Increased 3′‐Phosphoadenosine‐5′‐phosphosulfate Levels in Engineered Escherichia coli Cell Lysate Facilitate the In Vitro Synthesis of Chondroitin Sulfate A

Chondroitin sulfates (CSs) are linear glycosaminoglycans that have important applications in the medical and food industries. Engineering bacteria for the microbial production of CS will facilitate a one‐step, scalable production with good control over sulfation levels and positions in contrast to extraction from animal sources. To achieve this goal, Escherichia coli (E. coli) is engineered in this study using traditional metabolic engineering approaches to accumulate 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS), the universal sulfate donor. PAPS is one of the least‐explored components required for the biosynthesis of CS. The resulting engineered E. coli strain shows an ≈1000‐fold increase in intracellular PAPS concentrations. This study also reports, for the first time, in vitro biotransformation of CS using PAPS, chondroitin, and chondroitin‐4‐sulfotransferase (C4ST), all synthesized from different engineered E. coli strains. A 10.4‐fold increase is observed in the amount of CS produced by biotransformation by employing PAPS from the engineered PAPS‐accumulating strain. The data from the biotransformation experiments also help evaluate the reaction components that need improved production to achieve a one‐step microbial synthesis of CS. This will provide a new platform to produce CS.     

5,2′‐dibromo‐2,4′,5′‐trihydroxydiphenylmethanone attenuates LPS‐induced inflammation and ROS production in EA.hy926 cells via HMBOX1 induction

Inflammation and reactive oxygen species (ROS) are important factors in the pathogenesis of atherosclerosis (AS). 5,2′‐dibromo‐2,4′,5′‐trihydroxydiphenylmethanone (TDD), possess anti‐atherogenic properties; however, its underlying mechanism of action remains unclear. Therefore, we sought to understand the therapeutic molecular mechanism of TDD in inflammatory response and oxidative stress in EA.hy926 cells. Microarray analysis revealed that the expression of homeobox containing 1 (HMBOX1) was dramatically upregulated in TDD‐treated EA.hy926 cells. According to the gene ontology (GO) analysis of microarray data, TDD significantly influenced the response to lipopolysaccharide (LPS); it suppressed the LPS‐induced adhesion of monocytes to EA.hy926 cells. Simultaneously, TDD dose‐dependently inhibited the production or expression of IL‐6, IL‐1β, MCP‐1, TNF‐α, VCAM‐1, ICAM‐1 and E‐selectin as well as ROS in LPS‐stimulated EA.hy926 cells. HMBOX1 knockdown using RNA interference attenuated the anti‐inflammatory and anti‐oxidative effects of TDD. Furthermore, TDD inhibited LPS‐induced NF‐κB and MAPK activation in EA.hy926 cells, but this effect was abolished by HMBOX1 knockdown. Overall, these results demonstrate that TDD activates HMBOX1, which is an inducible protective mechanism that inhibits LPS‐induced inflammation and ROS production in EA.hy926 cells by the subsequent inhibition of redox‐sensitive NF‐κB and MAPK activation. Our study suggested that TDD may be a potential novel agent for treating endothelial cells dysfunction in AS.     

Magnesium methoxide‐induced chemiluminescent decomposition of bicyclic dioxetanes bearing a 2′‐alkoxy‐2‐hydroxy‐1,1′‐binaphthyl‐7‐yl moiety

Bicyclic dioxetanes 2a–c bearing a 2′‐alkoxy‐2‐hydroxy‐1,1′‐binaphthyl‐7‐yl moiety were effectively synthesized and their base‐induced chemiluminescent decomposition was investigated by the use of alkaline metal (Na⁺ and K⁺) or Mg²⁺ alkoxide in MeOH. When 2a–c were treated with tetrabutylammonium fluoride (TBAF) in dimethyl sulfoxide (DMSO) as a reference system, they showed chemiluminescence as a flash of orange light (maximum wavelength λ_max^CL = 573–577 nm) with efficiency Φ^CL = 6–8 × 10^–2. On the other hand, for an alkaline metal (Na⁺ or K⁺) alkoxide/MeOH system, 2a–c decomposed slowly to emit a glow of chemiluminescence, the spectra of which were shifted slightly toward red from the TBAF/DMSO system, and Φ^CL (= 1.4–2.3 × 10^–3) was considerably decreased. In addition, Mg(OMe)₂ was found to play a characteristic role as a base for the chemiluminescent decomposition of 2a–c through coordination to the intermediary oxidoaryl‐substituted dioxetanes 13. Thus, Mg²⁺ increased Φ^CL to more than twice those with Na⁺ or K⁺, while it shifted λ_max^CL considerably toward blue (λ_max^CL = 550–566 nm). Copyright © 2012 John Wiley & Sons, Ltd.     

