Polyethylene glycol‐based low generation dendrimers functionalized with β‐cyclodextrin as cryo‐ and dehydro‐protectant of catalase formulations

Polyethylene glycol (PEG)‐based low generation dendrimers are analyzed as single excipient or combined with trehalose in relation to their structure and efficiency as enzyme stabilizers during freeze‐thawing, freeze‐drying, and thermal treatment. A novel functional dendrimer (DG_o‐CD) based on the known PEG's ability as cryo‐protector and β‐CD as supramolecular stabilizing agent is presented. During freeze‐thawing, PEG and β‐CD failed to prevent catalase denaturation, while dendrimers, and especially DG_o‐CD, offered the better protection to the enzyme. During freeze‐drying, trehalose was the best protective additive but DG_o‐CD provided also an adequate catalase stability showing a synergistic behavior in comparison to the activities recovered employing PEG or β‐CD as unique additives. Although all the studied dendrimers improved the enzyme remaining activity during thermal treatment of freeze‐dried formulations, the presence of amorphous trehalose was critical to enhance enzyme stability. The crystallinity of the protective matrix, either of PEG derivatives or of trehalose, negatively affected catalase stability in the freeze‐dried systems. When humidified at 52% of relative humidity, the dendrimers delayed trehalose crystallization in the combined matrices, allowing extending the protection at those conditions in which normally trehalose fails. The results show how a relatively simple covalent combination of a polymer such as PEG with β‐CD could significantly affect the properties of the individual components. Also, the results provide further insights about the role played by polymer–enzyme supramolecular interactions (host–guest crosslink, hydrogen bonding, and hydrophobic interactions) on enzyme stability in dehydrated models, being the effect on the stabilization also influenced by the physical state of the matrix. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:786–795, 2013     

Epitope structure and binding affinity of single chain llama anti‐β‐amyloid antibodies revealed by proteolytic excision affinity‐mass spectrometry

ß‐Amyloid (Aß) immunotherapy has become a promising strategy for reducing the level of Aß in brain. New immunological approaches have been recently proposed for rapid, early diagnosis, and molecular treatment of neurodegenerative diseases related to Alzheimer's Disease (AD). The combination of proteolytic epitope excision and extraction and mass spectrometry using digestion with various proteases has been shown to be an efficient tool for the identification and molecular characterization of antigenic determinants. Here, we report the identification of the Aβ epitope recognized by the variable domain of single chain llama anti‐Aβ‐antibodies, termed Aβ‐nanobodies, that have been discovered in the blood of camelids and found to be promising candidates for immunotherapy of AD. The epitope recognized by two Aβ‐specific nanobodies was identified by proteolytic epitope extraction‐ and excision‐mass spectrometry using a series of proteases (trypsin, chymotrypsin, GluC‐protease, and LysC‐protease). Matrix‐assisted laser desorption ionization – mass spectrometric analysis of the affinity – elution fraction provided the epitope, Aβ(17–28), in the mid‐ to carboxy‐terminal domain of Aβ, which has been shown to exert an Aß‐fibril inhibiting effect. Affinity studies of the synthetic epitope confirmed that the Aβ(17–28) peptide is the minimal fragment that binds to the nanobodies. The interactions between the nanobodies and full length Aβ(1–40) or Aβ‐peptides containing or lacking the epitope sequence were further characterized by enzyme linked immunosorbent assay and bioaffinity analysis. Determinations of binding affinities between the Aβ‐nanobodies and Aβ(1–40) and the Aβ(17–28) epitope provided K_D values of approximately 150 and 700 nmol, respectively. Thus, the knowledge of the epitope may be highly useful for future studies of Aβ‐aggregation (oligomerization and fibril formation) and for designing new aggregation inhibitors. Copyright © 2012 John Wiley & Sons, Ltd.     

