Binding Pockets and Permeation Channels for Dioxygen through Cofactorless 3‐Hydroxy‐2‐methylquinolin‐4‐one 2,4‐Dioxygenase in Association with Its Natural Substrate, 3‐Hydroxy‐2‐methylquinolin‐4(1H)‐one. A Perspective from Molecular Dynamics Simulations

This work describes an investigation of pathways and binging pockets (BPs) for dioxygen (O₂) through the cofactorless oxygenase 3‐hydroxy‐2‐methylquinolin‐4‐one 2,4‐dioxygenase in complex with its natural substrate, 3‐hydroxy‐2‐methylquinolin‐4(1H)‐one, in aqueous solution. The investigation tool was random‐acceleration molecular dynamics (RAMD), whereby a tiny, randomly oriented external force is applied to O₂ in order to accelerate its movements. In doing that, care was taken that the external force only continues, if O₂ moves along a direction for a given period of time, otherwise the force changed direction randomly. Gates for expulsion of O₂ from the protein, which can also be taken as gates for O₂ uptake, were found throughout almost the whole external surface of the protein, alongside a variety of BPs for O₂. The most exploited gates and BPs were not found to correspond to the single gate and BP proposed previously from the examination of the static model from X‐ray diffraction analysis of this system. Therefore, experimental investigations of this system that go beyond the static model are urgently needed.     

Mapping of O‐linked β‐N‐acetylglucosamine modification sites in key contractile proteins of rat skeletal muscle

O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) is a widespread modification of serine/threonine residues of nucleocytoplasmic proteins. Recently, several key contractile proteins in rat skeletal muscle (i.e., myosin heavy and light chains and actin) were identified as O‐GlcNAc modified. Moreover, it was demonstrated that O‐GlcNAc moieties involved in contractile protein interactions could modulate Ca²⁺ activation parameters of contraction. In order to better understand how O‐GlcNAc can modulate the contractile activity of muscle fibers, we decided to identify the sites of O‐GlcNAc modification in purified contractile protein homogenates. Using an MS‐based method that relies on mild β‐elimination followed by Michael addition of DTT (BEMAD), we determined the localization of one O‐GlcNAc site in the subdomain four of actin and four O‐GlcNAc sites in the light meromyosin region of myosin heavy chains (MHC). According to previous reports concerning the role of these regions, our data suggest that O‐GlcNAc sites might modulate the actin–tropomyosin interaction, and be involved in MHC polymerization or interactions between MHC and other contractile proteins. Thus, the results suggest that this PTM might be involved in protein–protein interactions but could also modulate the contractile properties of skeletal muscle.     

Insight into a strategy for attenuating AmpC‐mediated β‐lactam resistance: Structural basis for selective inhibition of the glycoside hydrolase NagZ

NagZ is an exo‐N‐acetyl‐β‐glucosaminidase, found within Gram‐negative bacteria, that acts in the peptidoglycan recycling pathway to cleave N‐acetylglucosamine residues off peptidoglycan fragments. This activity is required for resistance to cephalosporins mediated by inducible AmpC β‐lactamase. NagZ uses a catalytic mechanism involving a covalent glycosyl enzyme intermediate, unlike that of the human exo‐N‐acetyl‐β‐glucosaminidases: O‐GlcNAcase and the β‐hexosaminidase isoenzymes. These latter enzymes, which remove GlcNAc from glycoconjugates, use a neighboring‐group catalytic mechanism that proceeds through an oxazoline intermediate. Exploiting these mechanistic differences we previously developed 2‐N‐acyl derivatives of O‐(2‐acetamido‐2‐deoxy‐D ‐glucopyranosylidene)amino‐N‐phenylcarbamate (PUGNAc), which selectively inhibits NagZ over the functionally related human enzymes and attenuate antibiotic resistance in Gram‐negatives that harbor inducible AmpC. To understand the structural basis for the selectivity of these inhibitors for NagZ, we have determined its crystallographic structure in complex with N‐valeryl‐PUGNAc, the most selective known inhibitor of NagZ over both the human β‐hexosaminidases and O‐GlcNAcase. The selectivity stems from the five‐carbon acyl chain of N‐valeryl‐PUGNAc, which we found ordered within the enzyme active site. In contrast, a structure determination of a human O‐GlcNAcase homologue bound to a related inhibitor N‐butyryl‐PUGNAc, which bears a four‐carbon chain and is selective for both NagZ and O‐GlcNAcase over the human β‐hexosamnidases, reveals that this inhibitor induces several conformational changes in the active site of this O‐GlcNAcase homologue. A comparison of these complexes, and with the human β‐hexosaminidases, reveals how selectivity for NagZ can be engineered by altering the 2‐N‐acyl substituent of PUGNAc to develop inhibitors that repress AmpC mediated β‐lactam resistance.     

