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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.43, #1)

Carnosine in exercise and disease: introduction to the International Congress held at Ghent University, Belgium, July 2011 by Wim Derave; Craig Sale (pp. 1-4).

Determinants of muscle carnosine content by R. C. Harris; J. A. Wise; K. A. Price; H. J. Kim; C. K. Kim; C. Sale (pp. 5-12).

The main determinant of muscle carnosine (M-Carn) content is undoubtedly species, with, for example, aerobically trained female vegetarian athletes [with circa 13 mmol/kg dry muscle (dm)] having just 1/10th of that found in trained thoroughbred horses. Muscle fibre type is another key determinant, as type II fibres have a higher M-Carn or muscle histidine containing dipeptide (M-HCD) content than type I fibres. In vegetarians, M-Carn is limited by hepatic synthesis of β-alanine, whereas in omnivores this is augmented by the hydrolysis of dietary supplied HCD’s resulting in muscle levels two or more times higher. β-alanine supplementation will increase M-Carn. The same increase in M-Carn occurs with administration of an equal molar quantity of carnosine as an alternative source of β-alanine. Following the cessation of supplementation, M-Carn returns to pre-supplementation levels, with an estimated t_1/2 of 5–9 weeks. Higher than normal M-Carn contents have been noted in some chronically weight-trained subjects, but it is unclear if this is due to the training per se, or secondary to changes in muscle fibre composition, an increase in β-alanine intake or even anabolic steroid use. There is no measureable loss of M-Carn with acute exercise, although exercise-induced muscle damage may result in raised plasma concentrations in equines. Animal studies indicate effects of gender and age, but human studies lack sufficient control of the effects of diet and changes in muscle fibre composition.

Keywords: Muscle carnosine; β-alanine; Species; Diet; Exercise and training; Age and gender

The influence of sex, age and heritability on human skeletal muscle carnosine content by Audrey Baguet; Inge Everaert; Erik Achten; Martine Thomis; Wim Derave (pp. 13-20).

The dipeptide carnosine is found in high concentrations in human skeletal muscle and shows large inter-individual differences. Sex and age are determining factors, however, systematic studies investigating the sex effects on muscle carnosine content throughout the human lifespan are lacking. Despite the large inter-individual variation, the intra-individual variation is limited. The question may be asked whether the carnosine content is a muscle characteristic which may be largely genetically determined. A total of 263 healthy male and female subjects of 9–83 years were divided into five different age groups: prepubertal children (PC), adolescents (A), young adults (YA), middle adults (MA) and elderly (E). We included 25 monozygotic and 22 dizygotic twin pairs among the entire study population to study the heritability. The carnosine content was measured non-invasively in the gastrocnemius medialis and soleus by proton magnetic resonance spectroscopy (¹H-MRS). In boys, carnosine content was significantly higher (gastrocnemius 22.9%; soleus 44.6%) in A compared to PC, while it did not differ in girls. A decrease (~16%) was observed both in males and females from YA to MA. However, elderly did not have lower carnosine levels in comparison with MA. Higher correlations were found in monozygotic (r = 0.86) compared to dizygotic (r = 0.51) twins, in soleus muscle, but not in gastrocnemius. In conclusion, this study found an effect of puberty on muscle carnosine content in males, but not in females. Muscle carnosine decreased mainly during early adulthood and hardly from adulthood to elderly. High intra-twin correlations were observed, but muscle-dependent differences preclude clear conclusions toward heritability.

Keywords: Muscle carnosine; Puberty; Aging; Heritability; ¹H-MRS

Reduced muscle carnosine content in type 2, but not in type 1 diabetic patients by Bruno Gualano; Inge Everaert; Sanne Stegen; Guilherme Giannini Artioli; Youri Taes; Hamilton Roschel; Eric Achten; Maria Concepción Otaduy; Antonio Herbert Lancha Junior; Roger Harris; Wim Derave (pp. 21-24).

Carnosine is present in high concentrations in skeletal muscle where it contributes to acid buffering and functions also as a natural protector against oxidative and carbonyl stress. Animal studies have shown an anti-diabetic effect of carnosine supplementation. High carnosinase activity, the carnosine degrading enzyme in serum, is a risk factor for diabetic complications in humans. The aim of the present study was to compare the muscle carnosine concentration in diabetic subjects to the level in non-diabetics. Type 1 and 2 diabetic patients and matched healthy controls (total n = 58) were included in the study. Muscle carnosine content was evaluated by proton magnetic resonance spectroscopy (3 Tesla) in soleus and gastrocnemius. Significantly lower carnosine content (−45%) in gastrocnemius muscle, but not in soleus, was shown in type 2 diabetic patients compared with controls. No differences were observed in type 1 diabetic patients. Type II diabetic patients display a reduced muscular carnosine content. A reduction in muscle carnosine concentration may be partially associated with defective mechanisms against oxidative, glycative and carbonyl stress in muscle.

