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.     

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.     

Determination of 3′-azido-2′,3′-dideoxyuridine in maternal plasma, amniotic fluid, fetal and placental tissues by high-performance liquid chromatography

3′-Azido-2′,3′-dideoxyuridine (AZDU, Azddu, CS-87) is a nucleoside analog of 3′-azido-3′-deoxythymidine (zidovudine, AZT) that has been shown to inhibit human immunodeficiency virus (HIV-1). AZDU is a potential candidate for treatment of pregnant mothers to prevent prenatal transmission of HIV/AIDS to their unborn children. A rapid and efficient high-performance liquid chromatography (HPLC) method for the determination of AZDU concentrations in rat maternal plasma, amniotic fluid, placental and fetal tissue samples has been developed and validated. Tissue samples were homogenized in distilled water, protein precipitated and extracted using a C-18 solid-phase extraction (SPE) method prior to analysis. Plasma and amniotic fluid samples were protein precipitated with 2 M perchloric acid prior to analysis. Baseline resolution was achieved using a 4.5% acetonitrile in 40 mM sodium acetate (pH 7) buffer mobile phase for amniotic fluid, placenta and fetus samples and with a 5.5% acetonitrile in buffer solution for plasma at flow-rates of 2.0 ml/min. The HPLC system consists of a Hypersil ODS column (150×4.6 mm) with a Nova-Pak C-18 guard column with detection at 263 nm. The method yields retention times of 6.2 and 12.2 min for AZDU and AZT in plasma and 8.3 and 17.6 min for AZDU and AZT in amniotic fluid, fetal and placental tissues. Limits of detection ranged from 0.01 to 0.075 μg/ml. Recoveries ranged from 81 to 96% for AZDU and from 82 to 96% for AZT in the different matrices. Intra-day (n=6) and inter-day (n=9) precision (% RSD) and accuracy (% Error) ranged from 1.48 to 6.25% and from 0.50 to 10.07%, respectively.     

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.     

Amyloid β‐induced astrogliosis is mediated by β1‐integrin via NADPH oxidase 2 in Alzheimer's disease

Astrogliosis is a hallmark of Alzheimer′s disease (AD) and may constitute a primary pathogenic component of that disorder. Elucidation of signaling cascades inducing astrogliosis should help characterizing the function of astrocytes and identifying novel molecular targets to modulate AD progression. Here, we describe a novel mechanism by which soluble amyloid‐β modulates β1‐integrin activity and triggers NADPH oxidase (NOX)‐dependent astrogliosis in vitro and in vivo. Amyloid‐β oligomers activate a PI3K/classical PKC/Rac1/NOX pathway which is initiated by β1‐integrin in cultured astrocytes. This mechanism promotes β1‐integrin maturation, upregulation of NOX2 and of the glial fibrillary acidic protein (GFAP) in astrocytes in vitro and in hippocampal astrocytes in vivo. Notably, immunochemical analysis of the hippocampi of a triple‐transgenic AD mouse model shows increased levels of GFAP, NOX2, and β1‐integrin in reactive astrocytes which correlates with the amyloid β‐oligomer load. Finally, analysis of these proteins in postmortem frontal cortex from different stages of AD (II to V/VI) and matched controls confirmed elevated expression of NOX2 and β1‐integrin in that cortical region and specifically in reactive astrocytes, which was most prominent at advanced AD stages. Importantly, protein levels of NOX2 and β1‐integrin were significantly associated with increased amyloid‐β load in human samples. These data strongly suggest that astrogliosis in AD is caused by direct interaction of amyloid β oligomers with β1‐integrin which in turn leads to enhancing β1‐integrin and NOX2 activity via NOX‐dependent mechanisms. These observations may be relevant to AD pathophysiology.     