Scavenging of reactive oxygen species by N‐substituted indole‐2 and 3‐carboxamides

Indole‐2 and 3‐carboxamides (IDs) are proposed to be selective cyclooxygenase inhibitors. Since cyclooxygenase‐1 may be involved in reactive oxygen species (ROS) production, we hypothesize that these indole derivatives have antioxidative properties. We have employed chemiluminescence (CL) and electron spin resonance (ESR) spin trapping to examine this hypothesis. We report here the results of a study of reactivity of 10 selected indole derivatives towards ROS. The following generators of ROS were applied: potassium superoxide (KO₂) as a source of superoxide radicals (O₂^·?), the Fenton reaction (Co‐EDTA/H₂O₂) for hydroxyl radicals (HO^·), and a mixture of alkaline aqueous H₂O₂ and acetonitrile for singlet oxygen (¹O₂). Hydroxyl radicals were detected as 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) spin adduct, whereas 2,2,6,6‐tetramethyl‐piperidine (TEMP) was used as a detector of ¹O₂. Using the Fenton reaction, 0.5 mmol/L IDs were found to inhibit DMPO‐?H radical formation in the range 7–37%. Furthermore the tested compounds containing the thiazolyl group also inhibited the ¹O₂‐dependent TEMPO radical, generated in the acetonitrile + H₂O₂ system. About 20% inhibition was obtained in the presence of 0.5 mmol/L IDs. 1 mmol/L IDs caused an approximately 13–70% decrease in the CL sum from the O₂^·? generating system (1 mmol/L). The aim of this paper is to evaluate these indole derivatives as antioxidants and their abilities to scavenge ROS. Copyright © 2004 John Wiley & Sons, Ltd.     

Resolution of 1‐n‐Butyl‐3‐Methyl‐3‐Phospholene 1‐Oxide With TADDOL Derivatives and Calcium Salts of O,O'‐Dibenzoyl‐(2R,3R)‐ or O,O'‐di‐p‐Toluoyl‐(2R,3R)‐tartaric Acid

The resolution methods applying (?)‐(4R,5R)‐4,5‐bis(diphenylhydroxymethyl)‐2,2‐dimethyldioxolane (“TADDOL”), (?)‐(2R,3R)‐α,α,α',α'‐tetraphenyl‐1,4‐dioxaspiro[4.5]decan‐2,3‐dimethanol (“spiro‐TADDOL”), as well as the acidic and neutral Ca²⁺ salts of (?)‐O,O'‐dibenzoyl‐ and (?)‐O,O'‐di‐p‐toluoyl‐(2R,3R)‐tartaric acid were extended for the preparation of 1‐n‐butyl‐3‐methyl‐3‐phospholene 1‐oxide in optically active form. In one case, the intermediate diastereomeric complex could be identified by single‐crystal X‐ray analysis. The absolute P‐configuration of the enantiomers of the phospholene oxide was also determined by comparing the experimentally obtained and calculated CD spectra. Chirality 26:174–182, 2014. © 2014 Wiley Periodicals, Inc.     

11α‐Ethoxy‐β‐boswellic Acid and Nizwanone,a New Boswellic Acid Derivative and a New Triterpene,Respectively, from Boswellia sacra

A new boswellic acid derivative, 11α‐ethoxy‐β‐boswellic acid (EBA; 1 ) and a new ursane‐type triterpene, named nizwanone ( 2 ), were isolated from Omani frankincense Boswellia sacra Flueck . together with two known compounds papyriogenin B and rigidenol. The structures of 1 and 2 were elucidated by detailed spectroscopic analysis using ¹H‐ and ¹³C‐NMR, ¹H,¹H‐COSY, HMQC, HMBC, and HR‐EI‐MS techniques. The relative configurations of 1 and 2 were assigned by comparative analysis of the NMR spectral data with those of known analogs together with NOESY experiments. Structures of known compounds were identified by comparison with the reported data.     

