (NBD‐Cl) in borate buffer (pH 9) to form a highly fluorescent derivative that was measured at 530 nm after excitation at 460 nm. Factors affecting the formation of the reaction product were studied and optimized, and the reaction mechanism was postulated. The fluorescence–concentration plot is rectilinear over the range of 0.6–8 µg/mL with limit of detection of 0.06 µg/mL and limit of quantitation of 0.19 µg/mL. The method was applied to the analysis of commercial tablets and the results were in good agreement with those obtained using the reference method. Validation of the analytical procedures was evaluated according to ICH guidelines. Copyright © 2015 John Wiley & Sons, Ltd.     

Dual labeling with 5‐bromo‐2'‐deoxyuridine and 5‐ethynyl‐2'‐deoxyuridine for estimation of cell migration rate in the small intestinal epithelium

Small intestinal epithelium is a self‐renewing system in which the entire sequence of cell proliferation, differentiation, and removal is coupled to cell migration along the crypt‐villus axis. We examined whether dual labeling with different thymidine analogues, 5‐bromo‐2'‐deoxyuridine (BrdU) and 5‐ethynyl‐2'‐deoxyuridine (EdU), can be used to estimate cell migration rates on the villi of small intestines in rats. Rats received a single intraperitoneal injection of BrdU and EdU within a time interval, and signals in tissue sections were examined by immunohistochemistry and the “click” reaction, respectively. We successfully observed BrdU‐ and EdU‐positive cells on the epithelium with no cross‐reaction. In addition, we observed an almost complete overlapping of BrdU‐ and EdU‐positive cells in rats administered simultaneously with BrdU and EdU. By calculating the cell migration rate by dividing the distance between the median cell positions of the distribution of BrdU‐ and EdU‐positive cells by the time between the injection of BrdU and EdU, we estimated approximately 9 and 5 μm/h for the cell migration rates on the villi in the jejunum and ileum, respectively. We propose that dual labeling with BrdU and EdU within a time interval, followed by detecting with immunohistochemistry and the click reaction, respectively, is useful to estimate accurately the cell migration rate in the intestinal epithelium in a single animal.     

Spectrofluorimetric determination of aluminium using 2‐hydroxy‐1‐naphthylidene‐(8‐aminoquinoline)

A sensitive and selective spectrofluorimetric method has been developed for the rapid determination of aluminium. This method is based on the complex formation between aluminium and 2‐hydroxy‐1‐naphthylidene‐(8‐aminoquinoline) (HNAQ). The optimum conditions for the complex formation were a metal‐to‐ligand (M : L) stoichiometric ratio of 1:1, a pH of 5.5 and a 0.20 m acetate buffer. The fluorescence of the complex was monitored at an emission wavelength of 502 nm with excitation at 438 nm. Under these conditions, linear calibration curves were obtained in the ranges 0.05–1 and 1–5 ppm. The detection limit was 3.4 ppb for the former and 13.5 ppb for the latter. The maximum relative standard deviation of the method for an aluminium standard of 200 ppb was 1.5% (n = 5). This method was successfully applied for the determination of aluminium in drinking water, pharmaceutical antacid tablets and suspension samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Facile removal of 4‐methoxybenzyl protecting group from selenocysteine

Historically, methods to remove the 4‐methoxybenzyl (Mob)–protecting group from selenocysteine (Sec) in peptides have used harsh and toxic reagents. The use of 2,2′‐dithiobis‐5‐nitropyridine (DTNP) is an improvement over these methods; however, many wash steps are required to remove the by‐product contaminant 5‐nitro‐2‐thiopyridine. Even with many washes, excess DTNP adheres to the peptide. The final product needs excess purification to remove these contaminants. It was recently discovered by our group that hindered hydrosilanes could be used to reduce Cys(Mob). We sought to apply a similar methodology to reduce Sec(Mob), which we expected to be even more labile. Here, we present a gentle and facile method for deprotection of Sec(Mob) using triethylsilane (TES), phenol, and a variety of other scavengers often used in deprotection cocktails. The different cocktails were all incubated at 40 °C for 4 hours. The combination of TFA/TES/thioanisole (96:2:2) appeared to be the most efficient of the cocktails tested, providing complete deprotection and yielded peptide that was mainly in the diselenide form. This cocktail also showed no evidence of side reactions or significant contaminants in the high‐performance liquid chromatography (HPLC) and mass spectral (MS) analyses. We envision that our new method will allow for a simple and gentle “one‐pot” deprotection of Sec(Mob) following solid‐phase peptide synthesis and will minimize the need for extensive purification steps.     

