Kinetic Modeling of Amino Acid Oxidation Catalyzed by a New D‐Amino Acid Oxidase from Arthrobacter protophormiae

Amino acid oxidases, which enantiospecifically catalyze the oxidative deamination of either D‐ or L‐amino acids, belong to the class of oxidoreductases functioning with a tightly bound cofactor. This cofactor favors industrial applications of D‐amino acid oxidases (D‐AAO). Hence, the enzyme is very important for the industrial application in the purification and determination of certain amino acids. In developing the enzyme‐catalyzed reaction for large‐scale production, modeling of the reaction kinetics plays an important role. Therefore, the subject of this study was the kinetics of the oxidative deamination, a very complex reaction system, which is catalyzed by D‐AAO from Arthrobacter protophormiae using its natural substrate D‐methionine and the aromatic amino acid 3,4‐dihydroxyphenyl‐D‐alanine (D‐DOPA). The kinetic parameters determined by the measurement of the initial rate and nonlinear regression were verified in batch reactor experiments by comparing calculated and experimental concentration‐time curves. It was found that the enzyme is highly specific towards D‐methionine (K_m = 0.24 mM) and not as specific to D‐DOPA as a substrate (K_m = 9.33 mM). The enzyme activity towards D‐methionine ( = 3.01 U/mL) was approx. seven times higher than towards D‐DOPA ( = 20.01 U/mL). The enzyme exhibited no activity towards L‐methionine and L‐DOPA. Batch and repetitive batch experiments were performed with both substrates in the presence and in the absence of catalase for hydrogen peroxide decomposition. Their comparison made it possible to conclude that hydrogen peroxide has no negative influence on the enzyme activity.     

Ultrastructural localization of oxidative and peroxidative activities in a carrot suspension cell culture

Summary Oxidative and peroxidative activities were localized at the ultrastructural level in suspension cells of an anthocyanin-producing strain of carrot after treatment with dihydroxyphenylalanine (DOPA) and diaminobenzidine (DAB). In DOPA-treated cells a reaction ascribed to polyphenoloxidase (PPO) occurred in the thylakoids of plastids. After DAB treatment at pH 9.0 reactions occurred in microbodies and plastid thylakoids; after treatment at pH 6.8 additional reactions occurred in the mitochondrial cristae and cytoplasmic ground substance. No reaction occurred in the cell walls at either pH. A reaction could not be unequivocally detected in the vacuoles because of the natural occurrence of osmiophilic material. Application of peroxidase and PPO inhibitors indicated that four distinct systems were involved in the DAB reactions: catalase was correlated with the reaction in the microbodies, peroxidase with the reaction in the cytoplasmic ground substance, cytochromes with the mitochondrial reaction, and PPO with the reaction in the thylakoids of the plastids.Contribution No. 1964 of the Rhode Island Agricultural Experiment Station.     

槐尺蠖多酚氧化酶的纯化及酶学特征

经40%饱和度硫酸铵分级沉淀,Sephadex G-100凝胶过滤等步骤,将槐尺蠖Semiothisa cinerearia Bremer et Grey 多酚氧化酶纯化,纯化倍数为6.96倍。该酶对焦性没食子酸,邻苯二酚和L多巴的Km值分别为0.23 mmol/L, 0.48 mmol/L和0.49 mmol/L。多酚氧化酶在pH 7.0,37℃时活性最高,并在40℃以上条件下,随着保温时间的延长酶活力下降。用槲皮苷和硫脲作抑制剂对该酶活性的抑制结果表明,这两种抑制剂分别属于竞争性和非竞争性抑制剂。     

Development and enzyme activity of protein bodies in proteinoplasts of tobacco root cells