Desensitization of β‐adrenergic receptors in lung injury induced by 2‐chloroethyl ethyl sulfide,a mustard analog

2‐Choloroethyl Ethyl Sulfide (CEES) exposure causes inflammatory lung diseases, including acute respiratory distress syndrome (ARDS) and pulmonary fibrosis. This may be associated with oxidative stress, which has been implicated in the desensitization of beta‐adrenergic receptors (β‐ARs). The objective of this study was to investigate whether lung injury induced by intratracheal CEES exposure (2 mg/kg body weight) causes desensitization of β‐ARs. The animals were sacrificed after 7 days and lungs were removed. Lung injury was established by measuring the leakage of iodinated‐bovine serum albumin ([¹²⁵I]‐BSA) into lung tissue. Receptor‐binding characteristics were determined by measuring the binding of [³H] dihydroalprenolol ([³H] DHA) (0.5–24 nM) to membrane fraction in the presence and absence of DLDL ‐propranolol (10 μ M). Both high‐ and low‐affinity β‐ARs were identified in the lung. Binding capacity was significantly higher in low‐affinity site in both control and experimental groups. Although CEES exposure did not change K_D and B_max at the high‐affinity site, it significantly decreased both K_D and B_max at low affinity sites. A 20% decrease in β₂‐AR mRNA level and a 60% decrease in membrane protein levels were observed in the experimental group. Furthermore, there was significantly less stimulation of adenylate cyclase activity by both cholera toxin and isoproterenol in the experimental group in comparison to the control group. Treatment of lungs with 3‐isobutyl‐1‐methylxanthine (IBMX), an inhibitor of phosphodiesterase (PDE) could not abolish the difference between the control group and the experimental group on the stimulation of the adenylate cyclase activity. Thus, our study indicates that CEES‐induced lung injury is associated with desensitization of β₂‐AR. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:59–70, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20265     

Self‐Assembled BiFeO3‐ε‐Fe2O3 Vertical Heteroepitaxy for Visible Light Photoelectrochemistry

Self‐assembled vertical heterostructure with a high interface‐to‐volume ratio offers tremendous opportunities to realize intriguing properties and advanced modulation of functionalities. Here, a heterostructure composed of two visible‐light photocatalysts, BiFeO₃ (BFO) and ε‐Fe₂O₃ (ε‐FO), is designed to investigate its photoelectrochemical performance. The structural characterization of the BFO‐FO heterostructures confirms the phase separation with BFO nanopillars embedded in the ε‐FO matrix. The investigation of band structure of the heterojunction suggests the assistance of photoexcited carrier separation, leading to an enhanced photoelectrochemical performance. The insights into the charge separation are further revealed by means of ultrafast dynamics and electrochemical impedance spectroscopies. This work shows a delicate design of the self‐assembled vertical heteroepitaxy by taking advantage of the intimate contact between two phases that can lead to a tunable charge interaction, providing a new configuration for the optimization of photoelectrochemical cell.     

Detection of nonopioid β‐endorphin receptor in the rat myocardium

Two selective agonists of nonopioid β‐endorphin receptor, synthetic peptides TPLVTLFK (octarphin) and SLTCLVKGFY (immunorphin), were labeled with tritium to specific activity of 29 and 25 Ci/mmol, respectively. Both labeled peptides were found to bind to high‐affinity naloxone‐insensitive binding sites on the membranes isolated from the rat myocardium (Kd = 2.0 ± 0.2 and 2.5 ± 0.3 nM, respectively). The [³H]octarphin specific binding to the myocardial membranes was inhibited by unlabeled β‐endorphin (Ki = 1.9 ± 0.2 nM) and immunorphin (Ki = 2.2 ± 0.3 nM). The [³H]immunorphin specific binding with the membranes was inhibited by unlabeled β‐endorphin (Ki = 2.3 ± 0.3 nM) and octarphin (Ki = 2.4 ± 0.3 nM). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but also to α‐endorphin, γ‐endorphin, [Met⁵]enkephalin and [Leu⁵]enkephalin. Thus, β‐endorphin, immunorphin and octarphin bind to the common high‐affinity naloxone‐insensitive receptor of the rat myocardial membranes. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Separation of Enantiomers by Inclusion Gas Chromatography: On the Influence of Water in the Molecular Complexation of Methyl 2‐Chloropropanoate Enantiomers and the Modified γ‐Cyclodextrin Lipodex‐E