Novel grafted agar disks for the covalent immobilization of β‐D‐galactosidase

Novel grafted agar disks were prepared for the covalent immobilization of β‐D‐galactosidase (β‐gal). The agar disks were activated through reacting with ethylenediamine or different molecular weights of Polyethyleneimine (PEI), followed by glutaraldehyde (GA). The modification of the agar gel and the binding of the enzyme were verified by Fourier Transform Infrared (FTIR) and elemental analysis. Moreover, the agar's activation process was optimized, and the amount of immobilized enzyme increased 3.44 folds, from 38.1 to 131.2 U/g gel, during the course of the optimization process. The immobilization of β‐gal onto the activated agar disks caused its optimum temperature to increase from 45°C to 45–55°C. The optimum pH of the enzyme was also shifted towards the acidic side (3.6–4.6) after its immobilization. Additionally, the Michaelis‐Menten constant (K_m) increased for the immobilized β‐gal as compared to its free counterpart whereas the maximum reaction rate (V_max) decreased. The immobilized enzyme was also shown to retain 92.99% of its initial activity after being used for 15 consecutive times. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 675–684, 2015.     

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

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

Hysteresis in poly‐2′‐deoxycytidine i‐motif folding is impacted by the method of analysis as well as loop and stem lengths

In DNA, i‐motif (iM) folds occur under slightly acidic conditions when sequences rich in 2′‐deoxycytidine (dC) nucleotides adopt consecutive dC self base pairs. The pH stability of an iM is defined by the midpoint in the pH transition (pH_T) between the folded and unfolded states. Two different experiments to determine pH_T values via circular dichroism (CD) spectroscopy were performed on poly‐dC iMs of length 15, 19, or 23 nucleotides. These experiments demonstrate two points: (1) pH_T values were dependent on the titration experiment performed, and (2) pH‐induced denaturing or annealing processes produced isothermal hysteresis in the pH_T values. These results in tandem with model iMs with judicious mutations of dC to thymidine to favor particular folds found the hysteresis was maximal for the shorter poly‐dC iMs and those with an even number of base pairs, while the hysteresis was minimal for longer poly‐dC iMs and those with an odd number of base pairs. Experiments to follow the iM folding via thermal changes identified thermal hysteresis between the denaturing and annealing cycles. Similar trends were found to those observed in the CD experiments. The results demonstrate that the method of iM analysis can impact the pH_T parameter measured, and hysteresis was observed in the pH_T and T_m values.     

Molecular modeling of the binding modes of the iron‐sulfur protein to the Jac1 co‐chaperone from Saccharomyces cerevisiae by all‐atom and coarse‐grained approaches

The iron‐sulfur protein 1 (Isu1) and the J‐type co‐chaperone Jac1 from yeast are part of a huge ATP‐dependent system, and both interact with Hsp70 chaperones. Interaction of Isu1 and Jac1 is a part of the iron‐sulfur cluster biogenesis system in mitochondria. In this study, the structure and dynamics of the yeast Isu1–Jac1 complex has been modeled. First, the complete structure of Isu1 was obtained by homology modeling using the I‐TASSER server and YASARA software and thereafter tested for stability in the all‐atom force field AMBER. Then, the known experimental structure of Jac1 was adopted to obtain initial models of the Isu1–Jac1 complex by using the ZDOCK server for global and local docking and the AutoDock software for local docking. Three most probable models were subsequently subjected to the coarse‐grained molecular dynamics simulations with the UNRES force field to obtain the final structures of the complex. In the most probable model, Isu1 binds to the left face of the Γ‐shaped Jac1 molecule by the β‐sheet section of Isu1. Residues L₁₀₅, L₁₀₉, and Y₁₆₃ of Jac1 have been assessed by mutation studies to be essential for binding (Ciesielski et al., J Mol Biol 2012; 417:1–12). These residues were also found, by UNRES/molecular dynamics simulations, to be involved in strong interactions between Isu1 and Jac1 in the complex. Moreover, N₉₅, T₉₈, P₁₀₂, H₁₁₂, V₁₅₉, L₁₆₇, and A₁₇₀ of Jac1, not yet tested experimentally, were also found to be important in binding. Proteins 2015; 83:1414–1426. © 2015 Wiley Periodicals, Inc.     