Cyclic adenosine 5′‐diphosphoribose (cADPR) cyclic guanosine 3′,5′‐monophosphate positively function in Ca2+ elevation in methyl jasmonate‐induced stomatal closure,cADPR is required for methyl jasmonate‐induced ROS accumulation NO production in guard cells

Methyl jasmonate (MeJA) signalling shares several signal components with abscisic acid (ABA) signalling in guard cells. Cyclic adenosine 5′‐diphosphoribose (cADPR) and cyclic guanosine 3′,5′‐monophosphate (cGMP) are second messengers in ABA‐induced stomatal closure. In order to clarify involvement of cADPR and cGMP in MeJA‐induced stomatal closure in Arabidopsis thaliana (Col‐0), we investigated effects of an inhibitor of cADPR synthesis, nicotinamide (NA), and an inhibitor of cGMP synthesis, LY83583 (LY, 6‐anilino‐5,8‐quinolinedione), on MeJA‐induced stomatal closure. Treatment with NA and LY inhibited MeJA‐induced stomatal closure. NA inhibited MeJA‐induced reactive oxygen species (ROS) accumulation and nitric oxide (NO) production in guard cells. NA and LY suppressed transient elevations elicited by MeJA in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) in guard cells. These results suggest that cADPR and cGMP positively function in [Ca²⁺]_cyt elevation in MeJA‐induced stomatal closure, are signalling components shared with ABA‐induced stomatal closure in Arabidopsis, and that cADPR is required for MeJA‐induced ROS accumulation and NO production in Arabidopsis guard cells.     

Synthesis and in Vitro Antiviral Activities of [(Dihydrofuran‐2‐yl)oxy]methyl‐phosphonate Nucleosides with 2‐Substituted Adenine as Base

The synthesis of [(2′,5′‐dihydrofuran‐2‐yl)oxy]methyl‐phosphonate nucleosides with a 2‐substituted adenine base moiety starting from 2‐deoxy‐3,5‐bis‐O‐(4‐methylbenzoyl)‐α‐L ‐ribofuranosyl chloride and 2,6‐dichloropurine is described. The key step is the regiospecific and stereoselective introduction of a phosphonate synthon at C(2) of the furan ring. None of the synthesized compounds showed significant in vitro activity against HIV, BVDV, and HBV.     

A method for rapid and sensitive negative staining of proteins in SDS‐PAGE using 2′,7′‐dichlorofluorescein

We report here a rapid and sensitive technique for negative visualization of protein in 1D and 2D SDS‐PAGE by using 2′, 7′‐dichlorofluorescein (DCF), which appeared as transparent and colorless bands in an opaque gel matrix background. For DCF stain, down to 0.1–0.2 ng protein could be easily visualized within 7 min by only two steps, and the staining is fourfold more sensitive than that of Eosin Y (EY) negative stain and glutaraldehyde (GA) silver stain, and eightfold more sensitive than that of the commonly used imidazole‐zinc (IZ) negative stain. Furthermore, DCF stain provided good reproducibility, linearity, and MS compatibility compared with those of IZ stain. In addition, the potential staining mechanism was investigated by colorimetric experiment and molecular docking, and the results demonstrated that the interaction between DCF and protein occurs mainly via van der waals force, electrostatic interaction, and hydrogen bonding.     