Cyclo‐oxygenase 2 up‐regulates the effect of multidrug resistance

COX‐2 (cyclo‐oxygenase 2), an inducible form of the enzyme that catalyses the first step in the synthesis of prostanoids, is associated with inflammatory diseases and carcinogenesis, which is suspected to promote angiogenesis and tissue invasion of tumours and resistance to apoptosis. COX‐2 is also involved in drug resistance and poor prognosis of many neoplastic diseases or cancers. The activation of the COX‐2/PGE₂ (prostaglandin E₂)/prostaglandin E receptor signal pathway can up‐regulate the expression of all three ABC (ATP‐binding‐cassette) transporters, MDR1/P‐gp (multidrug resistance/P‐glycoprotein), MRP1 (multidrug‐resistance protein 1) and BCRP (breast‐cancer‐resistance protein), which encode efflux pumps, playing important roles in the development of multidrug resistance. In addition, COX inhibitors inhibit the expression of MDR1/P‐gp, MRP1 and BCRP and enhance the cytotoxicity of anticancer drugs. Therefore we can use the COX inhibitors to potentialize the effects of chemotherapeutic agents and reverse multidrug resistance to facilitate the patient who may benefit from addition of COX inhibitors to standard cytotoxic therapy.     

Ameliorative Effects of Curcumin on Fibrinogen‐Like Protein‐2 Gene Expression,Some Oxido‐Inflammatory and Apoptotic Markers in a Rat Model of l‐Arginine‐Induced Acute Pancreatitis

The aim of the study was to investigate the ameliorative effects of curcumin on fibrinogen like protein‐2 (fgl‐2), some oxido‐inflammatory and apoptotic markers in rat‐induced acute pancreatitis (AP). Seventy‐five albino rats were divided into control group, l ‐arginine (l ‐Arg)‐induced AP group, curcumin pre‐treated group before AP induction, curcumin post‐treated group after AP induction, and curcumin injected group only. AP group showed severe necrotizing pancreatitis confirmed by histopathological changes and elevations in serum amylase and lipase activities, levels of epithelial neutrophil‐activating peptide 78, tissue content of protein carbonyls, levels of tumor necrosis factor α, and caspase‐3 as well as myeloperoxidase activity. Significant elevation in pancreatic fgl‐2 mRNA expression was detected in AP group. Improvement of all parameters was detected with increase of caspase‐3 in both curcumin‐treated groups that confirmed curcumin ameliorative effects against AP through induction of apoptosis and inhibition of micro‐thrombosis, inflammation, and oxidative stress.     

Nitric oxide‐mediated regulation of ‐amyloid clearance via alterations of MMP‐9/TIMP‐1

Fibrillar amyloid plaques are largely composed of amyloid‐beta (Aβ) peptides that are metabolized into products, including Aβ1‐16, by proteases including matrix metalloproteinase 9 (MMP‐9). The balance between production and degradation of Aβ proteins is critical to amyloid accumulation and resulting disease. Regulation of MMP‐9 and its endogenous inhibitor tissue inhibitor of metalloproteinase (TIMP)‐1 by nitric oxide (NO) has been shown. We hypothesize that nitric oxide synthase (NOS2) protects against Alzheimer's disease pathology by increasing amyloid clearance through NO regulation of MMP‐9/TIMP‐1 balance. We show NO‐mediated increased MMP‐9/TIMP‐1 ratios enhanced the degradation of fibrillar Aβ in vitro, which was abolished when silenced for MMP‐9 protein translation. The in vivo relationship between MMP‐9, NO and Aβ degradation was examined by comparing an Alzheimer's disease mouse model that expresses NOS2 with a model lacking NOS2. To quantitate MMP‐9 mediated changes, we generated an antibody recognizing the Aβ1‐16 fragment, and used mass spectrometry multi‐reaction monitoring assay for detection of immunoprecipitated Aβ1‐16 peptides. Aβ1‐16 levels decreased in brain lysates lacking NOS2 when compared with strains that express human amyloid precursor protein on the NOS2 background. TIMP‐1 increased in the APPSwDI/NOS2^?/? mice with decreased MMP activity and increased amyloid burden, thereby supporting roles for NO in the regulation of MMP/TIMP balance and plaque clearance.     

LncRNA‐SULT1C2A regulates Foxo4 in congenital scoliosis by targeting rno‐miR‐466c‐5p through PI3K‐ATK signalling

Congenital scoliosis (CS) is the result of anomalous vertebrae development, but the pathogenesis of CS remains unclear. Long non‐coding RNAs (lncRNAs) have been implicated in embryo development, but their role in CS remains unknown. In this study, we investigated the role and mechanisms of a specific lncRNA, SULT1C2A, in somitogenesis in a rat model of vitamin A deficiency (VAD)‐induced CS. Bioinformatics analysis and quantitative real‐time PCR (qRT‐PCR) indicated that SULT1C2A expression was down‐regulated in VAD group, accompanied by increased expression of rno‐miR‐466c‐5p but decreased expression of Foxo4 and somitogenesis‐related genes such as Pax1, Nkx3‐2 and Sox9 on gestational day (GD) 9. Luciferase reporter and small interfering RNA (siRNA) assays showed that SULT1C2A functioned as a competing endogenous RNA to inhibit rno‐miR‐466c‐5p expression by direct binding, and rno‐miR‐466c‐5p inhibited Foxo4 expression by binding to its 3′ untranslated region (UTR). The spatiotemporal expression of SULT1C2A, rno‐miR‐466c‐5p and Foxo4 axis was dynamically altered on GDs 3, 8, 11, 15 and 21 as detected by qRT‐PCR and northern blot analyses, with parallel changes in Protein kinase B (AKT) phosphorylation and PI3K expression. Taken together, our findings indicate that SULT1C2A enhanced Foxo4 expression by negatively modulating rno‐miR‐466c‐5p expression via the PI3K‐ATK signalling pathway in the rat model of VAD‐CS. Thus, SULT1C2A may be a potential target for treating CS.     