Keywords: Skeletal muscle; Glycation; AGEs; Oxidative stress; Carbonyl stress; Carnosine; Beta-alanine; Diabetic complications; MRS

Effects of β-alanine supplementation on exercise performance: a meta-analysis by R. M. Hobson; B. Saunders; G. Ball; R. C. Harris; C. Sale (pp. 25-37).

Due to the well-defined role of β-alanine as a substrate of carnosine (a major contributor to H⁺ buffering during high-intensity exercise), β-alanine is fast becoming a popular ergogenic aid to sports performance. There have been several recent qualitative review articles published on the topic, and here we present a preliminary quantitative review of the literature through a meta-analysis. A comprehensive search of the literature was employed to identify all studies suitable for inclusion in the analysis; strict exclusion criteria were also applied. Fifteen published manuscripts were included in the analysis, which reported the results of 57 measures within 23 exercise tests, using 18 supplementation regimes and a total of 360 participants [174, β-alanine supplementation group (BA) and 186, placebo supplementation group (Pla)]. BA improved (P = 0.002) the outcome of exercise measures to a greater extent than Pla [median effect size (IQR): BA 0.374 (0.140–0.747), Pla 0.108 (−0.019 to 0.487)]. Some of that effect might be explained by the improvement (P = 0.013) in exercise capacity with BA compared to Pla; no improvement was seen for exercise performance (P = 0.204). In line with the purported mechanisms for an ergogenic effect of β-alanine supplementation, exercise lasting 60–240 s was improved (P = 0.001) in BA compared to Pla, as was exercise of >240 s (P = 0.046). In contrast, there was no benefit of β-alanine on exercise lasting <60 s (P = 0.312). The median effect of β-alanine supplementation is a 2.85% (−0.37 to 10.49%) improvement in the outcome of an exercise measure, when a median total of 179 g of β-alanine is supplemented.

Keywords: β-Alanine supplementation; Meta-analysis; Exercise test and duration

Effect of beta-alanine supplementation on repeated sprint performance during the Loughborough Intermittent Shuttle Test by Bryan Saunders; Craig Sale; Roger C. Harris; Caroline Sunderland (pp. 39-47).

The aim of this study was to examine the effect of β-alanine supplementation on repeated sprint performance during an intermittent exercise protocol designed to replicate games play. Sixteen elite and twenty non-elite game players performed the Loughborough Intermittent Shuttle Test (LIST) on two separate occasions. Trials were separated by 4 weeks of supplementation with either β-alanine (BA) or maltodextrin (MD). There was no deterioration in sprint times from Set 1 to Set 6 of the LIST in either group prior to supplementation (elite: P = 0.92; non-elite: P = 0.12). Neither BA nor MD supplementation affected sprint times. Blood lactate concentrations were elevated during exercise in both groups, with no effect of supplementation. β-Alanine supplementation did not significantly improve sprint performance during the LIST. Neither group showed a performance decrement prior to supplementation, which might have masked any benefit from increased muscle buffering capacity due to β-alanine supplementation.

Keywords: Beta-alanine; Carnosine; Intermittent exercise; Repeated sprint performance

Beta-alanine (Carnosyn™) supplementation in elderly subjects (60–80 years): effects on muscle carnosine content and physical capacity by Serena del Favero; Hamilton Roschel; Marina Y. Solis; Ana P. Hayashi; Guilherme G. Artioli; Maria Concepción Otaduy; Fabiana B. Benatti; Roger C. Harris; John A. Wise; Cláudia C. Leite; Rosa M. Pereira; Ana L. de Sá-Pinto; Antonio Herbert Lancha-Junior; Bruno Gualano (pp. 49-56).