Kinetic studies of the effect of a 17‐nucleotide difference in the 0.3‐kb region containing the R‐U5‐5′ leader sequence of Friend murine leukemia virus on viral gene expression

In addition to the env gene, a 0.3‐kb fragment containing the R‐U5‐5′ leader sequence is essential for the induction of spongiform neurodegeneration by Friend murine leukemia virus (Fr‐MLV) clone A8 and it also influences expression of the Env protein. Kinetic studies were carried out using two recombinant viruses, R7f, carrying the A8 0.3‐kb fragment, and Rec5, carrying the 0.3‐kb fragment of the non‐neuropathogenic Fr‐MLV clone 57. These analyses suggested that the 0.3‐kb fragment influenced the expression level of the Env protein by regulating the amount of spliced env‐mRNA rather than the amount of total viral mRNA or viral production.     

Targeting human telomeric G‐quadruplex DNA with antitumour natural alkaloid aristololactam‐β‐D‐glucoside and its comparison with daunomycin

Study on anticancer agents that act via stabilization of telomeric G‐quadruplex DNA has emerged as novel and exciting field for anticancer drug discovery. The interaction of carbohydrate containing anticancer alkaloid aristololactam‐β‐D‐glucoside (ADG) with human telomeric G‐quadruplex DNA sequence was characterized by different biophysical techniques. The binding parameters were compared with daunomycin (DAN), a well‐known chemotherapeutic drug. The Scatchard binding isotherms revealed noncooperative binding for both with the binding affinity values of (1.01 ± 0.05) × 10⁶ and (1.78 ± 0.18) × 10⁶ M⁻¹ for ADG and DAN, respectively. Circular dichroism, ferrocyanide quenching study, anisotropy study, thiazole orange displacement, optical melting, differential scanning calorimetry study, and molecular docking study suggest significant stacking and stabilizing efficiency of ADG with comparison to DAN. The energetics of the interaction for ADG and DAN revealed that both reactions were predominantly entropy driven. Negative heat capacity values were obtained from the temperature dependence of the enthalpy change. The standard molar Gibbs energy change exhibited only marginal alterations with temperature suggesting the occurrence of enthalpy‐entropy compensation. These findings indicate that ADG can act as a stabilizer of telomeric G‐quadruplex DNA and thereby can be considered as a potential telomerase inhibitor.     

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.     

The role of 5′-methylthioadenosine phosphorylase in 5′-methylthioadenosine-mediated inhibition of lymphocyte transformation

To determine if increased 5′-methylthioadenosine phosphorylase activity in activated lymphocytes may be responsible for the decreased inhibitory effect noted when 5′-methylthioadenosine is added after stimulation, the activity of this enzyme was monitored during lymphocyte transformation. A direct correlation existed between the transformation process and 5′-methylthioadenosine phosphorylase activity; the longer the stimulation process progressed, the greater the enzyme activity. The 7-deaza analog of 5′-methylthioadenosine, 5′-methylthiotubercidin, was utilized to explore further the role that the phosphorylase may play in the reversal process. 5′-Methylthioadenosine acted as a potent inhibitor, but not a substrate, of the 5′-methylthioadenosine phosphorylase, and was an even more potent inhibitor of lymphocyte transformation than 5′-methylthioadenosine. However, in direct contrast to the 5′-methylthioadenosine effect, inhibition by 5′-methylthiotubercidin could not be completely reversed. These data suggest the 5′-methylthioadenosine phosphorylase plays an important role in reversing 5′-methylthioadenosine-mediated inhibition and that the potent, nonreversible inhibitory effects of 5′-methylthiotubercidin are due to its resistance to 5′-methylthioadenosine phosphorylase degradation.     

