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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.43, #2)

Modulating protein activity and cellular function by methionine residue oxidation by Zong Jie Cui; Zong Qiang Han; Zhi Ying Li (pp. 505-517).

The sulfur-containing amino acid residue methionine (Met) in a peptide/protein is readily oxidized to methionine sulfoxide [Met(O)] by reactive oxygen species both in vitro and in vivo. Methionine residue oxidation by oxidants is found in an accumulating number of important proteins. Met sulfoxidation activates calcium/calmodulin-dependent protein kinase II and the large conductance calcium-activated potassium channels, delays inactivation of the Shaker potassium channel ShC/B and L-type voltage-dependent calcium channels. Sulfoxidation at critical Met residues inhibits fibrillation of atherosclerosis-related apolipoproteins and multiple neurodegenerative disease-related proteins, such as amyloid beta, α-synuclein, prion, and others. Methionine residue oxidation is also correlated with marked changes in cellular activities. Controlled key methionine residue oxidation may be used as an oxi-genetics tool to dissect specific protein function in situ.

Keywords: Protein methionine oxidation; Methionine sulfoxide; Calcium/calmodulin-dependent protein kinase II; BK potassium channel; Protein fibrillation; Neurodegenerative diseases; Singlet oxygen

In sickness and in health: the widespread application of creatine supplementation by Bruno Gualano; Hamilton Roschel; Antonio Herbert Lancha-Jr.; Charles E. Brightbill; Eric S. Rawson (pp. 519-529).

There is an extensive and still growing body of the literature supporting the efficacy of creatine (Cr) supplementation. In sports, creatine has been recognized as the most effective nutritional supplement in enhancing exercise tolerance, muscle strength and lean body mass. From a clinical perspective, the application of Cr supplementation is indeed exciting. Evidences of benefits from this supplement have been reported in a broad range of diseases, including myopathies, neurodegenerative disorders, cancer, rheumatic diseases, and type 2 diabetes. In addition, after hundreds of published studies and millions of exposures creatine supplementation maintains an excellent safety profile. Thus, we contend that the widespread application of this supplement may benefit athletes, elderly people and various patient populations. In this narrative review, we aimed to summarize both the ergogenic and therapeutic effects of Cr supplementation. Furthermore, we reviewed the impact of Cr supplementation on kidney function.

Keywords: Nutritional supplements; Exercise performance; Sports; Chronic diseases

Structure-based prediction of protein–protein binding affinity with consideration of allosteric effect by Feifei Tian; Yonggang Lv; Li Yang (pp. 531-543).

The conformational change upon protein–protein binding is largely ignored for a long time in the affinity prediction community. However, it is widely recognized that allosteric effect does play an important role in biomolecular recognition and association. In this article, we describe a new quantitative structure–activity relationship (QSAR)-based strategy to capture the structural and nonbonding information relating to not only the direct noncovalent interactions between protein binding partners, but also the indirect allosteric effect associated with binding. This method is then employed to quantitatively model and predict the protein–protein binding affinities compiled in a recently published benchmark consisting of 144 functionally diverse protein complexes with their structures available in both bound and unbound states (Kastritis et al. Protein Sci 20:482–491, 2011). With incorporating genetic algorithm and partial least squares regression (GA-PLS) into this method, a significant linear relationship between structural information descriptors and experimentally measured affinities is readily emerged and, on this basis, detailed discussions of physicochemical properties and structural implications underlying protein binding process, as well as the contribution of allosteric effect to the binding are addressed. We also give an empirical estimation of the prediction limit r _pred ² = 0.80 for structure-based method used to determine protein–protein binding affinity.

Keywords: Protein–protein binding; Noncovalent interaction; Allosteric effect; Quantitative structure–activity relationship; Statistical modeling

Predicting protein submitochondria locations by combining different descriptors into the general form of Chou’s pseudo amino acid composition by Guo-Liang Fan; Qian-Zhong Li (pp. 545-555).

Knowledge of the submitochondria location of protein is integral to understanding its function and a necessity in the proteomics era. In this work, a new submitochondria data set is constructed, and an approach for predicting protein submitochondria locations is proposed by combining the amino acid composition, dipeptide composition, reduced physicochemical properties, gene ontology, evolutionary information, and pseudo-average chemical shift. The overall prediction accuracy is 93.57% for the submitochondria location and 97.79% for the three membrane protein types in the mitochondria inner membrane using the algorithm of the increment of diversity combined with the support vector machine. The performance of the pseudo-average chemical shift is excellent. For contrast, the method is also used to predict submitochondria locations in the data set constructed by Du and Li; an accuracy of 94.95% is obtained by our method, which is better than that of other existing methods.

Keywords: Submitochondria location; Increment of diversity; Average chemical shift; Support vector machine; Chou’s pseudo amino acid

Molecular dynamics simulation exploration of unfolding and refolding of a ten-amino acid miniprotein by Guang-Jiu Zhao; Chang-Li Cheng (pp. 557-565).

Steered molecular dynamics simulations are performed to explore the unfolding and refolding processes of CLN025, a 10-residue beta-hairpin. In unfolding process, when CLN025 is pulled along the termini, the force-extension curve goes back and forth between negative and positive values not long after the beginning of simulation. That is so different from what happens in other peptides, where force is positive most of the time. The abnormal phenomenon indicates that electrostatic interaction between the charged termini plays an important role in the stability of the beta-hairpin. In the refolding process, the collapse to beta-hairpin-like conformations is very fast, within only 3.6 ns, which is driven by hydrophobic interactions at the termini, as the hydrophobic cluster involves aromatic rings of Tyr1, Tyr2, Trp9, and Tyr10. Our simulations improve the understanding on the structure and function of this type of miniprotein and will be helpful to further investigate the unfolding and refolding of more complex proteins.Unfolding and refolding processes of CLN025, a 10-residue beta-hairpin, are explored by use of the steered molecular dynamics simulations.

Keywords: Protein folding; Protein unfolding; Hydrogen bond; Beta-hairpin; Steered molecular dynamics

Relationship between protein folding kinetics and amino acid properties by Jitao T. Huang; Dajie J. Xing; Wei Huang (pp. 567-572).

The successful prediction of protein-folding rates based on the sequence-predicted secondary structure suggests that the folding rates might be predicted from sequence alone. To pursue this question, we directly predict the folding rates from amino acid sequences, which do not require any information on secondary or tertiary structure. Our work achieves 88% correlation with folding rates determined experimentally for proteins of all folding types and peptide, suggesting that almost all of the information needed to specify a protein’s folding kinetics and mechanism is comprised within its amino acid sequence. The influence of residue on folding rate is related to amino acid properties. Hydrophobic character of amino acids may be an important determinant of folding kinetics, whereas other properties, size, flexibility, polarity and isoelectric point, of amino acids have contributed little to the folding rate constant.

Keywords: Protein folding kinetics; Folding rate constants; Amino acid composition; Hydrophobic character; Statistical analysis

AMS 4.0: consensus prediction of post-translational modifications in protein sequences by Dariusz Plewczynski; Subhadip Basu; Indrajit Saha (pp. 573-582).

We present here the 2011 update of the AutoMotif Service (AMS 4.0) that predicts the wide selection of 88 different types of the single amino acid post-translational modifications (PTM) in protein sequences. The selection of experimentally confirmed modifications is acquired from the latest UniProt and Phospho.ELM databases for training. The sequence vicinity of each modified residue is represented using amino acids physico-chemical features encoded using high quality indices (HQI) obtaining by automatic clustering of known indices extracted from AAindex database. For each type of the numerical representation, the method builds the ensemble of Multi-Layer Perceptron (MLP) pattern classifiers, each optimising different objectives during the training (for example the recall, precision or area under the ROC curve (AUC)). The consensus is built using brainstorming technology, which combines multi-objective instances of machine learning algorithm, and the data fusion of different training objects representations, in order to boost the overall prediction accuracy of conserved short sequence motifs. The performance of AMS 4.0 is compared with the accuracy of previous versions, which were constructed using single machine learning methods (artificial neural networks, support vector machine). Our software improves the average AUC score of the earlier version by close to 7 % as calculated on the test datasets of all 88 PTM types. Moreover, for the selected most-difficult sequence motifs types it is able to improve the prediction performance by almost 32 %, when compared with previously used single machine learning methods. Summarising, the brainstorming consensus meta-learning methodology on the average boosts the AUC score up to around 89 %, averaged over all 88 PTM types. Detailed results for single machine learning methods and the consensus methodology are also provided, together with the comparison to previously published methods and state-of-the-art software tools. The source code and precompiled binaries of brainstorming tool are available at http://code.google.com/p/automotifserver/ under Apache 2.0 licensing.

