Conformations of peptides containing a chiral cyclic α, α‐disubstituted α‐amino acid within the sequence of Aib residues

A single chiral cyclic α,α‐disubstituted amino acid, (3S,4S)‐1‐amino‐(3,4‐dimethoxy)cyclopentanecarboxylic acid [(S,S)‐Ac₅c^dOM], was placed at the N‐terminal or C‐terminal positions of achiral α‐aminoisobutyric acid (Aib) peptide segments. The IR and ¹H NMR spectra indicated that the dominant conformations of two peptides Cbz‐[(S,S)‐Ac₅c^dOM]‐(Aib)₄‐OEt ( 1) and Cbz‐(Aib)₄‐[(S,S)‐Ac₅c^dOM]‐OMe (2) in solution were helical structures. X‐ray crystallographic analysis of 1 and 2 revealed that a left‐handed (M) 3₁₀‐helical structure was present in 1 and that a right‐handed (P) 3₁₀‐helical structure was present in 2 in their crystalline states. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

β‐cyclodextrin encapsulated polyphenols as effective antioxidants

Formation of dityrosine (DT) cross‐linkages in proteins is one of the most widely used markers of oxidative stress. Ribonuclease A (RNase A) has 6 Tyr residues and shows a characteristic DT fluorescence peak upon oxidation in addition to major changes in its secondary structure. DT formation can be prevented by using polyphenols (GA, ECG, and EGCG) which are known to have strong antioxidant activity. However, it has been observed that ECG and EGCG initiate protein oligomerization due to protein‐polyphenol cross‐linkages. To prevent the formation of such cross‐linkages we have used β‐cyclodextrin (β‐CD) to encapsulate the polyphenols and studied its antioxidant properties along with that of free polyphenols. The polyphenol/β‐cyclodextrin (β‐CD) inclusion complexes not only prevent DT formation but also reduce protein oligomerization. This may be attributed to the fact that the quinone forming rings of ECG and EGCG become encapsulated in the cavity of β‐CD and are no longer available for protein cross‐linking.     

IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis

Inflammatory cytokines are closely related to pigmentary changes. In this study, the effects of IFN‐γ on melanogenesis were investigated. IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis in B16 melanoma cells and normal human melanocytes. MITF mRNA and protein expressions were significantly inhibited in response to IFN‐γ. IFN‐γ inhibited CREB binding to the MITF promoter but did not affect CREB phosphorylation. Instead, IFN‐γ inhibited the association of CBP and CREB through the increased association between CREB binding protein (CBP) and STAT1. These findings suggest that IFN‐γ inhibits both basal and α‐MSH‐induced melanogenesis by inhibiting MITF expression. The inhibitory action of IFN‐γ in α‐MSH‐induced melanogenesis is likely to be associated with the sequestration of CBP via the association between CBP and STAT1. These data suggest that IFN‐γ plays a role in controlling inflammation‐ or UV‐induced pigmentary changes.     

Characterization of an Importin α/β‐recognized nuclear localization signal in β‐dystroglycan

β‐dystroglycan (β‐DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of β‐DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of β‐DG, characterizing a functional nuclear localization signal (NLS) in the β‐DG cytoplasmic domain, within amino acids 776–782. The NLS either alone or in the context of the whole β‐DG protein was able to target the heterologous GFP protein to the nucleus, with site‐directed mutagenesis indicating that amino acids R⁷⁷⁹ and K⁷⁸⁰ are critical for NLS functionality. The nuclear transport molecules Importin (Imp)α and Impβ bound with high affinity to the NLS of β‐DG and were found to be essential for NLS‐dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of β‐DG may result in cytoplasmic retention, with Y⁸⁹² playing a key role. β‐DG thus follows a conventional Impα/β‐dependent nuclear import pathway, with important implications for its potential function in the nucleus. J. Cell. Biochem. 110: 706–717, 2010. © 2010 Wiley‐Liss, Inc.     

Chiral separation of catechin by capillary electrophoresis using mono‐, di‐, tri‐succinyl‐β‐cyclodextrin as chiral selectors

The chiral separation of (±)‐catechin was investigated by capillary electrophoresis using characterized succinyl‐β‐cyclodextrins (Suc‐β‐CDs) with one to three degree of substitution values. The effects of nature and concentration of Suc‐β‐CDs and running buffer pH on the migration time and resolution of (±)‐catechin are discussed. All three kinds of Suc‐β‐CDs show a clear baseline separation of (±)‐catechin in capillary electrophoresis. Mono‐Suc‐β‐CD effectively separated (±)‐catechin, and additional substituted CDs (di‐ and tri‐Suc‐β‐CD) were capable of chiral separation at a broad pH range. The optimum running conditions were found to be 100 mM borate buffer (pH 9.8) containing 5 mM mono‐Suc‐β‐CD with no methanol organic modifier. Chirality, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

Spectrofluorimetric determination of the antineoplastic agent lomustine based on the sensitizing effect of β‐cyclodextrin