The development of protein bodies in proteinoplasts of tobacco (Nicotiana tabacum L. var. Wis. 38) roots was investigated with TEM, HVEM, and enzyme cytochemistry. These plastids contain a three-dimensional network of fenestrated tubules which originate from invaginations of the inner membrane of the plastid envelope. Elaboration of the network occurs in parallel with cell differentiation: slender tubules common to plastids in meristematic cells undergo dilation as protein accumulates during cell differentiation; proteinoplasts of vacuolate and root cap cells usually contain a large protein body. The contents of the peripheral tubules, originating from the inner membrane, are less electron dense than the tubules making up the central network. Localized dilations within the tubular network result in the formation of dense spheroidal structures, protein bodies, apparently as a result of continued protein accumulation via tubules connecting to the central network. Protein might be imported from segments of rough ER attached to or apposed to the outer membrane of the proteinoplast envelope. The presence of catalase (E.C. 1.11.1.6), peroxidase (E.C. 1.11.1.7), and cytochrome oxidase (E.C. 1.9.3.1) was demonstrated by cytochemistry with diaminobenzidine (DAB) as substrate. Oxidized DAB was found in protein bodies after incubation in each of the specific reaction media. While aminotriazole and sodium azide inhibited oxidation of DAB by catalase and peroxidase, respectively, only potassium cyanide completely inhibited oxidation of DAB in protein bodies. We conclude that protein bodies of proteinoplasts in tobacco roots are not sites for storage of protein, rather protein bodies contain heme protein(s) with strong oxidase activity that may convey a specific function to proteinoplasts.     

Maize root peroxidases: relationship with polyphenol oxidases

Maize root peroxidases (POD) may also have polyphenol oxidase (PPO) activity as shown by using 3-amino-9-ethylcarbazole or DOPA as hydrogen donor to detect isoenzymes after disc gel electrophoresis. Copper chelators inhibited POD activity, and since PODs are haemoproteins, it can be concluded that copper chelators are not entirely specific for Cu enzymes. This raises the question whether PPO are only Cu enzymes. In POD preparations contaminated by catalase, POD activity could be over-estimated; this could be due to the auto-oxidation of the hydrogen donor or to stimulation of PPO activity by oxygen, as demonstrated with DOPA, dopamine and gallic acid. No correlation was found between the chemical nature of the substrate and the type of peroxidatic or oxidatic oxidation.     

Development and enzyme activity of protein bodies in proteinoplasts of tobacco root cells

Summary The development of protein bodies in proteinoplasts of tobacco (Nicotiana tabacum L. var. Wis. 38) roots was investigated with TEM, HVEM, and enzyme cytochemistry. These plastids contain a three-dimensional network of fenestrated tubules which originate from invaginations of the inner membrane of the plastid envelope. Elaboration of the network occurs in parallel with cell differentiation: slender tubules common to plastids in meristematic cells undergo dilation as protein accumulates during cell differentiation; proteinoplasts of vacuolate and root cap cells usually contain a large protein body. The contents of the peripheral tubules, originating from the inner membrane, are less electron dense than the tubules making up the central network. Localized dilations within the tubular network result in the formation of dense spheroidal structures, protein bodies, apparently as a result of continued protein accumulation via tubules connecting to the central network. Protein might be imported from segments of rough ER attached to or apposed to the outer membrane of the proteinoplast envelope.The presence of catalase (E.C. 1.11. 1.6), peroxidase (E.C. 1.11.1.7), and cytochrome oxidase (E.C. 1.9.3.1) was demonstrated by cytochemistry with diaminobenzidine (DAB) as substrate. Oxidized DAB was found in protein bodies after incubation in each of the specific reaction media. While aminotriazole and sodium azide inhibited oxidation of DAB by catalase and peroxidase, respectively, only potassium cyanide completely inhibited oxidation of DAB in protein bodies. We conclude that protein bodies of proteinoplasts in tobacco roots are not sites for storage of protein, rather protein bodies contain heme protein(s) with strong oxidase activity that may convey a specific function to proteinoplasts.Abbreviations used CAT catalase - CYT-OX cytochrome oxidase - DAB diaminobenzidine - ER endoplasmic reticulum - f filaments - HVEM high voltage electron microscopy - M mitochondrion - MT microtubule - P peroxisome - PB protein body - PER peroxidase - Pl plastid - Pg plastoglobuli - RER rough endoplasmic reticulum - RuBPcase ribulose-1,5-bisphosphate carboxylase - S starch - T tubule - V vacuole Scientific Article No. A3997, Contribution No. 6981, of the Maryland Agricultural Experiment StationThe scale bar on each micrograph is 0.1 , unless indicated otherwise     

Some kinetic properties of polyphenol oxidase obtained from dill (Anethum graveolens)