Keywords: Post-translational modifications; AMS-4; High quality indices; MLP; Consensus

Fuzzy clustering of physicochemical and biochemical properties of amino Acids by Indrajit Saha; Ujjwal Maulik; Sanghamitra Bandyopadhyay; Dariusz Plewczynski (pp. 583-594).

In this article, we categorize presently available experimental and theoretical knowledge of various physicochemical and biochemical features of amino acids, as collected in the AAindex database of known 544 amino acid (AA) indices. Previously reported 402 indices were categorized into six groups using hierarchical clustering technique and 142 were left unclustered. However, due to the increasing diversity of the database these indices are overlapping, therefore crisp clustering method may not provide optimal results. Moreover, in various large-scale bioinformatics analyses of whole proteomes, the proper selection of amino acid indices representing their biological significance is crucial for efficient and error-prone encoding of the short functional sequence motifs. In most cases, researchers perform exhaustive manual selection of the most informative indices. These two facts motivated us to analyse the widely used AA indices. The main goal of this article is twofold. First, we present a novel method of partitioning the bioinformatics data using consensus fuzzy clustering, where the recently proposed fuzzy clustering techniques are exploited. Second, we prepare three high quality subsets of all available indices. Superiority of the consensus fuzzy clustering method is demonstrated quantitatively, visually and statistically by comparing it with the previously proposed hierarchical clustered results. The processed AAindex1 database, supplementary material and the software are available at http://sysbio.icm.edu.pl/aaindex/ .

Keywords: Amino acids; AAindex database; Consensus fuzzy clustering; High-quality indices; Validity measures; Physico-chemical features

Study of the urinary and faecal excretion of N ^ε-carboxymethyllysine in young human volunteers by Cristina Delgado-Andrade; Frédéric J. Tessier; Céline Niquet-Leridon; Isabel Seiquer; M. Pilar Navarro (pp. 595-602).

The dietary habits of the adolescent population with a high intake of snack and fast foods mean that they consume a high rate of which in turn leads to the development of different degenerative disorders. There are few studies available on MRP absorption and metabolism. We investigated the effects of a MRP-high and a MRP-low diet on carboxymethyllysine (CML) intake and excretion in 11–14 years adolescent males. In a 2-period crossover trial, 20 healthy subjects were randomly assigned to two groups. The first group consumed the MRP-low diet for 2 weeks, observed a 40-day washout period, and then consumed the MRP-high diet for 2 weeks. The second group received the diets in the reverse order. Subjects collected urine and faeces on the last 3 days of each dietary period. The consumption of the MRP-high diet led to a higher CML input (P < 0.05) (11.28 vs. 5.36 mg/day CML for MRP-high and -low diet, respectively). In parallel, the faecal excretion was also greater (P < 0.05) (3.52 vs. 1.23 mg/day CML, respectively) and proportional to the dietary intake. The urinary elimination of CML was not increased significantly when the MRP-high diet was consumed compared to consumption of the MRP-low diet, and was not proportional to the dietary exposure of CML. In conclusion it was shown that CML absorption and faecal excretion were highly influenced by dietary CML levels. Since the compound has long-term effects on health, an excessive intake deserves attention, especially in a population nutritionally at risk as adolescents.

Keywords: Maillard reaction products; Adolescence; Intake; Urinary excretion; Faecal elimination; Carboxymethyllysine

In silico investigation of molecular mechanism of laminopathy caused by a point mutation (R482W) in lamin A/C protein by Vidya Rajendran; Rituraj Purohit; Rao Sethumadhavan (pp. 603-615).

Lamin A/C proteins are the major components of a thin proteinaceous filamentous meshwork, the lamina, that underlies the inner nuclear membrane. A few specific mutations in the lamin A/C gene cause a disease with remarkably different clinical features: FPLD, or familial partial lipodystrophy (Dunnigan-type), which mainly affects adipose tissue. Lamin A/C mutant R482W is the key variant that causes FPLD. Biomolecular interaction and molecular dynamics (MD) simulation analysis were performed to understand dynamic behavior of native and mutant structures at atomic level. Mutant lamin A/C (R482W) showed more interaction with its biological partners due to its expansion of interaction surface and flexible nature of binding residues than native lamin A/C. MD simulation clearly indicates that the flexibility of interacting residues of mutant are mainly due to less involvement in formation of inter and intramolecular hydrogen bonds. Our analysis of native and Mutant lamin A/C clearly shows that the structural and functional consequences of the mutation R482W causes FPLD. Because of the pivotal role of lamin A/C in maintaining dynamics of nuclear function, these differences likely contribute to or represent novel mechanisms in laminopathy development.

Keywords: Docking simulation; Laminopathy; RMSD; SASA

Design, synthesis and biological activity of new neurohypophyseal hormones analogues conformationally restricted in the N-terminal part of the molecule. Highly potent OT receptor antagonists by Anna Kwiatkowska; Monika Ptach; Lenka Borovičková; Jiřina Slaninová; Bernard Lammek; Adam Prahl (pp. 617-627).

In this study we present the synthesis and some pharmacological properties of fourteen new analogues of neurohypophyseal hormones conformationally restricted in the N-terminal part of the molecule. All new peptides were substituted at position 2 with cis-1-amino-4-phenylcyclohexane-1-carboxylic acid (cis-Apc). Moreover, one of the new analogues: [cis-Apc², Val⁴]AVP was also prepared in N-acylated forms with various bulky acyl groups. All the peptides were tested for pressor, antidiuretic, and in vitro uterotonic activities. We also determined the binding affinity of the selected compounds to human OT receptor. Our results showed that introduction of cis -Apc² in position 2 of either AVP or OT resulted in analogues with high antioxytocin potency. Two of the new compounds, [Mpa¹,cis-Apc²]AVP and [Mpa¹,cis-Apc²,Val⁴]AVP, were exceptionally potent antiuterotonic agents (pA₂ = 8.46 and 8.40, respectively) and exhibited higher affinities for the human OT receptor than Atosiban (K _i values 5.4 and 9.1 nM). Moreover, we have demonstrated for the first time that N -terminal acylation of AVP analogue can improve its selectivity. Using this approach, we obtained compound Aba[cis-Apc²,Val⁴]AVP (XI) which turned out to be a moderately potent and exceptionally selective OT antagonist (pA₂ = 7.26).

Keywords: Oxytocin antagonists; Atosiban; Neurohypophyseal hormones analogues; Arginine vasopressin (AVP); Antidiuretic hormone; Binding affinity

Modeling the acid–base properties of glutathione in different ionic media, with particular reference to natural waters and biological fluids by Rosalia Maria Cigala; Francesco Crea; Concetta De Stefano; Gabriele Lando; Demetrio Milea; Silvio Sammartano (pp. 629-648).

The acid–base properties of γ-l-glutamyl-l-cysteinyl-glycine (glutathione, GSH) were determined by potentiometry (ISE-H⁺, glass electrode) in pure NaI_(aq) and in NaCl_(aq)/MgCl_2(aq), and NaCl_(aq)/CaCl_2(aq) mixtures, at T = 298.15 K and different ionic strengths (up to I c ~ 5.0 mol L⁻¹). In addition, the activity coefficients of glutathione were also determined by the distribution method at the same temperature in various ionic media (LiCl_(aq), NaCl_(aq), KCl_(aq), CsCl_(aq), MgCl_2(aq), CaCl_2(aq), NaI_(aq)). The results obtained were also used to calculate the Specific ion Interaction Theory (SIT) and Pitzer coefficients for the dependence on medium and ionic strength of glutathione species, as well as the formation constants of weak Mg jH i(GSH)(i+2j⁻³⁾ and Ca jH i(GSH)(i+2j⁻³⁾ complexes. Direct calorimetric titrations were also carried out in pure NaCl_(aq) and in NaCl_(aq)/CaCl_2(aq) mixtures at different ionic strengths (0.25 ≤ I c/mol L⁻¹ ≤ 5.0) in order to determine the enthalpy changes for the protonation and complex formation equilibria in these media at T = 298.15 K. Results obtained are useful for the definition of glutathione speciation in any aqueous media containing the main cations of natural waters and biological fluids, such as Na⁺, K⁺, Mg²⁺, and Ca²⁺. Finally, this kind of systematic studies, where a series of ionic media (e.g., all alkali metal chlorides) is taken into account in the determination of various thermodynamic parameters, is useful for the definition of some trends in the thermodynamic behavior of glutathione in aqueous solution.

Keywords: Glutathione; Protonation; Activity coefficients; Distribution coefficients; Thermodynamic parameters; SIT and Pitzer models

Resolution of protected silaproline for a gram scale preparation by C. Martin; N. Vanthuyne; H. Miramon; J. Martinez; F. Cavelier (pp. 649-655).