Thermoluminescence properties of gamma‐irradiated nano‐structure hydroxyapatite

The suitability of nano‐structured hydroxyapatite (HAP) for use as a thermoluminescence dosimeter was investigated. HAP samples were synthesized using a hydrolysis method. The formation of nanoparticles was confirmed by X‐ray diffraction and average particle size was estimated to be ~30 nm. The glow curve exhibited a peak centered at around 200 °C. The additive dose method was applied and this showed that the thermoluminescence (TL) glow curves follow first‐order kinetics due to the non‐shifting nature of T_m after different doses. The numbers of overlapping peaks and related kinetic parameters were identified from T_m–T_stop through computerized glow curve deconvolution methods. The dependence of the TL responses on radiation dose was studied and a linear dose response up to 1000 Gy was observed for the samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Glycosaminoglycan and DNA Binding Induced Intra‐ and Intermolecular Exciton Coupling of the bis‐4‐Aminoquinoline Surfen

Despite the diverse biological activities of the glycosaminoglycan (GAG) antagonist surfen, the molecular details of its interaction with biomacromolecules remain poorly understood. Therefore, heparin and DNA binding properties of surfen were studied by circular dichroism (CD) and UV absorption spectroscopy methods. High‐affinity (K_a ~ 10⁷ M^‐1) association of surfen to the chiral heparin chain gives rise to a characteristic biphasic CD pattern due to the conformational twist of the aminoquinoline moieties around the central urea bridge. At higher drug loading, intermolecular stacking of surfen molecules alters the induced CD profile and also provokes strong UV hypochromism. In contrast to the right‐handed heparin template, binding of surfen to the left‐helicity chondroitin sulfate chains produces inverted CD pattern. Large UV hypochromism as well as polyphasic induced ellipticity bands indicate that surfen intercalates between the base pairs of calf‐thymus DNA. Extensive CD spectroscopic changes observed at higher drug binding ratios refer to cooperative binding interactions between the intercalated drug molecules. The inherent conformational flexibility of surfen demonstrated here for the first time is important in its binding to distinct macromolecular targets and should be considered for rational drug design of novel GAG antagonists. Chirality 27:605–612, 2015. © 2015 Wiley Periodicals, Inc.     

Chaperone‐client interactions between Hsp21 and client proteins monitored in solution by small angle X‐ray scattering and captured by crosslinking mass spectrometry

The small heat shock protein (sHsp) chaperones are important for stress survival, yet the molecular details of how they interact with client proteins are not understood. All sHsps share a folded middle domain to which is appended flexible N‐ and C‐terminal regions varying in length and sequence between different sHsps which, in different ways for different sHsps, mediate recognition of client proteins. In plants there is a chloroplast‐localized sHsp, Hsp21, and a structural model suggests that Hsp21 has a dodecameric arrangement with six N‐terminal arms located on the outside of the dodecamer and six inwardly‐facing. Here, we investigated the interactions between Hsp21 and thermosensitive model substrate client proteins in solution, by small‐angle X‐ray scattering (SAXS) and crosslinking mass spectrometry. The chaperone‐client complexes were monitored and the R_g‐values were found to increase continuously during 20 min at 45°, which could reflect binding of partially unfolded clients to the flexible N‐terminal arms of the Hsp21 dodecamer. No such increase in R_g‐values was observed with a mutational variant of Hsp21, which is mainly dimeric and has reduced chaperone activity. Crosslinking data suggest that the chaperone‐client interactions involve the N‐terminal region in Hsp21 and only certain parts in the client proteins. These parts are peripheral structural elements presumably the first to unfold under destabilizing conditions. We propose that the flexible and hydrophobic N‐terminal arms of Hsp21 can trap and refold early‐unfolding intermediates with or without dodecamer dissociation.     