Structural studies on KijD1, a sugar C‐3′‐methyltransferase

Kijanimicin is an antitumor antibiotic isolated from Actinomadura kijaniata. It is composed of three distinct moieties: a pentacyclic core, a monosaccharide referred to as d ‐kijanose, and a tetrasaccharide chain composed of l ‐digitoxose units. d ‐Kijanose is a highly unusual nitro‐containing tetradeoxysugar, which requires at least ten enzymes for its production. Here we describe a structural analysis of one of these enzymes, namely KijD1, which functions as a C‐3′‐methyltransferase using S‐adenosylmethionine as its cofactor. For this investigation, two ternary complexes of KijD1, determined in the presence of S‐adenosylhomocysteine (SAH) and dTDP or SAH and dTDP‐3‐amino‐2,3,6‐trideoxy‐4‐keto‐3‐methyl‐d ‐glucose, were solved to 1.7 or 1.6 Å resolution, respectively. Unexpectedly, these structures, as well as additional biochemical analyses, demonstrated that the quaternary structure of KijD1 is a dimer. Indeed, this is in sharp contrast to that previously observed for the sugar C‐3′‐methyltransferase isolated from Micromonospora chalcea. By the judicious use of site‐directed mutagenesis, it was possible to convert the dimeric form of KijD1 into a monomeric version. The quaternary structure of KijD1 could not have been deduced based solely on bioinformatics approaches, and thus this investigation highlights the continuing need for experimental validation.     

Superbanone,A New 2‐Aryl‐3‐benzofuranone and Other Bioactive Constituents from the Tube Roots of Butea superba

Phytochemical investigation from the tube roots of Butea superba, led to the isolation and identification of a new 2‐aryl‐3‐benzofuranone named superbanone ( 1 ), one benzoin, 2‐hydroxy‐1‐(2‐hydroxy‐4‐methoxyphenyl)‐2‐(4‐methoxyphenyl)ethanone ( 2 ), eight pterocarpans ( 3  –  10 ), and eleven isoflavonoids ( 11  –  21 ). Compound 2 was identified for the first time as a natural product. The structure of the isolated compounds was elucidated using spectroscopic methods, mainly 1D‐ and 2D‐NMR. The isolated compounds and their derivatives were evaluated for α‐glucosidase inhibitory and antimalarial activities. Compounds 3 , 7 , 8 , and 11 showed promising α‐glucosidase inhibitory activity (IC₅₀ = 13.71 ± 0.54, 23.54 ± 0.75, 28.83 ± 1.02, and 12.35 ± 0.36 μm , respectively). Compounds 3 and 11 were twofold less active than the standard drug acarbose (IC₅₀ = 6.54 ± 0.04 μm ). None of the tested compounds was found to be active against Plasmodium falciparum strain 94. On the basis of biological activity results, structure–activity relationships are discussed.     

Confirming therapeutic target of protopine using immobilized β2‐adrenoceptor coupled with site‐directed molecular docking and the target‐drug interaction by frontal analysis and injection amount–dependent method

Drug‐protein interaction analysis is pregnant in designing new leads during drug discovery. We prepared the stationary phase containing immobilized β₂‐adrenoceptor (β ₂‐ AR) by linkage of the receptor on macroporous silica gel surface through N ,N ′‐carbonyldiimidazole method. The stationary phase was applied in identifying antiasthmatic target of protopine guided by the prediction of site‐directed molecular docking. Subsequent application of immobilized β ₂‐ AR in exploring the binding of protopine to the receptor was realized by frontal analysis and injection amount–dependent method. The association constants of protopine to β ₂‐ AR by the 2 methods were (1.00 ± 0.06) × 10⁵M⁻¹ and (1.52 ± 0.14) × 10⁴M⁻¹. The numbers of binding sites were (1.23 ± 0.07) × 10⁻⁷M and (9.09 ± 0.06) × 10⁻⁷M, respectively. These results indicated that β ₂‐ AR is the specific target for therapeutic action of protopine in vivo. The target‐drug binding occurred on Ser¹⁶⁹ in crystal structure of the receptor. Compared with frontal analysis, injection amount–dependent method is advantageous to drug saving, improvement of sampling efficiency, and performing speed. It has grave potential in high‐throughput drug‐receptor interaction analysis.     