The effects of solvent dipolarity/polarizability and solvent–solute hydrogen bonding on the photophysical properties of the antineoplastic drug lomustine were analysed by means of the linear solvation energy relationship (LSER) concept proposed by Kamlet and Taft. The LSER method enabled the overall solvent effects to be quantitatively estimated and separated into specific and non‐specific contributions. The molecular encapsulation of lomustine by β‐cyclodextrin (β‐CD) has been studied using fluorescence spectroscopy. The results are discussed in terms of the binding parameter and the effect of the solvent used. It was concluded that β‐CD forms a 1:1 inclusional complex with lomustine in acetonitrile solution and its association constant was calculated to be 500 M^?1. In addition, and for the first time, a simple, rapid and high sensitive fluorimetric method for the determination of lomustine was developed based upon the enhancement effect produced through complex formation with β‐CD. The new approach for the quantification of lomustine in the presence of β‐CD was described in aqueous and organic solutions. Better limits of detection (0.31 µg ml^?1) and quantification (1.05 µg ml^?1) were obtained in aqueous solution with respect to those obtained in organic solvent. Copyright © 2015 John Wiley & Sons, Ltd.     

Synephrine and phenylephrine act as α‐amylase, α‐glycosidase,acetylcholinesterase, butyrylcholinesterase,and carbonic anhydrase enzymes inhibitors

In this paper, synephrine and phenylephrine compounds showed excellent inhibitory effects against human carbonic anhydrase (hCA) isoforms I and II, α‐amylase, α‐glycosidase, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE). Synephrine and phenylephrine had K_i values of 199.02 ± 16.01 and 65.01 ± 5.00 μM against hCA I and 336.02 ± 74.01 and 92.04  ±  18.03 μM against hCA II, respectively. On the other hand, their K_i values were found to be 169.10  ±  80.03 and 88.03  ±  5.01 nM against AChE and 177.06  ±  6.01 and 78.03  ±  3.05 nM against BChE, respectively. α‐Amylase and α‐glycosidase enzymes were easily inhibited by these compounds. α‐Glycosidase inhibitors, generally defined to as starch blockers, are anti‐diabetic drugs that help to decrease post comestible blood glucose levels.     

α1 adrenoceptor activation by norepinephrine inhibits LPS‐induced cardiomyocyte TNF‐α production via modulating ERK1/2 and NF‐κB pathway

Cardiomyocyte tumour necrosis factor α (TNF‐α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)‐induced cardiomyocyte TNF‐α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS‐induced TNF‐α production in a dose‐dependent manner. α₁‐ adrenoceptor (AR) antagonist (prazosin), but neither β₁‐ nor β₂‐AR antagonist, abrogated the inhibitory effect of NE on LPS‐stimulated TNF‐α production. Furthermore, phenylephrine (PE), an α₁‐AR agonist, also suppressed LPS‐induced TNF‐α production. NE inhibited p38 phosphorylation and NF‐κB activation, but enhanced extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation and c‐Fos expression in LPS‐treated cardiomyocytes, all of which were reversed by prazosin pre‐treatment. To determine whether ERK1/2 regulates c‐Fos expression, p38 phosphorylation, NF‐κB activation and TNF‐α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c‐Fos expression, p38 mitogen‐activated protein kinase (MAPK) phosphorylation and TNF‐α production, but not NF‐κB activation in LPS‐challenged cardiomyocytes. In addition, pre‐treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS‐induced TNF‐α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c‐Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF‐α production and prevented LPS‐provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁‐AR by NE suppresses LPS‐induced cardiomyocyte TNF‐α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF‐κB activation.     

Asymmetric Allylation of α‐Ketoester‐Derived N‐Benzoylhydrazones Promoted by Chiral Sulfoxides/N‐Oxides Lewis Bases: Highly Enantioselective Synthesis of Quaternary α‐Substituted α‐Allyl‐α‐Amino Acids

Chiral sulfoxides/N‐oxides (R)‐ 1 and (R,R)‐ 2 are effective chiral promoters in the enantioselective allylation of α‐keto ester N‐benzoylhydrazone derivatives 3a , 3b , 3c , 3d , 3e , 3f , 3g to generate the corresponding N‐benzoylhydrazine derivatives 4a , 4b , 4c , 4d , 4e , 4f , 4g , with enantiomeric excesses as high as 98%. Representative hydrazine derivatives 4a , 4b were subsequently treated with SmI₂, and the resulting amino esters 5a , 5b with LiOH to obtain quaternary α‐substituted α‐allyl α‐amino acids 6a , 6b , whose absolute configuration was assigned as (S), with fundament on chemical correlation and electronic circular dichroism (ECD) data. Chirality 25:529–540, 2013. © 2013 Wiley Periodicals, Inc.     