Characterizing the release of bioactive N‐glycans from dairy products by a novel endo‐β‐N‐acetylglucosaminidase

Endo‐β‐N‐acetylglucosaminidase isolated from B. infantis ATCC 15697 (EndoBI‐1) is a novel enzyme that cleaves N‐N′‐diacetyl chitobiose moieties found in the N‐glycan core of high mannose, hybrid, and complex N‐glycans. These conjugated N‐glycans are recently shown as a new prebiotic source that stimulates the growth of a key infant gut microbe, Bifidobacterium longum subsp. Infantis. The effects of pH (4.45–8.45), temperature (27.5–77.5°C), reaction time (15–475 min), and enzyme/protein ratio (1:3,000–1:333) were evaluated on the release of N‐glycans from bovine colostrum whey by EndoBI‐1. A central composite design was used, including a two‐level factorial design (2⁴) with four center points and eight axial points. In general, low pH values, longer reaction times, higher enzyme/protein ratio, and temperatures around 52°C resulted in the highest yield. The results demonstrated that bovine colostrum whey, considered to be a by/waste product, can be used as a glycan source with a yield of 20 mg N‐glycan/g total protein under optimal conditions for the ranges investigated. Importantly, these processing conditions are suitable to be incorporated into routine dairy processing activities, opening the door for an entirely new class of products (released bioactive glycans and glycan‐free milk). The new enzyme's activity was also compared with a commercially available enzyme, showing that EndoBI‐1 is more active on native proteins than PNGase F and can be efficiently used during pasteurization, streamlining its integration into existing processing strategies. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1331–1339, 2015     

Low‐Cost N,N′‐Bicarbazole‐Based Dopant‐Free Hole‐Transporting Materials for Large‐Area Perovskite Solar Cells

Despite the recent unprecedented development of efficient dopant‐free hole transporting materials (HTMs) for high‐performance perovskite solar cells (PSCs) on small‐area devices (≤0.1 cm²), low‐cost dopant‐free HTMs for large‐area PSCs (≥1 cm²) with high power conversion efficiencies (PCEs) have rarely been reported. Herein, two novel HTMs, 3,3′,6,6′ (or 2,2′,7,7′)‐tetrakis(N,N′‐di‐p‐methoxyphenylamine)‐N,N′‐bicarbazole (3,6 BCz‐OMeTAD or 2,7 BCz‐OMeTAD), are synthesized via an extremely simple route from very cheap raw materials. Owing to their excellent film‐forming abilities and matching energy levels, 3,6 BCz‐OMeTAD and 2,7 BCz‐OMeTAD can be successfully employed as a perfect ultrathin (≈30 nm) hole transporting layer in large‐area PSCs up to 1 cm². The 3,6 BCz‐OMeTAD and 2,7 BCz‐OMeTAD based large‐area PSCs show highest PCEs up to 17.0% and 17.6%, respectively. More importantly, high performance large‐area PSCs based on 2,7 BCz‐OMeTAD retain 90% of the initial efficiency after 2000 h storage in an ambient environment without encapsulation.     

A study on the interaction between 3‐spiro‐piperidones and bovine serum albumin using spectroscopic approaches

The interaction between 3‐spiro‐2′‐pyrrolidine‐3′‐spiro‐3″‐piperidine‐2,3″‐dione (PPD) and bovine serum albumin (BSA) in aqueous solution was studied using fluorescence and UV–vis spectroscopy. Fluorescence emission data revealed that BSA (1.00 × 10^‐5 mol/L) fluorescence was statically quenched by PPD at various concentrations, which implies that a PPD–BSA complex was formed. The binding constant (K_A), the number of binding sites (n) and the specific binding site of the PPD with BSA were determined. Energy‐transfer efficiency parameters were determined and the mechanism of the interaction discussed. The thermodynamic parameters, ΔG, ΔH and ΔS, were obtained according to van't Hoff's equation, showing the involvement of hydrophobic forces in these interactions. The effect of PPD acting on the BSA conformation was detected by synchronous fluorescence. Copyright © 2012 John Wiley & Sons, Ltd.     