Synthesis and discovery of potent carbonic anhydrase,acetylcholinesterase, butyrylcholinesterase,and α‐glycosidase enzymes inhibitors: The novel N,N′‐bis‐cyanomethylamine and alkoxymethylamine derivatives

During this investigation, N,N′‐bis‐azidomethylamines, N,N′‐bis‐cyanomethylamine, new alkoxymethylamine and chiral derivatives, which are considered to be a new generation of multifunctional compounds, were synthesized, functional properties were investigated, and anticholinergic and antidiabetic properties of those compounds were studied through the laboratory tests, and it was approved that they contain physiologically active compounds rather than analogues. Novel N‐bis‐cyanomethylamine and alkoxymethylamine derivatives were effective inhibitors of the α‐glycosidase, cytosolic carbonic anhydrase I and II isoforms, butyrylcholinesterase (BChE), and acetylcholinesterase (AChE) with K_i values in the range of 0.15–13.31 nM for α‐glycosidase, 2.77–15.30 nM for human carbonic anhydrase isoenzymes I (hCA I), 3.12–21.90 nM for human carbonic anhydrase isoenzymes II (hCA II), 23.33–73.23 nM for AChE, and 3.84–48.41 nM for BChE, respectively. Indeed, the inhibition of these metabolic enzymes has been considered as a promising factor for pharmacologic intervention in a diversity of disturbances.     

Rapid quantitation of (−)-2′-deoxy-3′-thiacytidine in human serum by high-performance liquid chromatography with ultraviolet detection

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed and validated for the measurement of (−)-2′-deoxy-3′-thiacytidine (3TC) in human serum. The method included precipitation of serum proteins by trichloroacetic acid (20%, w/v) treatment followed by centrifugation. The resulting supernatant was directly injected and 3TC was isocratically chromatographed on a reversed-phase C₁₈ column using a mixture of phosphate buffer and methanol (88.3:11.7, v/v) and monitored at 280 nm. The limit of quantitation was 20 ng/ml using 100 μl of serum. The standard curve was linear within the range of 20–10 000 ng/ml. Replicate analysis of three quality control samples (40–1500 ng/ml) led to satisfactory intra- and itner-assay precision (coefficient of variation from 3.0 to 12.9%) and accuracy (deviation from −6.3 to 9.7%). Moreover, sample treatment processes including human immunodeficiency virus (HIV) heat-inactivation, exposure at room temperature and freezing-thawing cycles did not influence the stability of the analyte. This assay was successfully applied to the determination of 3TC serum levels in HIV-infected patients. In addition, preliminary results indicated that this procedure may also be extended to the measurement of 3TC in human plasma and urine.     

Hyperthermia‐induced seizures alter adenosine A1 and A2A receptors and 5′‐nucleotidase activity in rat cerebral cortex

Febrile seizure is one of the most common convulsive disorders in children. The neuromodulator adenosine exerts anticonvulsant actions through binding adenosine receptors. Here, the impact of hyperthermia‐induced seizures on adenosine A₁ and A_2A receptors and 5′‐nucleotidase activity has been studied at different periods in the cerebral cortical area by using radioligand binding, real‐time PCR, and 5′‐nucleotidase activity assays. Hyperthermic seizures were induced in 13‐day‐old rats using a warmed air stream from a hair dryer. Neonates exhibited rearing and falling over associated with hindlimb clonus seizures (stage 5 on Racine scale criteria) after hyperthermic induction. A significant increase in A₁ receptor density was observed using [³H]DPCPX as radioligand, and mRNA coding A₁ was observed 48 h after hyperthermia‐induced seizures. In contrast, a significant decrease in A_2A receptor density was detected, using [³H]ZM241385 as radioligand, 48 h after hyperthermia‐evoked convulsions. These short‐term changes in A₁ and A_2A receptors were also accompanied by a loss of 5′‐nucleotidase activity. No significant variations either in A₁ or A_2A receptor density or 5′‐nucleotidase were observed 5 and 20 days after hyperthermic seizures. Taken together, both regulation of A₁ and A_2A receptors and loss of 5′‐nucleotidase in the cerebral cortex suggest the existence of a neuroprotective mechanism against seizures.