Enantiomeric resolution of some nonsteroidal antiinflammatory and anticoagulant drugs using β‐cyclodextrins by capillary electrophoresis

β‐cyclodextrin (CD) and its derivatives HP‐β‐CD, DM‐β‐CD, and TM‐β‐CD have been employed as chiral selectors for the separation of three nonsteroidal antiinflammatory drugs (NSAIDs) and anticoagulant at relatively low concentration (8–15 mM) by capillary zone electrophoresis (CZE). In this study, baseline separation was achieved for ibuprofen, ketoprofen, naproxen, and warfarin. It was found that the addition of 0.1% hydroxypropyl methyl cellulose (HPMC) was effective for separation. Under these conditions, the S‐(+) enantiomer eluted before R‐(−) in terms of ibuprofen; the calculated energy values obtained from the molecular modeling correlated well with the elution order. An equation for calculating the pK_a values by capillary electrophoresis was introduced, and the pK_a values of the four chiral drugs at 25°C were obtained based on the equation. The value pK_a + 0.5 is proposed to be the suitable pH of the background electrolyte for the separation of chiral compounds containing a carboxylic group. Chirality 11:56–62, 1999. © 1999 Wiley‐Liss, Inc.     

Dynamic mechanistic modeling of the multienzymatic one‐pot reduction of dehydrocholic acid to 12‐keto ursodeoxycholic acid with competing substrates and cofactors

Ursodeoxycholic acid (UDCA) is a bile acid which is used as pharmaceutical for the treatment of several diseases, such as cholesterol gallstones, primary sclerosing cholangitis or primary biliary cirrhosis. A potential chemoenzymatic synthesis route of UDCA comprises the two‐step reduction of dehydrocholic acid to 12‐keto‐ursodeoxycholic acid (12‐keto‐UDCA), which can be conducted in a multienzymatic one‐pot process using 3α‐hydroxysteroid dehydrogenase (3α‐HSDH), 7β‐hydroxysteroid dehydrogenase (7β‐HSDH), and glucose dehydrogenase (GDH) with glucose as cosubstrate for the regeneration of cofactor. Here, we present a dynamic mechanistic model of this one‐pot reduction which involves three enzymes, four different bile acids, and two different cofactors, each with different oxidation states. In addition, every enzyme faces two competing substrates, whereas each bile acid and cofactor is formed or converted by two different enzymes. First, the kinetic mechanisms of both HSDH were identified to follow an ordered bi–bi mechanism with EBQ‐type uncompetitive substrate inhibition. Rate equations were then derived for this mechanism and for mechanisms describing competing substrates. After the estimation of the model parameters of each enzyme independently by progress curve analyses, the full process model of a simple batch‐process was established by coupling rate equations and mass balances. Validation experiments of the one‐pot multienzymatic batch process revealed high prediction accuracy of the process model and a model analysis offered important insight to the identification of optimum reaction conditions. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:375–386, 2015     

miR‐145 is differentially regulated by TGF‐β1 and ischaemia and targets Disabled‐2 expression and wnt/β‐catenin activity

The effect of wnt/β‐catenin signalling in the response to acute myocardial infarction (AMI) remains controversial. The membrane receptor adaptor protein Disabled‐2 (Dab2) is a tumour suppressor protein and has a critical role in stem cell specification. We recently demonstrated that down‐regulation of Dab2 regulates cardiac protein expression and wnt/β‐catenin activity in mesenchymal stem cells (MSC) in response to transforming growth factor‐β₁ (TGF‐β₁). Although Dab2 expression has been shown to have effects in stem cells and tumour suppression, the molecular mechanisms regulating this expression are still undefined. We identified putative binding sites for miR‐145 in the 3′‐UTR of Dab2. In MSC in culture, we observed that TGF‐β₁ treatment led to rapid and sustained up‐regulation of pri–miR‐145. Through gain and loss of function studies we demonstrate that miR‐145 up‐regulation was required for the down‐regulation of Dab2 and increased β‐catenin activity in response to TGF‐β₁. To begin to define how Dab2 might regulate wnt/β‐catenin in the heart following AMI, we quantified myocardial Dab2 as a function of time after left anterior descending ligation. There was no significant Dab2 expression in sham‐operated myocardium. Following AMI, Dab2 levels were rapidly up‐regulated in cardiac myocytes in the infarct border zone. The increase in cardiac myocyte Dab2 expression correlated with the rapid and sustained down‐regulation of myocardial pri–miR‐145 expression following AMI. Our data demonstrate a novel and critical role for miR‐145 expression as a regulator of Dab2 expression and β‐catenin activity in response to TGF‐β₁ and hypoxia.     