Crystal structure of the protein from Arabidopsis thaliana gene At5g06450, a putative DnaQ‐like exonuclease domain‐containing protein with homohexameric assembly

Arabidopsis thaliana gene At5g06450 encodes a putative DnaQ‐like 3′‐5′ exonuclease domain‐containing protein (AtDECP). The DnaQ‐like 3′‐5′ exonuclease domain is often found as a proofreading domain of DNA polymerases. The overall structure of AtDECP adopts an RNase H fold that consists of a mixed β‐sheet flanked by α‐helices. Interestingly, AtDECP forms a homohexameric assembly with a central six fold symmetry, generating a central cavity. The ring‐shaped structure and comparison with WRN‐exo, the best structural homologue of AtDECP, suggest a possible mechanism for implementing its exonuclease activity using positively charged patch on the N‐terminal side of the homohexameric assembly. The homohexameric structure of AtDECP provides unique information about the interaction between the DnaQ‐like 3′‐5′ exonuclease and its substrate nucleic acids.Proteins 2013. © 2013 Wiley Periodicals, Inc.     

Concurrent inhibition of NF‐κB,cyclooxygenase‐2, and epidermal growth factor receptor leads to greater anti‐tumor activity in pancreatic cancer

Inactivation of survival pathways such as NF‐κB, cyclooxygenase (COX‐2), or epidermal growth factor receptor (EGFR) signaling individually may not be sufficient for the treatment of advanced pancreatic cancer (PC) as suggested by recent clinical trials. 3,3′‐Diindolylmethane (B‐DIM) is an inhibitor of NF‐κB and COX‐2 and is a well‐known chemopreventive agent. We hypothesized that the inhibition of NF‐κB and COX‐2 by B‐DIM concurrently with the inhibition of EGFR by erlotinib will potentiate the anti‐tumor effects of cytotoxic drug gemcitabine, which has been tested both in vitro and in vivo. Inhibition of viable cells in seven PC cell lines treated with B‐DIM, erlotinib, or gemcitabine alone or their combinations was evaluated using 3‐(4,5‐dimetylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Significant inhibition in cell viability was observed in PC cells expressing high levels of COX‐2, EGFR, and NF‐κB proteins. The observed inhibition was associated with an increase in apoptosis as assessed by ELISA. A significant down‐regulation in the expression of COX‐2, NF‐κB, and EGFR in BxPC‐3, COLO‐357, and HPAC cells was observed, suggesting that simultaneous targeting of EGFR, NF‐κB, and COX‐2 is more effective than targeting either signaling pathway separately. Our in vitro results were further supported by in vivo studies showing that B‐DIM in combination with erlotinib and gemcitabine was significantly more effective than individual agents. Based on our preclinical in vitro and in vivo results, we conclude that this multi‐targeted combination could be developed for the treatment of PC patients whose tumors express high levels of COX‐2, EGFR, and NF‐κB. J. Cell. Biochem. 110: 171–181, 2010. © 2010 Wiley‐Liss, Inc.     

Crystal structure of a putative pyridoxine 5′‐phosphate oxidase (Rv2607) from Mycobacterium tuberculosis

The three‐dimensional structure of Rv2607, a putative pyridoxine 5′‐phosphate oxidase (PNPOx) from Mycobacterium tuberculosis, has been determined by X‐ray crystallography to 2.5 Å resolution. Rv2607 has a core domain similar to known PNPOx structures with a flavin mononucleotide (FMN) cofactor. Electron density for two FMN at the dimer interface is weak despite the bright yellow color of the protein solution and crystal. The shape and size of the putative binding pocket is markedly different from that of members of the PNPOx family, which may indicate some significant changes in the FMN binding mode of this protein relative to members of the family. Proteins 2006. © 2005 Wiley‐Liss, Inc.     

N‐methyl‐N‐nitrosourea induces retinal degeneration in the rat via the inhibition of NF‐κB activation