     

Photoluminescence imaging of europium (III)‐doped γ‐Al2O3 nanofiber structures

Aluminium oxide (Al₂O₃) has widely been used for catalysts, insulators, and composite materials for diverse applications. Herein, we demonstrated if γ‐Al₂O₃ was useful as a luminescence support material for europium (Eu) (III) activator ion. The hydrothermal method and post‐thermal treatment at 800°C were employed to synthesize Eu(III)‐doped γ‐Al₂O₃ nanofibre structures. Luminescence characteristics of Eu(III) ions in Al₂O₃ matrix were fully understood by taking 2D and 3D‐photoluminescence imaging profiles. Various sharp emissions between 580 to 720 nm were assigned to the ⁵D₀→⁷F_J (J = 0, 1, 2, 3, 4) transitions of Eu(III) activators. On the basis of X‐ray diffraction crystallography, Auger elemental mapping and the asymmetry ratio, Eu(III) ions were found to be well doped into the γ‐Al₂O₃ matrix at a low (1 mol%) doping level. A broad emission at 460 nm was substantially increased upon higher (2 mol%) Eu(III) doping due to defect creation. The first 3D photoluminescence imaging profiles highlight detailed understanding of emission characteristics of Eu(III) ions in Al oxide‐based phosphor materials and their potential applications.     

Protein kinase D1 regulates ERα‐positive breast cancer cell growth response to 17β‐estradiol and contributes to poor prognosis in patients

About 70% of human breast cancers express and are dependent for growth on estrogen receptor α (ERα), and therefore are sensitive to antiestrogen therapies. However, progression to an advanced, more aggressive phenotype is associated with acquisition of resistance to antiestrogens and/or invasive potential. In this study, we highlight the role of the serine/threonine‐protein kinase D1 (PKD1) in ERα‐positive breast cancers. Growth of ERα‐positive MCF‐7 and MDA‐MB‐415 human breast cancer cells was assayed in adherent or anchorage‐independent conditions in cells overexpressing or depleted for PKD1. PKD1 induces cell growth through both an ERα‐dependent manner, by increasing ERα expression and cell sensitivity to 17β‐estradiol, and an ERα‐independent manner, by reducing cell dependence to estrogens and conferring partial resistance to antiestrogen ICI 182,780. PKD1 knockdown in MDA‐MB‐415 cells strongly reduced estrogen‐dependent and independent invasion. Quantification of PKD1 mRNA levels in 38 cancerous and non‐cancerous breast cell lines and in 152 ERα‐positive breast tumours from patients treated with adjuvant tamoxifen showed an association between PKD1 and ERα expression in 76.3% (29/38) of the breast cell lines tested and a strong correlation between PKD1 expression and invasiveness (P < 0.0001). In tamoxifen‐treated patients, tumours with high PKD1 mRNA levels (n = 77, 50.66%) were significantly associated with less metastasis‐free survival than tumours with low PKD1 mRNA expression (n = 75, 49.34%; P = 0.031). Moreover, PKD1 mRNA levels are strongly positively associated with EGFR and vimentin levels (P < 0.0000001). Thus, our study defines PKD1 as a novel attractive prognostic factor and a potential therapeutic target in breast cancer.     

Prokaryotic expression and antimicrobial mechanism of XPF‐St7‐derived α‐helical peptides

XPF‐St7 (GLLSNVAGLLKQFAKGGVNAVLNPK) is an antimicrobial peptide isolated from Silurana tropicalis. We developed an α‐helical segment of XPF‐St7 termed as XPF2. Using the XPF2 as a framework, we increased the positive net charge of XPF2 by amino acid substitutions, and thus obtained two novel antimicrobial peptides XPF4 and XPF6. These were each fused with an ubiquitin tag and successfully expressed in Escherichia coli. This ubiquitin fusion system may present a viable alternative for industrial production of antimicrobial peptides. XPF4 and XPF6 showed much better overall antimicrobial activity against both Gram‐negative and Gram‐positive bacteria than XPF2. The therapeutic index of XPF4 and XPF6 was 5.6‐fold and 6.7‐fold of XPF2, respectively. Bacterial cell membrane permeabilization and genomic DNA interaction assays were utilized to explore the mechanism of action of XPF serial peptides. The results revealed that the target of these antimicrobial peptides was the bacterial cytoplasmic membrane. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.