The aim of this study was to investigate the effects of beta-alanine supplementation on exercise capacity and the muscle carnosine content in elderly subjects. Eighteen healthy elderly subjects (60–80 years, 10 female and 4 male) were randomly assigned to receive either beta-alanine (BA, n = 12) or placebo (PL, n = 6) for 12 weeks. The BA group received 3.2 g of beta-alanine per day (2 × 800 mg sustained-release Carnosyn™ tablets, given 2 times per day). The PL group received 2 × (2 × 800 mg) of a matched placebo. At baseline (PRE) and after 12 weeks (POST-12) of supplementation, assessments were made of the muscle carnosine content, anaerobic exercise capacity, muscle function, quality of life, physical activity and food intake. A significant increase in the muscle carnosine content of the gastrocnemius muscle was shown in the BA group (+85.4%) when compared with the PL group (+7.2%) (p = 0.004; ES: 1.21). The time-to-exhaustion in the constant-load submaximal test (i.e., TLIM) was significantly improved (p = 0.05; ES: 1.71) in the BA group (+36.5%) versus the PL group (+8.6%). Similarly, time-to-exhaustion in the incremental test was also significantly increased (p = 0.04; ES 1.03) following beta-alanine supplementation (+12.2%) when compared with placebo (+0.1%). Significant positive correlations were also shown between the relative change in the muscle carnosine content and the relative change in the time-to-exhaustion in the TLIM test (r = 0.62; p = 0.01) and in the incremental test (r = 0.48; p = 0.02). In summary, the current data indicate for the first time, that beta-alanine supplementation is effective in increasing the muscle carnosine content in healthy elderly subjects, with subsequent improvement in their exercise capacity.

Keywords: Acidosis; Buffering capacity; Ergogenic aid; Elderly people

Optimizing human in vivo dosing and delivery of β-alanine supplements for muscle carnosine synthesis by Trent Stellingwerff; Jacques Decombaz; Roger C. Harris; Chris Boesch (pp. 57-65).

Interest into the effects of carnosine on cellular metabolism is rapidly expanding. The first study to demonstrate in humans that chronic β-alanine (BA) supplementation (~3–6 g BA/day for ~4 weeks) can result in significantly augmented muscle carnosine concentrations (>50%) was only recently published. BA supplementation is potentially poised for application beyond the niche exercise and performance-enhancement field and into other more clinical populations. When examining all BA supplementation studies that directly measure muscle carnosine (n = 8), there is a significant linear correlation between total grams of BA consumed (of daily intake ranges of 1.6–6.4 g BA/day) versus both the relative and absolute increases in muscle carnosine. Supporting this, a recent dose–response study demonstrated a large linear dependency (R ² = 0.921) based on the total grams of BA consumed over 8 weeks. The pre-supplementation baseline carnosine or individual subjects’ body weight (from 65 to 90 kg) does not appear to impact on subsequent carnosine synthesis from BA consumption. Once muscle carnosine is augmented, the washout is very slow (~2%/week). Recently, a slow-release BA tablet supplement has been developed showing a smaller peak plasma BA concentration and delayed time to peak, with no difference in the area under the curve compared to pure BA in solution. Further, this slow-release profile resulted in a reduced urinary BA loss and improved retention, while at the same time, eliciting minimal paraesthesia symptoms. However, our complete understanding of optimizing in vivo delivery and dosing of BA is still in its infancy. Thus, this review will clarify our current knowledge of BA supplementation to augment muscle carnosine as well as highlight future research questions on the regulatory points of control for muscle carnosine synthesis.

Keywords: β-Alanine; Carnosine; Muscle; Synthesis; Washout; Dose–response

Effect of slow-release β-alanine tablets on absorption kinetics and paresthesia by Jacques Décombaz; Maurice Beaumont; Jacques Vuichoud; Florilene Bouisset; Trent Stellingwerff (pp. 67-76).

Oral β-alanine (βA) doses larger than 800 mg commonly result in unpleasant sensory symptoms (paresthesia). However, the association of form (pure vs. slow-release) with side-effects has not been fully described. The aim of this single-blinded, randomized three-arm clinical trial was to compare plasma kinetics and symptoms following βA bolus administration in solution or in slow-release tablet form. Eleven healthy adults ingested 1.6 g of a pure βA reference solution (REF), 1.6 g in slow-release βA tablets (TAB) or a placebo (PLA) after an overnight fast. During the next 6 h, urinary and plasma βA concentrations were measured and questionnaires about intensity, nature (pins and needles, itching, flushing, irritation, numbness, soreness), and spatial distribution of unusual sensations were filled in. TAB resulted in a smaller peak plasma concentration than REF (82 vs. 248 μmol L⁻¹, p < 0.001), delayed time to peak (1.0 vs. 0.5 h, p < 0.01) no difference in area under the curve, reduced loss in urine (202 vs. 663 μmol, p < 0.0001), and improved retention (98.9 vs. 96.3%, p < 0.001). Symptoms described as “pins and needles” were perceived rapidly on the skin of the arms and trunk after REF (T _max = 15 min) and their time course nearly mimicked plasma concentrations. Maximum intensity scores were weaker with TAB (“very low”) than with REF (“low”, p < 0.001), while TAB and PLA did not differ with respect to side-effects. In summary, ingesting 1.6 g βA in slow-release tablets rather than pure in solution results in slower absorption kinetics, improved whole body retention and sensory side-effects that cannot be differentiated from PLA.