Poly(ADP‐ribose) polymerase inhibition with HYDAMTIQ reduces allergen‐induced asthma‐like reaction,bronchial hyper‐reactivity and airway remodelling

Activation of poly(ADP‐ribose) polymerases (PARPs) is considered a key event in the molecular and cellular processes leading from acute asthma attacks to bronchial hyper‐reactivity, leucocyte recruitment, chronic inflammation, airway remodelling and lung damage. The present investigation has been carried out to investigate the action of hydroxyl‐dimethylaminomethyl‐thieno[2,3‐c]isoquinolin‐5(4H)‐one (HYDAMTIQ), a new potent PARP inhibitor, in the process leading from asthma‐like events to airway damage. Ovalbumin‐sensitized guinea pigs exposed two times to allergen inhalation were treated for 8 days with vehicle or HYDAMTIQ. Asthma‐like signs, bronchial hyper‐reactivity to methacholine, cytokine production, histamine release from mast cells, airway remodelling, collagen deposition and lung damage were evaluated. Repeated HYDAMTIQ administration (1‐10 mg/kg/day i.p.) reduced lung PARP activity, delayed the appearance and reduced the severity of allergen‐induced cough and dyspnoea and dampened the increased bronchial responses to methacholine. HYDAMTIQ‐treated animals presented reduced bronchial or alveolar abnormalities, lower number of eosinophils and other leucocytes in the lung and decreased smooth muscle or goblet cell hyperplasia. The treatment also reduced lung oxidative stress markers, such as malondialdehyde or 8‐hydroxy‐2′‐deoxyguanosine and the lung content of pro‐inflammatory cytokines (TNF‐α, interleukin (IL)‐1β, IL‐5, IL‐6 and IL‐18). Finally, mast cells isolated from the peritoneal or pleural cavities of sensitized, HYDAMTIQ‐treated animals had a reduced ability to release histamine when exposed to ovalbumin in vitro. Our findings support the proposal that PARP inhibitors could have a therapeutic potential to reduce chronic lung inflammation, airway damage and remodelling in severe unresponsive asthmatic patients.     

Upregulation of miR‐874‐3p and miR‐874‐5p inhibits epithelial ovarian cancer malignancy via SIK2

Based on miR‐874 expression levels in the GSE47841 microarray, we hypothesized that the mature products of miR‐874, miR‐874‐3p, or miR‐874‐5p, would inhibit epithelial ovarian cancer (EOC) cell proliferation, metastasis, and chemoresistance. We first examined miR‐874‐3p and miR‐874‐5p expression levels in primary EOC tumor tissue samples and found that they were significantly decreased. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) cell proliferation and transwell assays revealed that miR‐874‐3p and miR‐874‐5p significantly inhibit EOC cell proliferation, migration, and invasion. Then, using MTT and soft agar assays of paclitaxel‐treated Caov3 and SKOV3 cells transfected with miR‐874‐3p and miR‐874‐5p, we found that miR‐874‐3p and miR‐874‐5p enhance EOC cell chemosensitivity. We then confirmed that serine/threonine‐protein kinase 2 (SIK2) was a target gene of miR‐874‐3p and miR‐874‐5p. Overall, the results of this study indicate that SIK2 expression can serve as a prognostic biomarker for EOC and that miR‐874‐3p and miR‐874‐5p have the potential to enhance clinical treatment of EOC.     

Insights into the helix‐sense inversion of poly(β‐phenethyl‐L‐aspartate) by two‐dimensional Raman correlation spectroscopy

In this study, the transition process of the helix‐sense inversion of poly(β‐phenethyl‐L‐aspartate) was investigated by Raman scattering and 2‐dimensional correlation spectroscopy. Temperature‐dependent Raman spectra were obtained during the helix‐sense inversion. The results of 2‐dimensional correlation analysis in the spectral regions of 1600‐1800 and 3200‐3400 cm^?1 showed that the intensity changes of the side‐chain ester C═O stretching bands occurred prior to those of amide A and amide I bands in the unwinding process of αR‐helix on heating. The sequential order of the intensity changes for amide A, amide I, and the side‐chain ester C═O stretching bands during the inversion process was determined. It was found that the conformation change of the side chain occurred prior to that of the main chain for the αR‐helix on heating. Thus, we concluded that the transformation of the backbone chain from right‐handed to left‐handed is triggered by the conformational change of the side chains.