Silaproline is an analogue of proline, which exhibits similar conformational properties. Moreover, the presence of dimethylsilyl group confers to silaproline a higher lipophilicity as well as an improved resistance to biodegradation. This report describes the comparison of two routes to obtain Fmoc-(l) Sip-OH on the gram scale using chiral HPLC resolution.

Keywords: Silaproline; Chiral HPLC resolution; Absolute configuration; Enantiomers

Wavelet images and Chou’s pseudo amino acid composition for protein classification by Loris Nanni; Sheryl Brahnam; Alessandra Lumini (pp. 657-665).

The last decade has seen an explosion in the collection of protein data. To actualize the potential offered by this wealth of data, it is important to develop machine systems capable of classifying and extracting features from proteins. Reliable machine systems for protein classification offer many benefits, including the promise of finding novel drugs and vaccines. In developing our system, we analyze and compare several feature extraction methods used in protein classification that are based on the calculation of texture descriptors starting from a wavelet representation of the protein. We then feed these texture-based representations of the protein into an Adaboost ensemble of neural network or a support vector machine classifier. In addition, we perform experiments that combine our feature extraction methods with a standard method that is based on the Chou’s pseudo amino acid composition. Using several datasets, we show that our best approach outperforms standard methods. The Matlab code of the proposed protein descriptors is available at http://bias.csr.unibo.it/nanni/wave.rar .

Keywords: Proteins classification; Machine learning; Ensemble of classifiers; Support vector machines

Post-transcriptional divergence in the regulation of CAT-2A, CAT-2B and iNOS expression by dexamethasone in vascular smooth muscle cells by Shori Thakur; Anwar R. Baydoun (pp. 667-676).

Upregulation of l-arginine transport by pro-inflammatory mediators is a widely reported phenomenon which accompanies the expression of the inducible nitric oxide synthase (iNOS) enzyme in various cells. Both processes require de novo protein synthesis which may be regulated differentially through diverging signalling pathways. This is particularly defined by observations that the glucocorticoid dexamethasone, acting potentially through NF-κB, selectively blocks the expression of iNOS whilst having little or no effect on transport; suggesting that this ubiquitous transcription factor may not be required for induced transporter activity. This notion is however controversial as is the suggestion that dexamethasone may regulate iNOS expression exclusively through NF-κB. Thus, to further understand the mechanisms that control these processes, we have examined the level at which dexamethasone acts, investigating whether this involves NF-κB and whether the latter selectively regulates iNOS induction. Our current data directly demonstrate that induced l-arginine transport is critically dependent on the activation of NF-κB, and further confirmed its role in the induction of iNOS in rat aortic smooth muscle cells. More importantly, dexamethasone enhanced both iNOS and CAT gene expression but repressed iNOS protein with no noticeable effects on transporter function or indeed NF-κB activation. These novel and unexpected findings reflect the complex nature of the regulation of iNOS by glucocorticoids and prove, contrary to previous assumptions, that dexamethasone can regulate CAT gene expression despite failing to alter transporter function. Moreover, the effects of dexamethasone occur through a non-NF-κB-mediated action even though NF-κB is required for both processes.

Keywords: Inducible nitric oxide synthase; Dexamethasone; NF-κB; Cationic amino acid transporters; Smooth muscle cells; Septic shock

Amphibian cathelicidin fills the evolutionary gap of cathelicidin in vertebrate by Xue Hao; Hailong Yang; Ling Wei; Shilong Yang; Wenjuan Zhu; Dongying Ma; Haining Yu; Ren Lai (pp. 677-685).

Cathelicidins comprise a family of antimicrobial peptides (AMPs) sharing a highly conserved cathelin domain, and play a central role in the innate defense against infection in most of vertebrates. But so far it has not yet been found in amphibians although a large number of other groups of AMPs have been identified. In the current work, the first amphibian cathelicidin (cathelicidin-AL) has been characterized from the frog skin of Amolops loloensis. Cathelicidin-AL (RRSRRGRGGGRRGGSGGRGGRGGGGRSGAGSSIAGVGSRGGGGGRHYA) is a cationic peptide containing 48 amino acid residues (aa) with 12 basic aa and no acidic aa. The chemical synthesized peptide efficiently killed bacteria and some fungal species including clinically isolated drug-resistance microorganisms. The cDNA encoding cathelicidin-AL precursor was cloned from the skin cDNA library of A. loloensis. As other cathelicidins, the precursor of cathelicidin-AL also contains highly conserved anionic cathelin domain of cysteine proteinase inhibitor followed by the AMP fragment at C-terminus. Phylogenetic analysis revealed that as connecting link, the amphibian cathelicidin predates reptilia but postdates fish cathelicidin. The peptide purification combined with gene cloning results confirms the presence of cathelicidin in amphibians and filled the evolutionary gap of cathelicidin in vertebrate, considering amphibians’ special niche as the animals bridging the evolutionary land-water gap.

Keywords: Cathelicidin; Amphibian; Evolution; Structure and function

Resolution and absolute configuration of some α-aminoacetals: en route to enantiopure N-protected α-aminoaldehydes by Muriel Albalat-Serradeil; Géraldine Primazot; Didier Wilhelm; Jean-Claude Vallejos; Nicolas Vanthuyne; Christian Roussel (pp. 687-696).

The first successful resolution of rac-α-aminoacetals via diastereoisomeric salt formation with optically pure N-protected aminoacids is reported. The absolute configuration assignment of α-aminoacetal enantiomers is performed by an entirely non-racemizing chemical correlation method involving N-protection and a new efficient hydrolysis step followed by a reduction of the resulting N-protected α-aminoaldehyde intermediates. A racemization method of optically enriched α-aminoacetals is exemplified to allow valorisation of both enantiomers.

Keywords: Chiral α-aminoalcohols; Chiral α-aminoaldehydes; Chiral HPLC; Resolving agents; Absolute configuration; Chemical correlation method

Stimulating effect of growth hormone on type IV collagen production by endothelial cells cultured in normal and high glucose by A. Bakillah; R. Guillot; P. Urios; A.-M. Grigorova-Borsos; M. Sternberg (pp. 697-707).

Collagen IV accumulation is characteristic of diabetic angiopathy. To test the possible contribution of GH, we studied its effects on collagen IV production by human umbilical vein endothelial cells at 5.5 and 16.7 mmol/l glucose. GH (100 ng/ml) markedly increased collagen IV level in the culture supernatant and in the insoluble extracellular matrix and cell fraction at both glucose concentrations. This stimulating effect of GH was additional to that of high glucose. It was more pronounced on collagen IV than on total protein synthesis. GH increased free latent gelatinase activity slightly at normal and markedly at high glucose. Using GF109203X, a PKC inhibitor, we observed that high glucose, but not GH, activated PKC. These two factors stimulating collagen IV production appear to work through different pathways, favoring an additivity of their effects. This supports the contribution of high plasma GH in diabetic vascular basement membrane thickening.

Keywords: Diabetes mellitus; Growth hormone; Endothelial cells; Type IV collagen; Gelatinase; Protein kinase C

Creatine supplementation reduces oxidative stress biomarkers after acute exercise in rats by Rafael Deminice; Alceu Afonso Jordao (pp. 709-715).

The objective of this study was to evaluate the effect of creatine supplementation on muscle and plasma markers of oxidative stress after acute aerobic exercise. A total of 64 Wistar rats were divided into two groups: control group (n = 32) and creatine-supplemented group (n = 32). Creatine supplementation consisted of the addition of 2% creatine monohydrate to the diet. After 28 days, the rats performed an acute moderate aerobic exercise bout (1-h swimming with 4% of total body weight load). The animals were killed before (pre) and at 0, 2 and 6 h (n = 8) after acute exercise. As expected, plasma and total muscle creatine concentrations were significantly higher (P < 0.05) in the creatine-supplemented group compared to control. Acute exercise increased plasma thiobarbituric acid reactive species (TBARS) and total lipid hydroperoxide. The same was observed in the soleus and gastrocnemius muscles. Creatine supplementation decreased these markers in plasma (TBARS: pre 6%, 0 h 25%, 2 h 27% and 6 h 20%; plasma total lipid hydroperoxide: pre 38%, 0 h 24%, 2 h 12% and 6 h 20%, % decrease). Also, acute exercise decreased the GSH/GSSG ratio in soleus muscle, which was prevented by creatine supplementation (soleus: pre 8%, 0 h 29%, 2 h 30% and 6 h 44%, % prevention). The results show that creatine supplementation inhibits increased oxidative stress markers in plasma and muscle induced by acute exercise.