Effects of hydroxy‐delta‐5‐steroid dehydrogenase, 3 beta‐ and steroid delta‐isomerase 1 polymorphisms on fat androstenone level and gene expression in Duroc pigs

A high level of androstenone in porcine adipose tissue is a major factor contributing to boar taint. Porcine hydroxy‐delta‐5‐steroid dehydrogenase, 3 beta‐ and steroid delta‐isomerase 1 (3β‐HSD, also known as HSD3B1) plays a key role in the hepatic metabolism that catalyzes androstenone to β‐androstenol. Therefore, 3β‐HSD is a candidate gene for boar taint. This study aimed to investigate functional 3β‐HSD polymorphisms in Duroc pigs. We found eight single nucleotide polymorphisms (SNPs) in the full‐length porcine 3β‐HSD. Four of the SNPs had restriction enzyme sites, and we genotyped them in 147 uncastrated male Duroc pigs using a polymerase chain reaction–restriction fragment length polymorphism method. Pigs with the GG genotype at the g.165262G>A locus (SNP5) had significantly lower androstenone levels than did those with other genotypes (P = 0.030). SNP5 also was associated with differences in 3β‐HSD mRNA levels: pigs with the GG genotype had higher levels than those with other genotypes (P = 0.019). The SNP5 polymorphism could affect the hepatic catabolism of androstenone and consequently impact androstenone accumulation in the adipose tissue. Therefore, SNP5 in the 3β‐HSD of Duroc pigs could be a useful selective marker for decreasing boar taint.     

HLA‐DQ7 β1 and β2 derived peptides as immunomodulators

Modulation of protein–protein interactions involved in the immune system by using small molecular mimics of the contact interfaces may lead to the blockage of the autoimmune response and the development of drugs for immunotherapy. The nonpolymorphic β‐regions, exposed to the microenvironment, of the modeled HLA‐DQ7, which is genetically linked to autoimmune diseases, were determined. Peptides 132–141 and 58–67, located at the β₁ and β₂ domains of HLA‐DQ7, respectively, were tested for their involvement in the interactions with CD4⁺ T lymphocytes. Linear, cyclic, and dimeric analogs that mimic the exposed surfaces of HLA‐DQ7 were designed and synthesized. Their immunosuppressory activities, found in the secondary, humoral immune response to sheep erythrocytes (SRBC) in mice in vitro, ranged from 11% to 53%. The significance of the total charge of the peptides, the pattern of the hydrogen bonding, and the presence of secondary structure were investigated in relation to the immunomodulatory effect of the peptides. Two dimeric analogs of the HLA‐DQ7 58–67 fragment, consisting of the two monomers covalently linked by a polyethylene glycol (PEG) spacer, able to mimic the superdimers, were also synthesized and studied. As the 58–67 segment is located at the β₁ region of HLA‐DQ7, close to the major histocompatibility complex (MHC) groove, one may assume that the 58–67 peptide could accommodate the association between T‐cell receptor (TCR) and human leukocyte antigen (HLA) by activating a co‐stimulatory molecule of the TCR/HLA interaction. This hypothesis is supported by the confocal laser image of the fluorescein‐labeled 58–67 peptide and by the fact that it is an immunostimulator at low concentration. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

The role of calcium in apoptosis induced by 7β‐hydroxycholesterol and cholesterol‐5β,6β‐epoxide