Polyphenol oxidase (PPO) was partially purified from dill by (NH4)(2)SO4 precipitation followed by dialysis and gel filtration chromatography. Polyphenol oxidase activity was measured spectrophotometrically at 420 nm using catechol, dopamine and chlorogenic acid as substrates. Optimum pH, temperature, and ionic strength were determined with three substrates. The best substrate of dill PPO was found to be chlorogenic acid. Some kinetic properties of the enzyme such as V(max,) K(M) and V(max)/K(M) were determined for all three substrates. The effects of various inhibitors on the reaction catalysed by the enzyme were tested and I(50) values calculated. The most effective inhibitor was L-cysteine. Activation energies, E(a), were determined from the Arrhenius equation. In addition, activation enthalpy, DeltaH(a), and Q(10) values of the enzyme were also calculated.     

Immunofluorescent demonstration of myeloperoxidase of phi bodies and rods in leukaemic leucocytes

Summary Previous studies employing enzyme histocytochemical methods based on the catalysis of the peroxidation of 3,3-diaminobenzidine (DAB) demonstrated the presence of hydroperoxidase activity in phi bodies and rods of immature leucocytes of patients with active acute myelogenous leukaemia. It could not be determined from these studies whether the DAB oxidation product was demonstrating a single hydroperoxidase, catalase or myeloperoxidase, or both. In the present study, immunofluorescence techniques for the two hydroperoxidases were applied in an attempt to identify this activity specifically. The results obtained indicate that myeloperoxidase is present in the phi bodies and rods, and that this enzyme may be the major on the only hydroperoxidase present. Its activity could account for the peroxidation of DAB under conditions which are more favourable for the demonstration of catalase.     

Enzymatic properties of phenoloxidase from Pieris rapae (Lepidoptera) larvae

The kinetic parameters of partially purified phenoloxidase (PO, EC. 1.14.18.1) from the 5th instar larvae of Pieris rapae (Lepidoptera) were determined, using L‐3, 4‐dihydroxyphenylalanine (L‐DOPA) as substrate. The optimal pH and temperature of the enzyme for the oxidation of L‐DOPA were determined to be at pH 7.0 and at 42°C, respectively. The enzyme was stable between pH 6.5 and 7.4 and at temperatures lower than 37°C. At pH 6.8 and 37°C, the Michaelis constant (K_m) and maximal velocity (V_m) of the enzyme for the oxidation of L‐DOPA were determined to be 0.80 μmol/L and 1.84 μmol/ L/min, respectively. Tetra‐hexylresorcinol and 4‐dodecylresorcinol effectively inhibited activity of phenoloxidase and this inhibition was reversible and competitive, with the IC₅₀ of 1.50 and 1.12 μmol/L, respectively. The inhibition constants were estimated to be 0.50 and 0.47 μmol/L, respectively.     

Unprecedented access of phenolic substrates to the heme active site of a catalase: Substrate binding and peroxidase‐like reactivity of Bacillus pumilus catalase monitored by X‐ray crystallography and EPR spectroscopy

Heme‐containing catalases and catalase‐peroxidases catalyze the dismutation of hydrogen peroxide as their predominant catalytic activity, but in addition, individual enzymes support low levels of peroxidase and oxidase activities, produce superoxide, and activate isoniazid as an antitubercular drug. The recent report of a heme enzyme with catalase, peroxidase and penicillin oxidase activities in Bacillus pumilus and its categorization as an unusual catalase‐peroxidase led us to investigate the enzyme for comparison with other catalase‐peroxidases, catalases, and peroxidases. Characterization revealed a typical homotetrameric catalase with one pentacoordinated heme b per subunit (Tyr340 being the axial ligand), albeit in two orientations, and a very fast catalatic turnover rate (k_cat = 339,000 s^?1). In addition, the enzyme supported a much slower (k_cat = 20 s^?1) peroxidatic activity utilizing substrates as diverse as ABTS and polyphenols, but no oxidase activity. Two binding sites, one in the main access channel and the other on the protein surface, accommodating pyrogallol, catechol, resorcinol, guaiacol, hydroquinone, and 2‐chlorophenol were identified in crystal structures at 1.65–1.95 Å. A third site, in the heme distal side, accommodating only pyrogallol and catechol, interacting with the heme iron and the catalytic His and Arg residues, was also identified. This site was confirmed in solution by EPR spectroscopy characterization, which also showed that the phenolic oxygen was not directly coordinated to the heme iron (no low‐spin conversion of the Fe^III high‐spin EPR signal upon substrate binding). This is the first demonstration of phenolic substrates directly accessing the heme distal side of a catalase. Proteins 2015; 83:853–866. © 2015 Wiley Periodicals, Inc.     