Keywords: Creatine supplementation; Acute exercise; Oxidative stress markers; Antioxidant

l-Leucine induces growth arrest and persistent ERK activation in glioma cells by Satoru Takeuchi; Hiroshi Nawashiro; Kojiro Wada; Namiko Nomura; Terushige Toyooka; Naoki Otani; Hideo Osada; Hirotaka Matsuo; Nariyoshi Shinomiya (pp. 717-724).

Glioma is the most common type of brain tumor, and has the worst prognosis in human malignancy. Experimental evidence suggests that the use of high concentrations of various amino acids may perturb neoplastic cell growth. Thus, the aim of this study was to investigate whether essential amino acids can alter the growth and proliferation of glioma cells. Studies were performed using C6 rat glioma cell lines. High concentration of l-leucine induced growth arrest of glioma cell lines. Terminal transferase uridyl nick end labeling assay and cell cycle analysis showed that the effect of l-leucine on glioma cells growth was not cytotoxic, but rather cytostatic. Additionally, the extracellular signal-regulated protein kinase was activated in l-leucine-treated glioma cells, and inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK) enhanced the effect of l-leucine on glioma cell growth. These data suggest that high concentration l-leucine combined with inhibition of MEK is a potential strategy for glioma cell growth arrest.

Keywords: Essential amino acid; l-Leucine; Glioma; Extracellular signal-regulated protein kinase; L-type amino acid transporter 1

Isoleucine or valine deprivation stimulates fat loss via increasing energy expenditure and regulating lipid metabolism in WAT by Ying Du; Qingshu Meng; Qian Zhang; Feifan Guo (pp. 725-734).

There has been a growing interest in controlling body weight by increasing dietary levels of leucine recently. By contrast, we have focused on studying the effect of deficiency of branched-chain amino acids (BCAAs) leucine on lipid metabolism. We previously have shown that mice fed a leucine-deficient diet for 7 days exhibit significant changes in lipid metabolism as demonstrated by suppressed lipogenesis in the liver and increased fat mobilization in white adipose tissue, the latter of which was found to be caused by increased lipolysis in WAT and uncoupling protein 1 expression in brown adipose tissue. The goal of our current study is to investigate whether the above effects of leucine deficiency can be generalized to the deficiency of other BCAAs including valine and isoleucine. In our current study, we show that valine or isoleucine deficiency has similar effects on reducing fat mass to leucine deprivation, in a similar manner as those observed during leucine deprivation.

Keywords: Isoleucine; Valine; Deprivation; Fat loss

Exploiting diverse stereochemistry of β-amino acids: toward a rational design of sheet-forming β-peptide systems by Gábor Pohl; Tamás Beke-Somfai; Imre G. Csizmadia; András Perczel (pp. 735-749).

Due to the two methylene groups in their backbone, β-amino acids can adopt numerous secondary structures, including helices, sheets and nanotubes. Chirality introduced by the additional side chains can significantly influence the folding preference of β-peptides composed of chiral β-amino acids. However, only conceptual suggestions are present in the literature about the effect of chirality on folding preferences. Summarizing both the experimental and computational results, Seebach (Chem Biodivers 1:1111–1240, 2004) has proposed the first selection rule on the effect of side chain chirality, on the folding preference of β-peptides. In order to extend and fine-tune the aforementioned predictions of Seebach, we have investigated its validity to the novel type of apolar sheet proposed recently (Pohl et al. in J Phys Chem B 114:9338–9348, 2010). In order to facilitate the rational design of sheet-like structures, a systematic study on the effect of chirality on “apolar” sheet stability is presented on disubstituted [HCO-β-Ala-β^2,3-hAla-β-Ala-NH₂]₂ model peptides calculated at the M05-2X/6-311++G(d,p)//M05-2X/6-31G(d) and B3LYP/6-311++G(d,p)//B3LYP/6-31G(d) levels of theory both in vacuum and in polar and apolar solvents. In addition, both types of “apolar” sheets were investigated; the one with two strands of identical (AA) and enantiomeric (AB) backbone structure. Our results show that heterochirally disubstituted sheets have the greatest preference for sheet formation (ΔG ~ −11 kcal mol⁻¹). However, in contrast to Seebach’s predictions, “homochiral disubstitution” itself does not necessarily disrupt the sheet structure, rather it could result stable fold (ΔG ~ −5 kcal mol⁻¹). Results indicate that both the methyl group orientation and the local conformational effect of substitution affects sheet stability, as point chirality was found to have influence only on the backbone torsional angles. These results enabled us to extend and generalize Seebach’s predictions and to propose a more general and accurate “rule of thumb” describing the effect of chirality on sheet stability. This offers an easy-to-use summary on how to design β-peptide sheet structures. We conclude that heterochirally disubstituted models are the best candidates for sheet formation, if the two strands are substituted in a way to create identical torsional angle sets on the two backbones for ideal hydrogen-bonding pattern. With adequately selected side chains, homochirally disubtituted derivatives may also form sheet structures, and the position of methyl groups would prevent assembly of more than two strands making it ideal to create hairpins.

Keywords: β-amino acid; β-peptide; Sheet structures; Computational chemistry; Chirality; Foldamer

Lasiocepsin, a novel cyclic antimicrobial peptide from the venom of eusocial bee Lasioglossum laticeps (Hymenoptera: Halictidae) by Lenka Monincová; Jiřina Slaninová; Vladimír Fučík; Oldřich Hovorka; Zdeněk Voburka; Lucie Bednárová; Petr Maloň; Jitka Štokrová; Václav Čeřovský (pp. 751-761).

In the venom of eusocial bee Lasioglossum laticeps, we identified a novel unique antimicrobial peptide named lasiocepsin consisting of 27 amino acid residues and two disulfide bridges. After identifying its primary structure, we synthesized lasiocepsin by solid-phase peptide synthesis using two different approaches for oxidative folding. The oxidative folding of fully deprotected linear peptide resulted in a mixture of three products differing in the pattern of disulfide bridges. Regioselective disulfide bond formation significantly improved the yield of desired product. The synthetic lasiocepsin possessed antimicrobial activity against both Gram-positive and -negative bacteria, antifungal activity against Candida albicans, and no hemolytic activity against human erythrocytes. We synthesized two lasiocepsin analogs cyclized through one native disulfide bridge in different positions and having the remaining two cysteines substituted by alanines. The analog cyclized through a Cys8–Cys25 disulfide bridge showed reduced antimicrobial activity compared to the native peptide while the second one (Cys17–Cys27) was almost inactive. Linear lasiocepsin having all four cysteine residues substituted by alanines or alkylated was also inactive. That was in contrast to the linear lasiocepsin with all four cysteine residues non-paired, which exhibited remarkable antimicrobial activity. The shortening of lasiocepsin by several amino acid residues either from the N- or C-terminal resulted in significant loss of antimicrobial activity. Study of Bacillus subtilis cells treated by lasiocepsin using transmission electron microscopy showed leakage of bacterial content mainly from the holes localized at the ends of the bacterial cells.

Keywords: Antimicrobial peptides; Analogs; Disulfide bridge; Peptide synthesis; Wild-bee venom; CD spectroscopy

Heterologous expression and characterization of the hydrophobin HFBI in Pichia pastoris and evaluation of its contribution to the food industry by Baolong Niu; Dandan Wang; Yanyan Yang; Haijin Xu; Mingqiang Qiao (pp. 763-771).

The class II hydrophobin HFBI from Trichoderma reesei was heterologously expressed by Pichia pastoris using pPIC9 vector under the control of the promoter AOX1. The recombinant HFBI (rHFBI) was purified by ultrafiltration and reverse-phase high performance liquid chromatography. Tricine-SDS-PAGE and Western blotting demonstrated that rHFBI with the expected molecular weight of 7.5 kDa was secreted into the culture medium. X-ray photoelectron spectroscopy and water contact angle measurements indicated that rHFBI could lead to the conversion of the wettability of the hydrophobic siliconized glass and hydrophilic mica surfaces relying on the self-assembly membrane on hydrophobic/hydrophilic interfaces. It was demonstrated that rHFBI had the ability to stabilize oil droplets, which was far excess of the class I hydrophobin HGFI heterologously expressed in P. pastoris (rHGFI) and the typical food emulsifier sodium caseinate. In gushing experiments, it was shown that rHFBI was a strong gushing inducer in beer, whereas rHGFI did not display any signs of gushing. This provided the potential of rHFBI to be used as a novel emulsifying agent and a predictor of gushing risk.

Keywords: Hydrophobin; Self-assembly; Heterologous expression; Emulsion; Gushing

The synthesis and role of taurine in the Japanese eel testis by Masato Higuchi; Fritzie T. Celino; Ayako Tamai; Chiemi Miura; Takeshi Miura (pp. 773-781).