Retinitis pigmentosa (RP) is a group of inherited neurodegenerative diseases characterized by the loss of photoreceptor cells through apoptosis. N‐methyl‐N‐nitrosourea (MNU) is an alkylating toxicant that induces photoreceptor cell death resembling hereditary RP. This study aimed to investigate the role of nuclear factor κB (NF‐κB) in MNU‐induced photoreceptor degeneration. Adult rats received a single intraperitoneal injection of MNU (60 mg/kg bodyweight). Hematoxylin and eosin staining demonstrated progressive outer nuclear layer (ONL) loss after MNU treatment. Transmission electron microscopy revealed nuclear pyknosis, chromatin margination in the photoreceptors, increased secondary lysosomes, and lobulated retinal‐pigmented epithelial cells in MNU‐treated rats. Numerous photoreceptor cells in the ONL showed positive TUNEL staining and apoptosis rate peaked at 24 hours. Enhanced depth imaging spectral‐domain optical coherence tomography showed ONL thinning and decreased choroid thickness. Electroretinograms showed decreased A wave amplitude that predominated in scotopic conditions. Western blot analysis showed that nuclear IκBα level increased, whereas nuclear NF‐κB p65 decreased significantly in the retinas of MNU‐treated rats. These findings indicate that MNU leads to selective photoreceptor degradation, and this is associated with the inhibition of NF‐κB activation.     

An Alternative Standard for Trolox‐Equivalent Antioxidant‐Capacity Estimation Based on Thiol Antioxidants. Comparative 2,2′‐Azinobis[3‐ethylbenzothiazoline‐6‐sulfonic Acid] Decolorization and Rotational Viscometry Study Regarding Hyaluronan Degradation

Comparison of the effectiveness of antioxidant activity of three thiol compounds, D ‐penicillamine, reduced L ‐glutathione, and 1,4‐dithioerythritol, expressed as a radical‐scavenging capacity based on the two independent methods, namely a decolorization 2,2′‐azinobis[3‐ethylbenzothiazoline‐6‐sulfonic acid] assay and a rotational viscometry, is reported. Particular concern was focused on the testing of potential free‐radical scavenging effects of thiols against hyaluronan degradation, induced by hydroxyl radicals. A promising, solvent‐independent, antioxidative function of 1,4‐dithioerythritol, comparable to that of a standard compound, Trolox^®, was confirmed by the 2,2′‐azinobis[3‐ethylbenzothiazoline‐6‐sulfonic acid] assay. The new potential antioxidant 1,4‐dithioerythritol exhibited very good solubility in a variety of solvents (e.g., H₂O, EtOH, and DMSO) and could be widely accepted and used as an effective antioxidant standard instead of a routinely used Trolox^® on 2,2′‐azinobis[3‐ethylbenzothiazoline‐6‐sulfonic acid] assay.     

Determination of the Absolute Configuration of Two Pairs of C‐8 − C‐9′ Linked Neolignan Enantiomers

Two pairs of new neolignan enantiomers, (±)‐torreyayunan A ( 1a / 1b ) and (±)‐torreyayunan B ( 2a / 2b ), featuring a rare C‐8 ? C‐9′ linked skeleton, were isolated from leaves and twigs of Torreya yunnanensis. Their absolute configuration involving two chiral centers was determined by combined spectral and Density Functional Theory (DFT) calculation. This is the first report of the absolute configuration of this group of neolignans. Chirality 26:825–828, 2014. © 2014 Wiley Periodicals, Inc.     

Genistein‐3′‐sodium sulphonate protects against lipopolysaccharide‐induced lung vascular endothelial cell apoptosis and acute lung injury via BCL‐2 signalling

Under septic conditions, Lipopolysaccharide (LPS)‐induced apoptosis of lung vascular endothelial cells (ECs) triggers and aggravates acute lung injury (ALI), which so far has no effective therapeutic options. Genistein‐3′‐sodium sulphonate (GSS) is a derivative of native soy isoflavone, which has neuro‐protective effects through its anti‐apoptotic property. However, whether GSS protects against sepsis‐induced lung vascular endothelial cell apoptosis and ALI has not been determined. In this study, we found that LPS‐induced Myd88/NF‐κB/BCL‐2 signalling pathway activation and subsequent EC apoptosis were effectively down‐regulated by GSS in vitro. Furthermore, GSS not only reversed the sepsis‐induced BCL‐2 changes in expression in mouse lungs but also blocked sepsis‐associated lung vascular barrier disruption and ALI in vivo. Taken together, our results demonstrated that GSS might be a promising candidate for sepsis‐induced ALI via its regulating effects on Myd88/NF‐κB/BCL‐2 signalling in lung ECs.