Characterisation of phenol oxidase and peroxidase from maize silk

Silk of some maize genotypes contains a high level of phenolics that undergo enzymatic oxidation to form quinones, which condense among themselves or with proteins to form brown pigments. Two phenolic oxidizing enzymes, peroxidase (POD; EC 1.11.1.7) and polyphenol oxidase (PPO; EC 1.10.3.1), from maize (Zea mays L.) silk were characterised with respect to their preferred substrate, different isoforms and specific effectors. One browning silk sample with high, and two non‐browning samples with low phenolic content were investigated. Although POD oxidizes a wide range of phenolic substrates in vitro, its activity rate was independent of silk phenolic content. PPO activity, detected with o‐diphenolic substrates, was abundant only in browning silk, and low or absent in non‐browning silk. Pollination increased POD but not PPO activity. Isoelectric‐focusing (IEF) and specific staining for POD and PPO showed a high degree of polymorphism that varied with silk origin. The IEF pattern of POD revealed a number of anionic and several cationic isoenzymes, with the most pronounced having neutral pI 7 and a basic isoform with pI 10. Detected isoforms of PPO were anionic, except for one neutral form found only in browning silk, and occupied positions different from those of POD. Different inhibitory effects of NaN₃, EDTA, KCN, and L‐cysteine, as well as different impacts of a variety of cations on the oxidation of chlorogenic acid, mediated by PPO or POD, were detected. The findings are discussed in terms of a possible roles of these enzymes in defence and pollination.     

A reliable method with good cell preservation for the demonstration of peroxidase activity in human platelets and megakaryocytes

Summary We have tried to improve existing methods for demonstration of platelet peroxidase (PPO) in human platelets and megakaryocytes by introducing a fixation in 0.1% glutaraldehyde prior to incubation in the DAB medium. This prefixation with low concentration of glutaraldehyde preserves excellent morphological detail and does not inhibit PPO activity. All 23 platelet-rich plasma samples show PPO reaction product in the dense tubular system after incubation in DAB medium with 0.003% H₂O₂. When 0.01% H₂O₂ is used in excessive DAB medium, PPO activity can also be demonstrated in platelets and megakaryocytes of bone-marrow cell suspensions. This method can be used for the identification of megakaryoblasts in acute non-lymphocytic leukemia, myelodysplastic syndromes and in blastic crisis of chronic myeloid leukemia. PPO cytochemistry can be combined with postfixation in a OsO₄-ruthenium red mixture. This method reveals -granules, dense bodies, microtubul,, glycogen, mitochondria, dense tubular system and invaginated membrane system in the same platelet and is useful for investigation of platelet ultrastructure.     

Evolution of albinism in cave planthoppers by a convergent defect in the first step of melanin biosynthesis

Albinism, the reduction or loss of melanin pigment, is found in many diverse cave‐dwelling animals. The mechanisms responsible for loss of melanin pigment are poorly understood. In this study we use a melanogenic substrate assay to determine the position where melanin synthesis is blocked in independently evolved cave planthoppers from Hawaii and Croatia. In this assay, substrates of enzymes responsible for melanin biosynthesis are added to fixed specimens in vitro and their ability to rescue black melanin pigmentation is determined. L‐tyrosine, the first substrate in the pathway, did not produce melanin pigment, whereas L‐DOPA, the second substrate, restored black pigment. Substrates in combination with enzyme inhibitors were used to test the possibility of additional downstream defects in the pathway. The results showed that downstream reactions leading from L‐DOPA and dopamine to DOPA‐melanin and dopamine‐melanin, the two types of insect melanin, are functional. It is concluded that albinism is caused by a defect in the first step of the melanin synthesis pathway in cave‐adapted planthoppers from widely separated parts of the world. However, Western blots indicated that tyrosine hydroxylase (TH), the only enzyme shown to operate at the first step in insects, is present in Hawaiian cave planthoppers. Thus, an unknown factor(s) operating at this step may be important in the evolution of planthopper albinism. In the cavefish Astyanax mexicanus, a genetic defect has also been described at the first step of melanin synthesis suggesting convergent evolution of albinism in both cave‐adapted insects and teleosts.     