In teleost fish, the progestin 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) is an essential component of the spermatogenesis pathway. In a series of investigations on the mechanisms underlying progestin-stimulated spermatogenesis, we have found that DHP up-regulates the expression of cysteine dioxygenase1 (CDO1) in the Japanese eel testis. CDO1 is one of the enzymes involved in the taurine biosynthesis pathway. To evaluate whether taurine is synthesized in the eel testis, cysteine sulfinate decarboxylase (CSD), another enzyme involved in taurine synthesis, was isolated from this species. RT-PCR and in vitro eel testicular culture revealed that although CSD was also expressed in eel testis, neither DHP nor other sex steroids affect CSD mRNA expression in a similar manner to CDO1. Using an in vitro eel testicular culture system, we further investigated the effects of DHP on taurine synthesis in the eel testis. HPLC analysis showed that DHP treatment significantly increases the taurine levels in the eel testis. These results suggest that DHP promotes taurine synthesis via the up-regulation of CDO1 mRNA expression during eel spermatogenesis. Furthermore, we observed from our analysis that although taurine does not induce complete spermatogenesis, it promotes spermatogonial DNA synthesis and the expression of Spo11, a meiosis-specific marker. These data thus suggest that taurine augments the effects of sex steroids in the promotion of spermatogonial proliferation and/or meiosis and hence that taurine plays important roles in spermatogenesis.

Keywords: Taurine; Progestin; Cysteine dioxygenase; Spermatogenesis; In vitro culture

Intraperitoneal injection of saline modulates hippocampal brain receptor complex levels but does not impair performance in the Morris Water Maze by Ajinkya Sase; Deeba Khan; Harald Höger; Gert Lubec (pp. 783-792).

The involvement of the hippocampus in pain has been demonstrated but key players, i.e. the major brain receptors have not been shown to be modulated by pain. It was therefore the aim of the study to show the concerted action and pattern of brain receptor complex levels in a non-invasive model of moderate pain. C57BL/6J mice were divided into four groups of 14 animals each: trained injected, trained non-injected, yoked injected and yoked non-injected. Animals were tested in the open field and the elevated plus maze for behavioural evaluation and cognitive functions were tested using the Morris Water Maze. Hippocampi were taken 6 h following sacrification. Membrane proteins were prepared by ultracentrifugation and run on blue native gels to keep the native state, blotted to membranes and western blotting was carried out using the primary antibodies against serotonin receptor 5HT1A, muscarinic acetylcholine receptor M1 (mAChR-M1), nicotinic acetylcholine receptor alpha7 (nAChR-alpha7), glutamate (AMPA) receptor (GluR1) and neurokinin receptor 1 (NK-1). There was no difference between performance in behaviour or in the MWM between groups. Brain receptor level changes involved all receptors given above. Pain affected mAChR-M1, GluR1 and NK-1 complex levels when yoked-injected were compared with yoked non-injected animals. Memory mechanisms affected mAChR-M1 complex levels when trained non-injected animals were compared with yoked non-injected controls. Taken together, the neurochemical basis for testing receptor agonists/antagonists on the role of pain and the hippocampus was generated that may be useful for interpretations of the role of this complex area in moderate pain.

Keywords: Intraperitoneal injection; Pain; Morris Water Maze; Serotonin; Muscarinic receptor; Nicotinergic receptor

Classification of G proteins and prediction of GPCRs-G proteins coupling specificity using continuous wavelet transform and information theory by Zhanchao Li; Xuan Zhou; Zong Dai; Xiaoyong Zou (pp. 793-804).

The coupling between G protein-coupled receptors (GPCRs) and guanine nucleotide-binding proteins (G proteins) regulates various signal transductions from extracellular space into the cell. However, the coupling mechanism between GPCRs and G proteins is still unknown, and experimental determination of their coupling specificity and function is both expensive and time consuming. Therefore, it is significant to develop a theoretical method to predict the coupling specificity between GPCRs and G proteins as well as their function using their primary sequences. In this study, a novel four-layer predictor (GPCRsG_CWTIT) based on support vector machine (SVM), continuous wavelet transform (CWT) and information theory (IT) is developed to classify G proteins and predict the coupling specificity between GPCRs and G proteins. SVM is used for construction of models. CWT and IT are used to characterize the primary structure of protein. Performance of GPCRsG_CWTIT is evaluated with cross-validation test on various working dataset. The overall accuracy of the G proteins at the levels of class and family is 98.23 and 85.42%, respectively. The accuracy of the coupling specificity prediction varies from 74.60 to 94.30%. These results indicate that the proposed predictor is an effective and feasible tool to predict the coupling specificity between GPCRs and G proteins as well as their functions using only the protein full sequence. The establishment of such an accurate prediction method will facilitate drug discovery by improving the ability to identify and predict protein–protein interactions. GPCRsG_CWTIT and dataset can be acquired freely on request from the authors.

Keywords: G protein-coupled receptors; G proteins; Support vector machine; Continuous wavelet transform; Information theory

Increased asymmetric dimethylarginine (ADMA) dimethylaminohydrolase (DDAH) activity in childhood hypercholesterolemia type II by Kristine Chobanyan-Jürgens; Anne-Jule Fuchs; Dimitrios Tsikas; Nele Kanzelmeyer; Anibh M. Das; Sabine Illsinger; Bernhard Vaske; Jens Jordan; Thomas Lücke (pp. 805-811).

Asymmetric dimethylarginine (ADMA) systemic concentrations are elevated in hypercholesterolemic adults and contribute to nitric oxide (NO) dependent endothelial dysfunction. Decreased activity of the key ADMA-hydrolyzing enzyme dimethylarginine dimethylaminohydrolase (DDAH) may be involved. Yet, the ADMA/DDAH/NO pathway has not been investigated in childhood hypercholesterolemia. We studied 64 children with hypercholesterolemia type II (HCh-II) and 54 normocholesterolemic (NCh) children (mean ± SD; age, years: 11.1 ± 3.5 vs. 11.9 ± 4.6). Plasma and urine ADMA was measured by GC–MS/MS. Dimethylamine (DMA), the ADMA metabolite, creatinine, nitrite and nitrate in urine were measured by GC–MS. The DMA/ADMA molar ratio in urine was calculated to estimate whole body DDAH activity. ADMA plasma concentration (mean ± SD; nM: 571 ± 85 vs. 542 ± 110, P = 0.17) and ADMA urinary excretion rate (mean ± SD: 7.1 ± 2 versus 7.2 ± 3 μmol/mmol creatinine, P = 0.6) were similar in HCh-II and NCh children. Both DMA excretion rate [median (25th–75th percentile): 56.3 (46.4–109.1) vs. 45.2 (22.2–65.5) μmol/mmol creatinine, P = 0.0004] and DMA/ADMA molar ratio [median (25th–75th percentile): 9.2 (6.0–16.3) vs. 5.4 (3.8–9.4), P = 0.0004] were slightly but statistically significantly increased in HCh-II children compared to NCh children. Plasma and urinary nitrite and nitrate were similar in both groups. In HCh-II whole body DDAH activity is elevated as compared to NCh. HCh-II children treated with drugs for hypercholesterolemia had lower plasma ADMA levels than untreated HCh-II or NCh children, presumably via increased DDAH activity. Differences between treated and untreated HCh-II children were not due to differences in age. In conclusion, HCh-II children do not have elevated ADMA plasma levels, largely due to an apparent increase in DDAH activity. While this would tend to limit development of endothelial dysfunction, it is not clear whether this might be medication-induced or represent a primary change in HCh-II children.

Keywords: ADMA; Children; DDAH; DMA; Hypercholesterolemia; Nitric oxide

Preventive oral supplementation with glutamine and arginine has beneficial effects on the intestinal mucosa and inflammatory cytokines in endotoxemic rats by Xihong Zhou; Xin Wu; Yulong Yin; Cui Zhang; Liuqin He (pp. 813-821).