A profound influence of water has previously been detected in the complexation of the enantiomers of methyl 2‐chloropropanoate (MCP) and the chiral selector octakis(3‐O‐butanoyl‐2,6‐di‐O‐pentyl)‐γ‐cyclodextrin (Lipodex‐E) in NMR and sensor experiments. We therefore investigated the retention behavior of MCP enantiomers on Lipodex‐E by gas chromatography (GC) under hydrous conditions. Addition of water to the N₂ carrier gas modestly reduced the retention factors k of the enantiomers, notably for the second eluted enantiomer (S)‐MCP. This resulted in an overall decrease of enantioselectivity ‐Δ_S,R(ΔG) in the presence of water. The effect was fully reversible. Consequently, for a conditioned column in the absence of residual water, the determined thermodynamic data, i.e. Δ_S,R(ΔH) = –12.64 ± 0.08 kJ mol^‐1 and Δ_S,R(ΔS) = –28.18 ± 0.23 J K^‐1 mol^‐1, refer to a true 1:1 complexation process devoid of hydrophobic hydration. Chirality 28:124–131, 2015. © 2015 Wiley Periodicals, Inc.     

17β‐Estradiol Protects Human Eyelid‐Derived Adipose Stem Cells against Cytotoxicity and Increases Transplanted Cell Survival in Spinal Cord injury

Stem cell transplantation represents a promising strategy for the repair of spinal cord injury (SCI). However, the low survival rate of the grafted cells is a major obstacle hindering clinical success because of ongoing secondary injury processes, which includes excitotoxicity, inflammation and oxidative stress. Previous studies have shown that 17b‐estradiol (E2) protects several cell types against cytotoxicity. Thus, we examined the effects of E2 on the viability of human eyelid adipose‐derived stem cells (hEASCs) in vitro with hydrogen peroxide (H₂O₂)‐induced cell model and in vivo within a rat SCI model. Our results showed that E2 protected hEASCs against H₂O₂‐induced cell death in vitro, and enhanced the survival of grafted hEASCs in vivo by reducing apoptosis. Additionally, E2 also enhanced the secretion of growth factors by hEASCs, thereby making the local microenvironment more conducive for tissue regeneration. Overall, E2 administration enhanced the therapeutic efficacy of hEASCs transplantation and facilitated motor function recovery after SCI. Hence, E2 administration may be an intervention of choice for enhancing survival of transplanted hEASCs after SCI.     

Role of γ‐glutamyltranspeptidase in the pathogenesis of Helicobacter pylori infection

γ‐Glutamyltranspeptidase and asparaginase have been shown to play important roles in Helicobacter pylori colonization and cell death induced by H. pylori infection. In this study, the association of γ‐glutamyltranspeptidase and asparaginase was elucidated by comparing activities of both deamidases in H. pylori strains from patients with chronic gastritis, gastric and duodenal ulcers, and gastric cancer. γ‐Glutamyltranspeptidase activities in H. pylori strains from patients with gastric cancer were significantly higher than in those from patients with chronic gastritis or gastric ulcers. There was a wide range of asparaginase activities in H. pylori strains from patients with gastric cancer and these were not significantly than those from patients with other diseases. To identify the contributions of γ‐glutamyltranspeptidase and asparaginase to gastric cell inflammation, human gastric epithelial cells (AGS line) were infected with H. pylori wild‐type and knockout strains and inflammatory responses evaluated by induction of interleukin‐8 (IL‐8). IL‐8 response was significantly decreased by knockout of the γ‐glutamyltranspeptidase‐encoding gene but not by knockout of the asparaginase‐encoding gene. Additionally, IL‐8 induction by infection with the H. pylori wild‐type strain was significantly decreased by adding glutamine during infection. These findings indicate that IL‐8 induction caused by γ‐glutamyltranspeptidase activity in H. pylori is mainly attributable to depletion of glutamine. These data suggest that γ‐glutamyltranspeptidase plays a significant role in the chronic inflammation caused by H. pylori infection.     