Revealing interaction between sulfobutylether‐β‐cyclodextrin and reserpine by chemiluminescence and site‐directed molecular docking

The host–guest interaction between sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD) and reserpine (RSP) is described using flow injection‐chemiluminescence (FI‐CL) and site‐directed molecular docking methods. It was found that RSP could inhibit the CL intensity produced by a luminol/SBE‐β‐CD system. The decrease in CL intensity was logarithmic over an RSP concentration range of 0.03 to 700.0 nM, giving a regression equation of ?I = 107.1lgC_RES + 186.1 with a detection limit of 10 pM (3σ). The CL assay was successfully applied in the determination of RSP in injection, saliva and urine samples with recoveries in the range 93.5–106.1%. Using the proposed CL model, the binding constant (K_CD‐R) and the stoichiometric ratio of SBE‐β‐CD/RSP were calculated to be 7.4 × 10⁶ M^‐1 and 1 : 1, respectively. Using molecular docking, it was confirmed that luminol binds to the small cavity of SBE‐β‐CD with a nonpolar interaction, while RSP targeted the larger cavity of SBE‐β‐CD and formed a 1 : 1 complex with hydrogen bonds. The proposed new CL method has the potential to become a powerful tool for revealing the host–guest interaction between CDs and drugs, as well as monitoring drugs with high sensitivity. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

DIGESTION IN ADULT FEMALES OF THE LEAF‐FOOTED BUG Leptoglossus zonatus (HEMIPTERA: COREIDAE) WITH EMPHASIS ON THE GLYCOSIDE HYDROLASES α‐AMYLASE, α‐GALACTOSIDASE,AND α‐GLUCOSIDASE

The leaffooted bug, Leptoglossus zonatus (Hemiptera: Coreidae) is an emerging pest of several crops around the World and up to now very little is known of its digestive system. In this article, glycoside hydrolase (carbohydrase) activities in the adult midgut cells and in the luminal contents of L. zonatus adult females were studied. The results showed the distribution of digestive carbohydrases in adults of this heteropteran species in the different intestinal compartments. Determination of the spatial distribution of α‐glucosidase activity in L. zonatus midgut showed only one major molecular form, which was not equally distributed between soluble and membrane‐bound isoforms, being more abundant as a membrane‐bound enzyme. The majority of digestive carbohydrases were found in the soluble fractions. Activities against starch, maltose and the synthetic substrate NPαGlu were found to show the highest levels of activity, followed by enzymes active against galactosyl oligosaccharides. Based on ion‐exchange chromatography elution profiles and banding patterns in mildly denaturing electrophoresis, both midgut α‐amylases and α‐galactosidases showed at least two isoforms. The data suggested that the majority of carbohydrases involved in initial digestion were present in the midgut lumen, whereas final digestion of starch and of galactosyl oligosaccharides takes place partially within the lumen and partially at the cell surface. The complex of carbohydrases here described was qualitatively appropriate for the digestion of free oligosaccharides and oligomaltodextrins released by α‐amylases acting on maize seed starch granules.     

Antidiabetic potential: in vitro inhibition effects of some natural phenolic compounds on α‐glycosidase and α‐amylase enzymes

α‐Glycosidase is a catalytic enzyme and it destroys the complex carbohydrates into simple absorbable sugar units. The natural phenolic compounds were tested for their antidiabetic properties as α‐glycosidase and α‐amylase inhibitors. The phenolic compounds investigated in this study have been used as antidiabetic common medicines. This paper aimed to consider their capability to inhibit α‐amylase and α‐glycosidase, two significant enzymes defined in serum glucose adjustment. These examination recorded impressive inhibition profiles with IC₅₀ values in the range of 137.36–737.23 nM against α‐amylase and 29.01–157.96 nM against α‐glycosidase.     

An efficient and enantioselective Michael addition of aromatic oximes to α,β‐unsaturated aldehydes promoted by a chiral diamine catalyst derived from α,α‐diphenyl prolinol

Chiral diamine catalysts 11a–e derived from α ,α ‐diphenyl prolinol were prepared and successfully applied to the Michael addition of aromatic oximes to α ,β ‐unsaturated aldehydes in mediocre to good yields (up to 78%) and good to high enantioselectivities (up to 93% ee ).     

The importance of starch and sucrose digestion in nutritive biology of synanthropic acaridid mites: α‐Amylases and α‐glucosidases are suitable targets for inhibitor‐based strategies of mite control

The adaptation of nine species of mites that infest stored products for starch utilization was tested by (1) enzymatic analysis using feces and whole mite extracts, (2) biotests, and (3) inhibition experiments. Acarus siro, Aleuroglyphus ovatus, and Tyroborus lini were associated with the starch‐type substrates and maltose, with higher enzymatic activities observed in whole mite extracts. Lepidoglyphus destructor was associated with the same substrates but had higher activities in feces. Dermatophagoides farinae, Chortoglyphus arcuatus, and Caloglyphus redickorzevi were associated with sucrose. Tyrophagus putrescentiae and Carpoglyphus lactis had low or intermediate enzymatic activity on the tested substrates. Biotests on starch additive diets showed accelerated growth of species associated with the starch‐type substrates. The inhibitor acarbose suppressed starch hydrolysis and growth of the mites. We suggest that the species with higher starch hydrolytic activity in feces were more tolerant to acarbose, and α‐amylase and α‐glucosidase of synanthropic mites are suitable targets for inhibitor‐based strategies of mite control. © 2009 Wiley Periodicals, Inc.     

Folding type-specific secondary structure propensities of amino acids,derived from α-helical, β-sheet, α/β, and α+β proteins of known structures

Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998