The objective of this study was to evaluate the effect of oral supplementation with a combination of arginine and glutamine on the intestinal mucosa and inflammatory cytokines of lipopolysaccharide (LPS)-induced adult rats. Fifty Sprague–Dawley rats (average weight of 185 ± 15 g) were randomly divided into five groups: control group A (CA) and control group B (CB), both orally supplemented with 0.9% saline; group Arg, supplemented with 300 mg/kg day⁻¹ arginine; group Gln, supplemented with 300 mg/kg day⁻¹ glutamine; group AG, supplemented with 150 mg/kg day⁻¹ arginine and 150 mg/kg day⁻¹ glutamine. The experiment lasted for 2 weeks. Food intake and body weight were measured during the experiment. At 10.00 h of day 15, animals were injected with 4 mg/kg LPS (group CB, Arg, Gln, and AG) or sterile saline (group CA) after supplementation. Then at 14.00 h, all animals were killed and blood and tissue collected. The results showed that compared with group CB, arginine concentration tended to be increased (P > 0.05) in group Arg and AG, while there was no significant difference in glutamine concentration among the groups challenged with LPS. Oral supplementation with arginine or/and glutamine mitigated morphology impairment (lower villus height, P < 0.05) in the jejunum and ileum induced by LPS challenge. LPS administration resulted in a significant increase in TNF-α, IL-1β, IL-6 and IL-10 mRNA abundance. Arginine only significantly decreased TNF-α mRNA abundance in the ileum, while glutamine significantly decreased both TNF-α and IL-10 mRNA in the ileum. A combination of arginine and glutamine significantly decreased TNF-α and IL-1β mRNA abundance in both the jejunum and ileum, while they also significantly decreased anti-inflammatory IL-10 in the ileum. These results revealed that an oral supply of combined arginine and glutamine had more favorable effects on the intestinal mucosa and inflammatory cytokines than a supply of arginine or glutamine alone.

Keywords: Arginine; Glutamine; Mucosal morphology; Inflammatory cytokines; Intestinal mucosa

Chemically modified diamond-like carbon (DLC) for protein enrichment and profiling by MALDI-MS by M. Najam-ul-Haq; M. Rainer; C. W. Huck; M. N. Ashiq; G. K. Bonn (pp. 823-831).

The development of new high throughput methods based on different materials with chemical modifications for protein profiling of complex mixtures leads towards biomarkers; used particularly for early diagnosis of a disease. In this work, diamond-like carbon (DLC) is developed and optimized for serum protein profiling by matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS). This study is carried out in connection with a material-based approach, termed as material-enhanced laser desorption ionization mass spectrometry. DLC is selected as carrier surface which provides large surface to volume ratio and offers high sensitivity. DLC has a dual role of working as MALDI target while acting as an interface for protein profiling by specifically binding peptides and proteins out of serum samples. Serum constituents are bound through immobilized metal ion affinity chromatography (IMAC) functionality, created through glycidyl methacrylate polymerization under ultraviolet light followed by further derivatization with iminodiacetic acid and copper ion loading. Scanning electron microscopy highlights the morphological characteristics of DLC surface. It could be demonstrated that IMAC functionalized DLC coatings represent a powerful material in trapping biomolecules for their further analysis by MALDI-MS resulting in improved sensitivity, specificity and capacity in comparison to other protein-profiling methods.

Keywords: Nanomaterials; Diamond-like carbon; IMAC; Protein profiling; MALDI-MS; Proteomics

Involvement of AtoSC two-component system in Escherichia coli flagellar regulon by Marina C. Theodorou; Evaggelos C. Theodorou; Dimitrios A. Kyriakidis (pp. 833-844).

The AtoSC two-component system in Escherichia coli is a key regulator of many physiological processes. We report here the contribution of AtoSC in E. coli motility and chemotaxis. AtoSC locus deletion in ΔatoSC cells renders cells not motile or responsive against any chemoattractant or repellent independently of the AtoSC inducer’s presence. AtoSC expression through plasmid complemented the ΔatoSC phenotype. Cells expressing either AtoS or AtoC demonstrated analogous motility and chemotactic phenotypes as ΔatoSC cells, independently of AtoSC inducer’s presence. Mutations of AtoC phosphate-acceptor sites diminished or abrogated E. coli chemotaxis. trAtoC, the AtoC constitutive active form which lacks its receiver domain, up-regulated E. coli motility. AtoSC enhanced the transcription of the flhDC and fliAZY operons and to a lesser extent of the flgBCDEFGHIJKL operon. The AtoSC-mediated regulation of motility and chemotactic response required also the expression of the CheAY system. The AtoSC inducers enhanced the AtoSC-mediated motility and chemotaxis. Acetoacetate or spermidine further promoted the responses of only AtoSC-expressing cells, while Ca²⁺ demonstrated its effects independently of AtoSC. Histamine regulated bacterial chemotaxis only in atoSC ⁺ cells in a concentration-dependent manner while reversed the AtoSC-mediated effects when added at high concentrations. The trAtoC-controlled motility effects were enhanced by acetoacetate or spermidine, but not by histamine. These data reveal that AtoSC system regulates the motility and chemotaxis of E. coli, participating in the transcriptional induction of the main promoters of the chemotactic regulon and modifying the motility and chemotactic phenotypes in an induction-dependent mechanism.

Keywords: AtoSC; Two-component system; Chemotaxis; Histamine; Ca²⁺ ; Motility

Beneficial effect of taurine on hypoxia- and glutamate-induced endoplasmic reticulum stress pathways in primary neuronal culture by Chunliu Pan; Howard Prentice; Allison L. Price; Jang-Yen Wu (pp. 845-855).

Stroke (hypoxia) is one of the leading causes of mortality in the developed countries, and it can induce excessive glutamate release and endoplasmic reticulum (ER) stress. Taurine, as a free amino acid, present in high concentrations in a range of organs in mammals, can provide protection against multiple neurological diseases. Here, we present a study to investigate the potential protective benefits of taurine against ER stress induced by glutamate and hypoxia/reoxygenation in primary cortical neuronal cultures. We found that taurine suppresses the up-regulation of caspase-12 and GADD153/CHOP induced by hypoxia/reoxygenation, suggesting that taurine may exert a protective function against hypoxia/reoxygenation by reducing the ER stress. Moreover, taurine can down-regulate the ratio of cleaved ATF6 and full length ATF6, and p-IRE1 expression, indicating that taurine inhibits the ER stress induced by hypoxia/reoxygenation and glutamate through suppressing ATF6 and IRE1 pathways.

Keywords: Taurine; Neuroprotection; Hypoxia; Endoplasmic reticulum stress; Glutamate

Stable triazolylphosphonate analogues of phosphohistidine by Shin Mukai; Gavin R. Flematti; Lindsay T. Byrne; Paul G. Besant; Paul V. Attwood; Matthew J. Piggott (pp. 857-874).

Histidine-phosphorylated proteins and the corresponding kinases are important components of bacterial and eukaryotic cell-signalling pathways, and are therefore potential drug targets. The study of these biomolecules has been hampered by the lability of the phosphoramidate functional group in the phosphohistidines and the lack of generic antibodies. Herein, the design and concise synthesis of stable triazolylphosphonate analogues of N1- and N3-phosphohistidine, and derivatives suitable for bioconjugation, are described.

Keywords: Phosphohistidine; Azide-alkyne cycloaddition; Triazolylalanine; Stable analogues; Synthesis; Haptens

Oxidative and nitrative modifications of enkephalins by human neutrophils: effect of nitroenkephalin on leukocyte functional responses by Elisabetta Capuozzo; Laura Pecci; Federica Giovannitti; Alessia Baseggio Conrado; Mario Fontana (pp. 875-884).

Neutrophils play a major role in acute inflammation by generating reactive oxygen/nitrogen species. Opioid peptides, including enkephalins, are present at inflammation sites. Neutrophils contribute to protect against inflammatory pain by releasing opioid peptides. In this investigation, the ability of human polymorphonuclear cells to induce oxidative and nitrative modifications of Leu-enkephalin has been investigated in vitro. Activated human neutrophils mediate the oxidation of Leu-enkephalin resulting in the production of dienkephalin. In the presence of nitrite at concentrations observed during inflammatory and infectious process (10–50 μM), nitroenkephalin, a nitrated derivative of Leu-enkephalin, is additionally formed. The yield of nitroenkephalin increases with nitrite concentration and is significantly inhibited by the addition of catalase or 4-aminobenzoic acid hydrazide (ABAH), a specific inhibitor of peroxidases. These results suggest that neutrophils induce nitration of Leu-enkephalin by a mechanism that is dependent on myeloperoxidase activity and hydrogen peroxide. Oxidative/nitrative modifications of Leu-enkephalin have been also evidenced when cells were treated with the NO-donor molecule, DEANO. The nitrated enkephalin has been examined for its effect on leukocyte functional responses. The data reveal that nitroenkephalin at micromolar concentrations inhibits superoxide anion generation and degranulation of azurophilic granules of human polymorphonuclear cells. Moreover, nitroenkephalin inhibits spontaneous apoptosis of neutrophils, as evaluated by measuring caspase-3 activity. Collectively, our data indicate that the nitrated enkephalin attenuates neutrophil activation and promotes the short-term survival of these cells, suggesting a possible role of the nitrocompound in the efficiency and resolution of inflammatory processes.

Keywords: Enkephalin; Nitroenkephalin; Neutrophils; Protein nitration; Inflammation; Apoptosis

Molecular properties of lysozyme-microbubbles: towards the protein and nucleic acid delivery by Sonia Melino; Meifang Zhou; Mariarosaria Tortora; Maurizio Paci; Francesca Cavalieri; Muthupandian Ashokkumar (pp. 885-896).