5α‐Androst‐16‐en‐3α‐ol β‐D‐Glucuronide,Precursor of 5α‐Androst‐16‐en‐3α‐ol in Human Sweat

5α‐Androst‐16‐en‐3α‐ol (α‐androstenol) is an important contributor to human axilla sweat odor. It is assumed that α‐andostenol is excreted from the apocrine glands via a H₂O‐soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H₂O‐soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α‐androstenol, β‐androstenol sulfates, 5α‐androsta‐5,16‐dien‐3β‐ol (β‐androstadienol) sulfate, α‐androstenol β‐glucuronide, α‐androstenol α‐glucuronide, β‐androstadienol β‐glucuronide, and α‐androstenol β‐glucuronide furanose. The occurrence of α‐androstenol β‐glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative‐ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α‐androstenol was observed after incubation of the sterile human sweat or α‐androstenol β‐glucuronide with a commercial glucuronidase enzyme, the urine‐isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have β‐glucuronidase activities. We demonstrated that if α‐ and β‐androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H₂O‐soluble precursor of α‐androstenol in apocrine secretion should be a β‐glucuronide.     

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.     

Non‐apoptotic function of caspases in a cellular model of hydrogen peroxide‐associated colitis

Oxidative stress, caused by reactive oxygen species (ROS), is a major contributor to inflammatory bowel disease (IBD)‐associated neoplasia. We mimicked ROS exposure of the epithelium in IBD using non‐tumour human colonic epithelial cells (HCEC) and hydrogen peroxide (H₂O₂). A population of HCEC survived H₂O₂‐induced oxidative stress via JNK‐dependent cell cycle arrests. Caspases, p21^WAF1 and γ‐H2AX were identified as JNK‐regulated proteins. Up‐regulation of caspases was linked to cell survival and not, as expected, to apoptosis. Inhibition using the pan‐caspase inhibitor Z‐VAD‐FMK caused up‐regulation of γ‐H2AX, a DNA‐damage sensor, indicating its negative regulation via caspases. Cell cycle analysis revealed an accumulation of HCEC in the G₁‐phase as first response to oxidative stress and increased S‐phase population and then apoptosis as second response following caspase inhibition. Thus, caspases execute a non‐apoptotic function by promoting cells through G₁‐ and S‐phase by overriding the G₁/S‐ and intra‐S checkpoints despite DNA‐damage. This led to the accumulation of cells in the G₂/M‐phase and decreased apoptosis. Caspases mediate survival of oxidatively damaged HCEC via γ‐H2AX suppression, although its direct proteolytic inactivation was excluded. Conversely, we found that oxidative stress led to caspase‐dependent proteolytic degradation of the DNA‐damage checkpoint protein ATM that is upstream of γ‐H2AX. As a consequence, undetected DNA‐damage and increased proliferation were found in repeatedly H₂O₂‐exposed HCEC. Such features have been associated with neoplastic transformation and appear here to be mediated by a non‐apoptotic function of caspases. Overexpression of upstream p‐JNK in active ulcerative colitis also suggests a potential importance of this pathway in vivo.     

Conformational flexibility of the glycosidase NagZ allows it to bind structurally diverse inhibitors to suppress β‐lactam antibiotic resistance

NagZ is an N‐acetyl‐β‐d ‐glucosaminidase that participates in the peptidoglycan (PG) recycling pathway of Gram‐negative bacteria by removing N‐acetyl‐glucosamine (GlcNAc) from PG fragments that have been excised from the cell wall during growth. The 1,6‐anhydromuramoyl‐peptide products generated by NagZ activate β‐lactam resistance in many Gram‐negative bacteria by inducing the expression of AmpC β‐lactamase. Blocking NagZ activity can thereby suppress β‐lactam antibiotic resistance in these bacteria. The NagZ active site is dynamic and it accommodates distortion of the glycan substrate during catalysis using a mobile catalytic loop that carries a histidine residue which serves as the active site general acid/base catalyst. Here, we show that flexibility of this catalytic loop also accommodates structural differences in small molecule inhibitors of NagZ, which could be exploited to improve inhibitor specificity. X‐ray structures of NagZ bound to the potent yet non‐selective N‐acetyl‐β‐glucosaminidase inhibitor PUGNAc (O‐(2‐acetamido‐2‐deoxy‐d ‐glucopyranosylidene) amino‐N‐phenylcarbamate), and two NagZ‐selective inhibitors – EtBuPUG, a PUGNAc derivative bearing a 2‐N‐ethylbutyryl group, and MM‐156, a 3‐N‐butyryl trihydroxyazepane, revealed that the phenylcarbamate moiety of PUGNAc and EtBuPUG completely displaces the catalytic loop from the NagZ active site to yield a catalytically incompetent form of the enzyme. In contrast, the catalytic loop was found positioned in the catalytically active conformation within the NagZ active site when bound to MM‐156, which lacks the phenylcarbamate extension. Displacement of the catalytic loop by PUGNAc and its N‐acyl derivative EtBuPUG alters the active site conformation of NagZ, which presents an additional strategy to improve the potency and specificity of NagZ inhibitors.     