Some kinetic properties of polyphenol oxidase from Thymbra spicata L. var. spicata

Polyphenol oxidase (PPO) of Thymbra (Thymbra spicata L. var. spicata) was isolated by (NH₄)₂SO₄ precipitation and dialysis. A diphenolase from Thymbra plant, active against 4-methylcatechol, catechol and pyrogallol was characterized in detail in terms of pH and temperature optima, stability, kinetic parameters and inhibition behaviour towards some general PPO inhibitors. 4-Methylcatechol was the most suitable substrate, due to the lowest K_m and the biggest V_max/K_m values, followed by catechol and pyrogallol. The Thymbra PPO had maximum activity at pH 5.0, 7.0 and 8.0 with 4-methylcatechol, catechol and pyrogallol substrates, respectively. The optimum temperature of activity for Thymbra PPO was 30, 40 and 50 °C for 4-methylcatechol, catechol and pyrogallol substrates, respectively. It was found that optimum temperature and pH were substrate-dependent studied. The enzyme activity decreased due to heat denaturation of the enzyme with increasing temperature and inactivation time. Inhibition of Thymbra PPO was investigated with inhibitors such as l-cysteine and glutathione using 4-methylcatechol, catechol and pyrogallol as substrates. It was found that l-cysteine was a more effective inhibitor than glutathione owing to lower K_i. The type of inhibition depended on the origin of the PPO studied and also on the substrate used. Furthermore, the IC₅₀ values of inhibitors sudied on PPO were determined by means of activity percentage (I) diagrams.     

Catalase Activities of Hydrocarbon-utilizing Candida Yeasts

The catalase activities of the Candida cells grown on hydrocarbons were generally much higher than those of the cells grown on Iauryl alcohol, glucose or ethanol. Km values for hydrogen peroxide of the enzymes from the glucose- and the hydrocarbon-grown cells of Candida tropicalis were the same level. The enzyme activities of the yeasts were higher at the exponential growth phase, especially of the hydrocarbon-grown cells, than at the stationary phase. Profuse appearance of microbodies having homogeneous matrix surrounded by a single-layer membrane has also been observed electronmicroscopically in the hydrocarbon- grown cells of several Candida yeasts. Cytochemical studies using 3,3′-diaminobenzidine (DAB) revealed that the catalase activity was located in microbodies. These facts suggest that the catalase activities would be related to the hydrocarbon metabolism in the yeasts.     

VARIATIONS IN AROMATIC AMINO ACID DECARBOXYLASE ACTIVITY TOWARDS DOPA AND 5-HYDROXYTRYPTOPHAN CAUSED BY pH CHANGES AND DENATURATION

—DOPA and 5-hydroxytryptophan (5-HTP) are generally supposed to be decarboxylated in mammalian tissues by a single enzyme, the two activities being present in constant ratio through a variety of purification procedures. It has now been shown that the ratio of activity of the liver enzyme towards the two substrates can be altered by mild treatments, such as might be used in solubilization of brain preparations. DOPA decarboxylase activity was preferentially inactivated by sodium dodecyl sulphate treatment, and 5-HTP decarboxylation by urea. Previous reports that the two substrates show different pH optima but are mutually competitive, have been confirmed. The K_m of the enzyme towards 5-HTP was lowest at pH 7.8 (the optimum pH for decarboxylation of this amino acid), but the variation with pH of the K_m towards DOPA was unrelated to the pH optimum for decarboxylation. There appeared to be no relation between the probable ionization state of the substrates and the pH dependence of the enzyme. Studies on the binding characteristics of the enzyme for the two products, dopamine and serotonin, did not show any specific saturable binding. It is proposed that the enzyme has a complex active site, with separate affinity sites for the two substrates, adjacent to a single catalytic site.     