Microbubbles (MBs) have specific acoustic properties that make them useful as contrast agents in ultrasound imaging. The use of the MBs in clinical practice led to the development of more sensitive imaging techniques both in cardiology and radiology. Protein-MBs are typically obtained by dispersing a gas phase in the protein solution and the protein deposited/cross-linked on the gas–liquid interface stabilizes the gas core. Innovative applications of protein-MBs prompt the investigation on the properties of MBs obtained using different proteins that are able to confer them specific properties and functionality. Recently, we have synthesized stable air-filled lysozyme-MBs (LysMBs) using high-intensity ultrasound-induced emulsification of a partly reduced lysozyme in aqueous solutions. The stability of LysMBs suspension allows for post-synthetic modification of MBs surface. In the present work, the protein folded state and the biodegradability property of LysMBs were investigated by limited proteolysis. Moreover, LysMBs were coated and functionalized with a number of biomacromolecules (proteins, polysaccharides, nucleic acids). Remarkably, LysMBs show a high DNA-binding ability and protective effects of the nucleic acids from nucleases and, further, the ability to transform the bacteria cells. These results highlight on the possibility of using LysMBs for delivery of proteins and nucleic acids in prophylactic and therapeutic applications.

Keywords: Microbubbles; Lysozyme; Lactoferrin; Nucleic acid delivery; Ultrasounds; Proteolysis

Overexpression of the CHRNA5/A3/B4 genomic cluster in mice increases the sensitivity to nicotine and modifies its reinforcing effects by Xavier Gallego; Susanna Molas; Alejandro Amador-Arjona; Michael J. Marks; Noemí Robles; Patricia Murtra; Lluís Armengol; Rubén D. Fernández-Montes; Mònica Gratacòs; Martí Pumarola; Roberto Cabrera; Rafael Maldonado; Josefa Sabrià; Xavier Estivill; Mara Dierssen (pp. 897-909).

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated pentameric ion channels that account for the effects of nicotine. Recent genetic studies have highlighted the importance of variants of the CHRNA5/A3/B4 genomic cluster in human nicotine dependence. Among these genetic variants those found in non-coding segments of the cluster may contribute to the pathophysiology of tobacco use through alterations in the expression of these genes. To discern the in vivo effects of the cluster, we generated a transgenic mouse overexpressing the human CHRNA5/A3/B4 cluster using a bacterial artificial chromosome. Transgenic mice showed increased functional α3β4-nAChRs in brain regions where these subunits are highly expressed under normal physiological conditions. Moreover, they exhibited increased sensitivity to the pharmacological effects of nicotine along with higher activation of the medial habenula and reduced activation of dopaminergic neurons in the ventral tegmental area after acute nicotine administration. Importantly, transgenic mice showed increased acquisition of nicotine self-administration (0.015 mg/kg per infusion) and a differential response in the progressive ratio test. Our study provides the first in vivo evidence of the involvement of the CHRNA5/A3/B4 genomic cluster in nicotine addiction through modifying the activity of brain regions responsible for the balance between the rewarding and the aversive properties of this drug.

Keywords: α5; α3; β4 Nicotinic receptor subunits; CHRNA5/A3/B4 genomic cluster; VTA; MHb; Nicotine addiction

Relationship between digestibility and secondary structure of raw and thermally treated legume proteins: a Fourier transform infrared (FT-IR) spectroscopic study by M. Carbonaro; P. Maselli; A. Nucara (pp. 911-921).

The secondary structure of proteins in legumes, cereals, milk products and chicken meat was studied by diffuse reflectance infrared spectroscopy in the region of the amide I band. Major secondary structure components ( β-sheets, random coil, α-helix, turns), together with the low- and high-frequency side contributions, were resolved and related to the in vitro digestibility behaviour of the different foods. A strong inverse correlation between the relative spectral weights of the β-sheet structures and in vitro protein digestibility values was measured. Structural modifications in legume proteins induced by autoclaving were monitored by the changes in the amide I spectra. The results indicate that the β-sheet structures of raw legume proteins and the intermolecular β-sheet aggregates, arising upon heating, are primary factors in adversely affecting the digestibility.

Keywords: FT-IR; Diffuse reflectance; Food protein structure; Thermal aggregation; Legumes; Digestibility

SAR study of tyrosine–chlorambucil hybrid regioisomers; synthesis and biological evaluation against breast cancer cell lines by Caroline Descôteaux; Kevin Brasseur; Valérie Leblanc; Sophie Parent; Éric Asselin; Gervais Bérubé (pp. 923-935).

Amino acids were transformed and coupled to chlorambucil, a well-known chemotherapeutic agent, in an attempt to create new anticancer drugs with selectivity for breast cancer cells. Among the amino acids available, tyrosine was selected to act as an estrogenic ligand. It is hypothesized that tyrosine, which shows some structural similitude with estradiol, could possibly mimic the natural hormone and, subsequently, bind to the estrogen receptor. In this exploratory study, several tyrosine-drug conjugates have been designed. Thus, ortho-, meta- and para-tyrosine–chlorambucil analogs were synthesized in order to generate new anticancer drugs with structural diversity, more specifically in regards to the phenol group location. These new analogs were produced in good yield following efficient synthetic methodology. All the tyrosine–chlorambucil hybrids were more effective than the parent drug, chlorambucil. In vitro biological evaluation on estrogen receptor positive and estrogen receptor negative (ER⁺ and ER⁻) breast cancer cell lines revealed an enhanced cytotoxic activity for compounds with the phenol function located at position meta. Molecular docking calculations were performed for the pure l-ortho, l-meta- and l-para-tyrosine phenolic regioisomers. The synthesis of all tyrosine–chlorambucil hybrid regioisomers and their biological activity are reported herein. Possible orientations within the targeted protein [estrogen receptor alpha (ERα)] are discussed in relation to the biological activity.

Keywords: Ortho-tyrosine–chlorambucil hybrid; Meta-tyrosine–chlorambucil hybrid; Para-tyrosine–chlorambucil hybrid; Tyrosine–chlorambucil hybrid; Breast cancer

Proliferation potential of human amniotic fluid stem cells differently responds to mercury and lead exposure by C. Gundacker; M. Scheinast; L. Damjanovic; C. Fuchs; M. Rosner; M. Hengstschläger (pp. 937-949).

There are considerable gaps in our knowledge on cell biological effects induced by the heavy metals mercury (Hg) and lead (Pb). In the present study we aimed to explore the effects of these toxicants on proliferation and cell size of primary human amniotic fluid stem (AFS) cells. Monoclonal human AFS cells were incubated with three dosages of Hg and Pb (single and combined treatment; ranging from physiological to cytotoxic concentrations) and the intracellular Hg and Pb concentrations were analyzed, respectively. At different days of incubation the effects of Hg and Pb on proliferation, cell size, apoptosis, and expression of cyclins and the cyclin-dependent kinase inhibitor p27 were investigated. Whereas we found Hg to trigger pronounced effects on proliferation of human AFS cells already at low concentrations, anti-proliferative effects of Pb could only be detected at high concentrations. Exposure to high dose of Hg induced pronounced downregulation of cyclin A confirming the anti-proliferative effects observed for Hg. Co-exposure to Hg and Pb did not cause additive effects on proliferation and size of AFS cells, and on cyclin A expression. Our here presented data provide evidence that the different toxicological effects of Pb and Hg on primary human stem cells are due to different intracellular accumulation levels of these two toxicants. These findings allow new insights into the functional consequences of Pb and Hg for mammalian stem cells and into the cell biological behavior of AFS cells in response to toxicants.

Keywords: Amniotic fluid stem cells; Cell size; Lead acetate; Methyl mercury; Proliferation; Cyclin A

The impact of photo-induced molecular changes of dairy proteins on their ACE-inhibitory peptides and activity by Barbara Kerkaert; Frédéric Mestdagh; Tatiana Cucu; Kshitij Shrestha; John Van Camp; Bruno De Meulenaer (pp. 951-962).

Among all dietary proteins, dairy proteins are the most important source of bio-active peptides which can, however, be affected by modifications upon processing and storage. Since it is still unknown to which extent the biological activity of dairy proteins is altered by chemical reactions, this study focuses on the effect of photo-induced molecular changes on the angiotensin I converting enzyme (ACE) inhibitory activity. Milk proteins were dissolved in phosphate buffer containing riboflavin and stored under light at 4°C for one month during which the molecular changes and the ACE-inhibitory activity were analysed. An increase in the total protein carbonyls and the N-formylkynurenine content was observed, besides a decrease in the free thiol, tryptophan, tyrosine and histidine content. These changes were more severe in caseins compared with whey proteins and resulted moreover in the aggregation of caseins. Due to these photo-induced molecular changes, a significant loss of the ACE-inhibitory activity was observed for casein peptides. A peptide analysis moreover illustrated that the decreased activity was not attributed to a reduced digestibility but to losses of specific ACE-inhibitory peptides. The observed molecular changes, more specifically the degradation of specific amino acids and the casein aggregation, could be assigned as the cause of the altered peptide pattern and as such of the loss in ACE-inhibitory activity.