Oxysterols, such as 7β‐hydroxy‐cholesterol (7β‐OH) and cholesterol‐5β,6β‐epoxide (β‐epoxide), may have a central role in promoting atherogenesis. This is thought to be predominantly due to their ability to induce apoptosis in cells of the vascular wall and in monocytes/macrophages. Although there has been extensive research regarding the mechanisms through which oxysterols induce apoptosis, much remains to be clarified. Given that experimental evidence has long associated alterations of calcium (Ca²⁺) homeostasis to apoptotic cell death, the aim of the present study was to determine the influence of intracellular Ca²⁺ changes on apoptosis induced by 7β‐OH and β‐epoxide. Ca²⁺ responses in differentiated U937 cells were assessed by epifluorescence video microscopy, using the ratiometric dye fura‐2. Over 15‐min exposure of differentiated U937 cells to 30 μM of 7β‐OH induced a slow but significant rise in fura‐2 ratio. The Ca²⁺ channel blocker nifedipine and the chelating agent EGTA blocked the increase in cytoplasmic Ca²⁺. Moreover, dihydropyridine (DHP) binding sites identified with BODIPY‐FLX‐DHP were blocked following pretreatment with nifedipine, indicating that the influx of Ca²⁺ occurred through L‐type channels. However, following long‐term incubation with 7β‐OH, elevated levels of cytoplasmic Ca²⁺ were not maintained and nifedipine did not provide protection against apoptotic cell death. Our results indicate that the increase in Ca²⁺ may be an initial trigger of 7β‐OH–induced apoptosis, but following chronic exposure to the oxysterol, the influence of Ca²⁺ on apoptotic cell death appears to be less significant. In contrast, Ca²⁺ did not appear to be involved in β‐epoxide–induced apoptosis. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:324–332, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20295     

Preparative Enantioseparation of β‐Substituted‐2‐Phenylpropionic Acids by Countercurrent Chromatography With Substituted β‐Cyclodextrin as Chiral Selectors

Preparative enantioseparation of four β‐substituted‐2‐phenylpropionic acids was performed by countercurrent chromatography with substituted β‐cyclodextrin as chiral selectors. The two‐phase solvent system was composed of n‐hexane‐ethyl acetate‐0.10 mol L‐1 of phosphate buffer solution at pH 2.67 containing 0.10 mol L^‐1 of hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) or sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD). The influence factors, including the type of substituted β‐cyclodextrin, composition of organic phase, concentration of chiral selector, pH value of the aqueous phase, and equilibrium temperature were optimized by enantioselective liquid–liquid extraction. Under the optimum separation conditions, 100 mg of 2‐phenylbutyric acid, 100 mg of tropic acid, and 50 mg of 2,3‐diphenylpropionic acid were successfully enantioseparated by high‐speed countercurrent chromatography, and the recovery of the (±)‐enantiomers was in the range of 90–91% for (±)‐2‐phenylbutyric acid, 91–92% for (±)‐tropic acid, 85–87% for (±)‐2,3‐diphenylpropionic acid with purity of over 97%, 96%, and 98%, respectively. The formation of 1:1 stoichiometric inclusion complex of β‐substituted‐2‐phenylpropionic acids with HP‐β‐CD was determined by UV spectrophotometry and the inclusion constants were calculated by a modified Benesi‐Hildebrand equation. The results showed that different enantioselectivities among different racemates were mainly caused by different enantiorecognition between each enantiomer and HP‐β‐CD, while it might be partially caused by different inclusion capacity between racemic solutes and HP‐β‐CD. Chirality 27:795–801, 2015. © 2015 Wiley Periodicals, Inc.     

Effects of fluidity on the ensemble structure of a membrane embedded α‐helical peptide

Melittin, the main hemolytic component of honeybee venom, is unfolded in an aqueous environment and folds into an α‐helical conformation in a lipid environment. Membrane fluidity is known to affect the activity and structure of melittin. By combining two structurally sensitive optical methods, circular dichroism (CD) and deep‐ultraviolet resonance Raman spectroscopy (dUVRR), we have identified distinct structural fluctuations in melittin correlated with increased and decreased 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine bilayer fluidities. CD spectra have reduced intensity at temperatures above 22°C and high concentrations of the cholesterol analog 5α‐cholestan‐3β‐ol indicating distortions in the α‐helical structure under these conditions. No increase in the amide S is observed in the temperature‐dependent dUVRR spectra, suggesting an increase in 3₁₀‐helical structure with increasing temperatures above 22°C. However, incorporation of 25 mol% 5α‐cholestan‐3β‐ol resulted in a small increase in the amide S intensity indicating partial unfolding of melittin. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 895–902, 2014.     