Ultrastructural cytochemistry of the diaminobenzidine reaction in the aquatic fungus Entophlyctis variabilis

Ultrastructural localization of peroxidatic activity was investigated in the chytrid Entophlyctis variabilis with the 3,3-diaminobenzidine (DAB) cytochemical prodedure. The subcellular distribution of reaction product varied with changes in pH of the DAB medium and with the developmental stage of the fungus. Incubations in the DAB reaction medium at pH 9.2 produced an electron dense reaction product within single membrane bounded organelles which resembled microbodies but which varied in shapes from elongate to oval. At this pH the cell wall also stained darkly. When the pH of the DAB medium was lowered to pH 8.2 or 7.0, DAB oxidation product was localized within mitochondrial cristae as well as in microbodies and zoosporangial walls. As soon as zoospores were completely cleaved out of the zoosporangial cytoplasm, endoplasmic reticulum (ER) also stained. When the wall appeared around the encysted zoospore, ER staining was no longer found. The influence of the catalase inhibitor, aminotriazole, and the inhibitors of heme enzymes, sodium azide and sodium cyanide, on the staining patterns within cells incubated in the DAB media indicates that microbody staining is due to both catalase and peroxidase, mitochondrial staining is due to cytochrome c, and ER staining is due to peroxidase.Abbreviations DAB 3,3-diaminobenzidine-HCl - ER endoplasmic reticulum     

Actin localization and function in higher plants

Summary Two different cytochemical methods were used to study the localization of uricase (EC 1.7.3.3) and catalase (EC 1.11.1.6) in developing root nodules of soybean (Glycine max) inoculated as seeds withBradyrhizobium japonicum. One of the methods employs DAB (3,3-diaminobenzidine) and detects uricase activity indirectly by coupling it to endogenous catalase activity. The other method utilizes cerium chloride to detect uricase activity directly. These methods were modified to obtain not only a strong staining reaction but also improved ultrastructural preservation. With the indirect DAB method, intense staining indicative of both uricase and catalase activity was obtained in the enlarged peroxisomes of older uninfected cells. Similar staining was observed in enlarging peroxisomes of younger uninfected cells, and in the material of associated sacs whose bounding membranes appear to arise as distensions of the ER. The observations are discussed in relation to the controversial role of the ER in peroxisome biogenesis. Although the small peroxisome-like organelles of infected cells did not give a clearly positive reaction in the indirect DAB method, they reacted positively in the cerium chloride method, and are considered to be peroxisomes.Abbreviations DAB 3,3-diaminobenzidine - ER endoplasmic reticulum     

Caspase sensitive gold nanoparticle for apoptosis imaging in live cells

We developed a new apoptosis imaging probe with gold nanoparticles (AuNPs). A near-infrared fluorescence dye was attached to AuNP surface through the bridge of peptide substrate (DEVD). The fluorescence was quenched in physiological conditions due to the quenching effect of AuNP, and the quenched fluorescence was recovered after the DEVD had been cleaved by caspase-3, the enzyme involved in apoptotic process. The adhesion of DEVD substrates on AuNP surface was accomplished by conjugation of the 3,4-dihydroxy phenylalanine (DOPA) groups which are adhesive to inorganic surface and rich in mussels. This surface modification with DEVD substrates by DOPA groups resulted in increased stability of AuNP in cytosol condition for hours. Moreover, the cleavage of substrate and the dequenching process are very fast, and the cells did not need to be fixed for imaging. Therefore, the real-time monitoring of caspase activity could be achieved in live cells, which enabled early detection of apoptosis compared to a conventional apoptosis kit such as Annexin V-FITC. Therefore, our apoptosis imaging has great potential as a simple, inexpensive, and efficient apoptosis imaging probe for biomedical applications.     

Adsorption of chemically synthesized mussel adhesive peptide sequences containing DOPA on stainless steel

The adsorption of proteins at solid–liquid interfaces is important in biosensor and biomaterial applications. Marine mussels affix themselves to surfaces using a highly cross‐linked, protein‐based adhesive containing a high proportion of L‐3,4‐dihydroxyphenylalanine (DOPA) residues. In this work, the effect of DOPA residues on protein adhesion on stainless steel surfaces was studied using a quartz crystal microbalance with dissipation system. The adsorption of two repetitive peptide motifs, KGYKYYGGSS and KGYKYY, from the mussel Mytilus edulis foot protein 5 on stainless steel was studied before and after chemo‐enzymatic modification of tyrosine residues to DOPA using mushroom tyrosinase. Conversion from tyrosine to DOPA, evaluated by HPLC, was in the range 70–99%. DOPA‐modified sequences showed fourfold greater adhesion than unmodified M. edulis foot protein 5 motifs. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.