Keywords: Milk proteins; Photo-oxidation; ACE-inhibitory activity; ACE-inhibitory peptides; LC–TOF–MS

The steroid hormone 20-hydroxyecdysone upregulated the protein phosphatase 6 for the programmed cell death in the insect midgut by Chuan-Xu Wang; Wei-Wei Zheng; Peng-Cheng Liu; Jin-Xing Wang; Xiao-Fan Zhao (pp. 963-971).

Programmed cell death (PCD) plays an important role in insect midgut remodeling during metamorphosis. Insect midgut PCD is triggered by the steroid hormone 20-hydroxyecdysone (20E) and it is mediated by a series of genes. However, the mechanism by which 20E triggers midgut PCD is still unclear. Here, we report a protein phosphatase 6 (PP6) from Helicoverpa armigera playing roles in midgut PCD. PP6 was expressed in the midgut during larval growth and it is significantly increased during metamorphosis. The increase was proven to be regulated by 20E. The juvenile hormone analog methoprene has no effect on PP6 expression. RNA interference analysis suggests that 20E upregulated the PP6 transcript levels through the ecdysone receptor EcRB1. PP6 knockdown by larval feeding or PP6 dsRNA injection resulted in the repression of the midgut PCD during the metamorphic stage. The mechanism was demonstrated to be through the suppression of genes such as Broad (Br), E74a, E75b, HR3, E93, rpr, and caspase, which are involved in 20E signaling pathway or midgut PCD. These findings suggest that PP6 is involved in the 20E signal transduction pathway and participates in the PCD in midgut.

Keywords: Phosphatase 6; Programmed cell death; 20-hydroxyecdysone; Midgut

Balsamin, a novel ribosome-inactivating protein from the seeds of Balsam apple Momordica balsamina by Inderdeep Kaur; Santosh K. Yadav; Gururao Hariprasad; R. C. Gupta; Alagiri Srinivasan; Janendra K. Batra; Munish Puri (pp. 973-981).

Plant seeds, a rich source of proteins, are considered important for their application as functional ingredients in a food system. A novel ribosome-inactivating protein (RIP), balsamin was purified from the seeds of Balsam apple, Momordica balsamina. Balsamin was purified by ion exchange chromatography on CM Sepharose and gel filtration on superdex-75. It has a molecular weight of 28 kDa as shown by SDS-PAGE analysis. Balsamin inhibits protein synthesis in a rabbit reticulocyte lysate-based cell free translation assay with an IC₅₀ of 90.6 ng ml⁻¹. It has RNA N-glycosidase activity and releases a 400-base long fragment termed the Endo fragment from 28S rRNA in the same manner as does saporin-6 from Saponaria officinalis. The N-terminal sequence analysis of the first 12 amino acids of balsamin revealed that it shares 83% similarity with type I RIP α-MMC from Momordica charantia and 50% similarity with β-MMC (from Momordica charantia), bryodin I (from Bryonia dioica) and luffin a (from Luffa cylindrica). Balsamin was further characterized by mass spectrometry. CD spectroscopic studies indicate that secondary structure of balsamin contains helix (23.5%), β-strand (24.6%), turn (20%) and random coil (31.9%). Thus RIPs activity expressed in vegetables like Momordica sp. advocates its usage in diet.

Keywords: Ribosome inactivating protein (RIP); Momordica balsamina ; RNA N-glycosidase; Balsamin; Cucurbitaceae

Site-specific DOTA/europium-labeling of recombinant human relaxin-3 for receptor-ligand interaction studies by Wei-Jie Zhang; Xiao Luo; Ya-Li Liu; Xiao-Xia Shao; John D. Wade; Ross A. D. Bathgate; Zhan-Yun Guo (pp. 983-992).

Relaxin-3 (also known as INSL7) is a recently identified neuropeptide belonging to the insulin/relaxin superfamily. It has putative roles in the regulation of stress responses, food intake, and reproduction by activation of its cognate G-protein-coupled receptor RXFP3. It also binds and activates the relaxin family peptide receptors RXFP1 and RXFP4 in vitro. To obtain a europium-labeled relaxin-3 as tracer for studying the interaction of these receptors with various ligands, in the present work we propose a novel site-specific labeling strategy for the recombinant human relaxin-3 that has been previously prepared in our laboratory. First, the N-terminal 6×His-tag of the single-chain relaxin-3 precursor was removed by Aeromonas aminopeptidase and all of the primary amines of the resultant peptide were reversibly blocked by citroconic anhydride. Second, the A-chain N-terminus of the blocked peptide was released by endoproteinase Asp-N cleavage that removed the linker peptide between the B- and A-chains. Third, an alkyne moiety was introduced to the newly released A-chain N-terminus by reaction with the highly active primary amine-specific N-hydroxysuccinimide ester. Fourth, after removal of the reversible blockage under mild acidic condition, europium-loaded DOTA with an azide moiety was introduced to the two-chain relaxin-3 carrying the alkyne moiety through click chemistry. Using this site-specific labeling strategy, homogeneous monoeuropium-labeled human relaxin-3 could be obtained with good overall yield. In contrast, conventional random labeling resulted in a complex mixture that was poorly resolved because human relaxin-3 has four primary amine moieties that all react with the modification reagent. Both saturation and competition binding assays demonstrated that the DOTA/Eu³⁺-labeled relaxin-3 retained high binding affinity for human RXFP3, RXFP4, and RXFP1 and was therefore a suitable non-radioactive and stable tracer to study the interaction of various natural or designed ligands with these receptors. Using this site-specific labeling strategy, other functional probes, such as fluorescent dyes, biotin, or nanoparticles could also be introduced to the A-chain N-terminal of the recombinant human relaxin-3. Additionally, we improved the time-resolved fluorescence assay for the DOTA-bound europium ion which paves the way for the use of DOTA as a lanthanide chelator for protein and peptide labeling in future studies.

Keywords: Click chemistry; Europium; INSL7; Relaxin-3; Site-specific labeling

A simple screening method using ion chromatography for the diagnosis of cerebral creatine deficiency syndromes by Takahito Wada; Hiroko Shimbo; Hitoshi Osaka (pp. 993-997).

Cerebral creatine deficiency syndromes (CCDS) are caused by genetic defects in l-arginine:glycine amidinotransferase, guanidinoacetate methyltransferase or creatine transporter 1. CCDS are characterized by abnormal concentrations of urinary creatine (CR), guanidinoacetic acid (GA), or creatinine (CN). In this study, we describe a simple HPLC method to determine the concentrations of CR, GA, and CN using a weak-acid ion chromatography column with a UV detector without any derivatization. CR, GA, and CN were separated clearly with the retention times (mean ± SD, n = 3) of 5.54 ± 0.0035 min for CR, 6.41 ± 0.0079 min for GA, and 13.53 ± 0.046 min for CN. This new method should provide a simple screening test for the diagnosis of CCDS.

Keywords: Cerebral creatine deficiency syndromes; HPLC; Creatine; Guanidinoacetic acid; Creatinine

The suppression of thymic stromal lymphopoietin expression by selenium by Phil-Dong Moon; Hyung-Min Kim (pp. 999-1004).

Thymic stromal lymphopoietin (TSLP) is a key mediator of allergic diseases such as allergic rhinitis, asthma, and atopic dermatitis. Selenium (Se) has various effects such as antioxidant, antitumor, antiulcer, and anti-inflammatory effects. However, the effect of Se on the production of TSLP has not been clarified. Thus, we investigated how Se inhibits the production of TSLP in the human mast cell line, HMC-1 cells. Se suppressed the production and mRNA expression of TSLP in HMC-1 cells. The maximal inhibition rate of TSLP production by Se (10 μM) was 59.14 ± 1.10%. In addition, Se suppressed the nuclear factor-κB luciferase activity induced by phorbol myristate acetate plus A23187. In the activated HMC-1 cells, the activation of caspase-1 was increased; whereas the activation of caspase-1 was decreased by pretreatment with Se. These results suggest that Se can be used to treat inflammatory and atopic diseases through the suppression of TSLP.

Keywords: Thymic stromal lymphopoietin; Selenium; Nuclear factor-κB; Caspase-1

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.43, #2)