Biophysical properties and thermal stability of oligonucleotides of RNA containing 7,8‐dihydro‐8‐hydroxyadenosine

Circular dichroism (CD) was used to assess the stabilization/destabilization imposed by oxidative lesion 7,8‐dihydro‐8‐hydroxyadenosine (8‐oxoA) on strands of RNA with different structural motifs. RNA:RNA homoduplex destabilization was observed in a position dependent manner using 10‐mers as models that displayed differences between 12.7 and 15.1°C. We found that increasing the number of modifications resulted in depressed T_m values of about 12–15°C per lesion. The same effect was observed on RNA:DNA heteroduplex samples. We also tested the effects of this lesion in short hairpins containing the tetraloop UUCX (X = A, 8‐oxoA). We found that the stem was hypersensitive to substitution of A by 8‐oxoA and that it destabilized the structure by >23°C. Concomitant substitution at the stem and loop prevented formation of this secondary structure or lead to other less‐stable hairpins. Incorporation of this lesion at the first base of the loop had no effect on either structure. Overall, we found that the effects of 8‐oxoA on RNA structure are position dependent and that its stabilization may vary from sharp decreases to small increments, in some cases, leading to the formation of other more/less stable structures. These structural changes may have larger biological implications, particularly if the oxidatively modified RNA persists, thus leading to changes in RNA reactivity and function. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 167–174, 2015.     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin‐binding protein G from Streptococcus. IV. Implication for the mechanism of folding of the parent protein

A 34‐residue α/β peptide [IG(28–61)], derived from the C‐terminal part of the B3 domain of the immunoglobulin binding protein G from Streptoccocus, was studied using CD and NMR spectroscopy at various temperatures and by differential scanning calorimetry. It was found that the C‐terminal part (a 16‐residue‐long fragment) of this peptide, which corresponds to the sequence of the β‐hairpin in the native structure, forms structure similar to the β‐hairpin only at T = 313 K, and the structure is stabilized by non‐native long‐range hydrophobic interactions (Val47–Val59). On the other hand, the N‐terminal part of IG(28–61), which corresponds to the middle α‐helix in the native structure, is unstructured at low temperature (283 K) and forms an α‐helix‐like structure at 305 K, and only one helical turn is observed at 313 K. At all temperatures at which NMR experiments were performed (283, 305, and 313 K), we do not observe any long‐range connectivities which would have supported packing between the C‐terminal (β‐hairpin) and the N‐terminal (α‐helix) parts of the sequence. Such interactions are absent, in contrast to the folding pathway of the B domain of protein G, proposed recently by Kmiecik and Kolinski (Biophys J 2008, 94, 726–736), based on Monte‐Carlo dynamics studies. Alternative folding mechanisms are proposed and discussed. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 469–480, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Out‐of‐season production of 17,20β‐dihydroxypregn‐4‐en‐3‐one in the roach Rutilus rutilus

In this study, although the highest production of two physiologically significant progestins in teleosts [17,20β‐dihydroxypregn‐4‐en‐3‐one (17,20β‐P) and 17,20β,21‐trihydroxypregn‐4‐en‐3‐one (17,20β,21‐P)] was observed in the period just prior to spawning in both male and female roach Rutilus rutilus, there was also a substantial production (mean levels of 5–10 ng ml^?1 in blood; and a rate of release of 5–20 ng fish^?1 h^?1 into the water) in males and females in the late summer and early autumn (at least 7 months prior to spawning). During this period, the ovaries were increasing rapidly in size and histological sections were dominated by oocytes in the secondary growth phase [i.e. incorporation of vitellogenin (VTG)]. At the same time, the testes were also increasing rapidly in size and histological sections were dominated by cysts containing mainly spermatogonia type B. Measurements were also made of 11‐ketotestosterone (11‐KT) in males and 17β‐oestradiol and VTG in females. The 3 months with the highest production of 11‐KT coincided with the period that spermatozoa were present in the testes. In females, the first sign of a rise in 17β‐oestradiol concentrations coincided with the time of the first appearance of yolk globules in the oocytes (in August). The role of the progestins during the late summer and autumn has not been established.