Synthesis and Deprotection of Biodegradably and Thermally Protected Dinucleoside‐2′,5′‐Monophosphate Prodrug Model of 2‐5A

Protected dinucleoside‐2′,5′‐monophosphate has been prepared to develop a prodrug strategy for 2‐5A. The removal of enzymatically and thermally labile 4‐(acetylthio)‐2‐(ethoxycarbonyl)‐3‐oxo‐2‐methylbutyl phosphate protecting group and enzymatically labile 3′‐O‐pivaloyloxymethyl group was followed at pH 7.5 and 37 °C by HPLC from the fully protected dimeric adenosine‐2′,5′‐monophosphate 1 used as a model compound for 2‐5A. The desired unprotected 2′,3′‐O‐isopropylideneadenosine‐2′,5′‐monophosphate ( 9 ) was observed to accumulate as a major product. Neither the competitive isomerization of 2′,5′‐ to a 3′,5′‐linkage nor the P–O5′ bond cleavage was detected. The phosphate protecting group was removed faster than the 3′‐O‐protection and, hence, the attack of the neighbouring 3′‐OH on phosphotriester moiety did not take place.     

Synthesis,binding affinities and metabolic stability of dimeric dermorphin analogs modified with β3‐homo‐amino acids

In this study, proteinogenic amino acids residues of dimeric dermorphin pentapeptides were replaced by the corresponding β³‐homo‐amino acids. The potency and selectivity of hybrid α/β dimeric dermorphin pentapeptides were evaluated by competetive receptor binding assay in the rat brain using [3H]DAMGO (a μ ligand) and [3H]DELT (a δ ligand). Tha analog containing β³‐homo‐Tyr in place of Tyr (Tyr‐d ‐Ala‐Phe‐Gly‐β³‐homo‐Tyr‐NH‐)₂ showed good μ receptor affinity and selectivity (IC₅₀ = 0.302, IC₅₀ ratio μ/δ = 68) and enzymatic stability in human plasma. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Differential responses to oxidative stress and calcium influx on expression of the transforming growth factor‐β family in myoblasts and myotubes

Changes in gene expression of TGF‐β family members and their receptors in response to treatment with H₂O₂ and a calcium ionophore, A23187, were examined in C2C12 myoblasts and myotubes. The expression of Myf5, an initial regulator of myogenesis, was increased by A23187, and H₂O₂ inhibited the up‐regulation of Myf5. Treatment with H₂O₂ decreased the expression of MHC IIb, a protein component of the myofibrils, irrespective of the presence of A23187, suggesting an inhibitory role of oxidative stress for myogenesis. Expression of ligands and receptors for the TGF‐β family was modulated in response to H₂O₂ and A23187. Treatment with H₂O₂ decreased expression of TGF‐β3, BMP‐4, ALK4, ALK5, and ActRIIB, and increased expression of inhibin α and inhibin βA in either the myoblast stage or the myotube stage, or both. A23187 potentiated down‐regulation of BMP‐4 and ALK4 expression, and up‐regulation of TGF‐β1, TGF‐β2, inhibin α, inhibin βA, ALK2, and ALK3 expression. These results indicate that oxidative stress and Ca²⁺ influx affect expression of the TGF‐β family in C2C12 myoblasts and myotubes. Copyright © 2009 John Wiley & Sons, Ltd.