Keywords: β-Alanine; Supplementation; Nociception; Sensory; Side-effect

Exercise-induced oxidative stress: the effects of β-alanine supplementation in women by A. E. Smith; J. R. Stout; K. L. Kendall; D. H. Fukuda; J. T. Cramer (pp. 77-90).

The purpose of this study was to evaluate the effects of β-alanine supplementation on markers of oxidative stress. Twenty-four women (age: 21.7 ± 2.1 years; VO₂max: 2.6 ± 0.3 l min⁻¹) were randomly assigned, in a double-blind fashion, to a β-alanine (BA, 2 × 800 mg tablets, 3× daily; CarnoSyn^®; n = 13) or placebo (PL, 2 × 800 mg maltodextrin tablets, 3× daily; n = 11) group. A graded oxygen consumption test (VO₂max) was performed to evaluate VO₂max, time to exhaustion, ventilatory threshold and establish peak velocity (PV). A 40-min treadmill run was used to induce oxidative stress. Total antioxidant capacity, superoxide dismutase, 8-isoprostane (8ISO) and reduced glutathione were measured. Heart rate and ratings of perceived exertion were recorded during the 40 min run. Separate three- [4 × 2 × 2; acute (base vs. IP vs. 2 vs. 4 h) × chronic (pre- vs. post-) × treatment (BA vs. PL)] and two- [2 × 2; time (pre-supplement vs. post-supplement) × treatment (BA vs. PL)] way ANOVAs were used for analyses. There was a significant increase in VO₂max (p = 0.009), independent of treatment, with no significant changes in TTE (p = 0.074) or VT (p = 0.344). Ratings of perceived exertion values were significantly improved from pre- to post-supplementation for the BA group only at 40 min (p = 0.02). The ANOVA model demonstrated no significant treatment effects on oxidative stress. The chronic effects of BA supplementation demonstrated little antioxidant potential, in women, and little influence on aerobic performance assessments.

Keywords: Carnosine; Antioxidant; Sex; Running; Aerobic capacity; Supplement

Carnosine protects neurons against oxidative stress and modulates the time profile of MAPK cascade signaling by Konstantin Kulebyakin; Larisa Karpova; Ekaterina Lakonsteva; Mikhail Krasavin; Alexander Boldyrev (pp. 91-96).

Carnosine is a known protector of neuronal cells against oxidative injury which prevents both apoptotic and necrotic cellular death. It was shown earlier that carnosine serves as an intracellular buffer of free radicals. Using the model of ligand-dependent oxidative stress in neurons, we have shown that homocysteine (HC) initiates long-term activation of extracellular signal regulated kinase, isoforms 1 and 2 (ERK 1/2) and Jun N-terminal kinase (JNK) which corresponds to exitotoxic effect resulting in cellular death. l-Carnosine (β-alanyl-l-histidine) protects neurons from both excitotoxic effect of homocysteine and cellular death. Its analogs, β-alanyl-d-histidine (d-carnosine) and l-histidyl-β-alanine, restricted accumulation of free radicals and delayed activation of ERK1/2 and JNK in neuronal cells, but did not promote neuronal viability.

Keywords: Hyperhomocysteinemia; Carnosine; Excitotoxicity; MAPK; Neuroprotection

Role of l-carnosine in the control of blood glucose, blood pressure, thermogenesis, and lipolysis by autonomic nerves in rats: involvement of the circadian clock and histamine by Katsuya Nagai; Mamoru Tanida; Akira Niijima; Nobuo Tsuruoka; Yoshinobu Kiso; Yuko Horii; Jiao Shen; Nobuaki Okumura (pp. 97-109).

l-Carnosine (β-alanyl-l-histidine; CAR) is synthesized in mammalian skeletal muscle. Although the physiological roles of CAR have not yet been clarified, there is evidence that the release of CAR from skeletal muscle during physical exercise affects autonomic neurotransmission and physiological functions. In particular, CAR affects the activity of sympathetic and parasympathetic nerves innervating the adrenal glands, liver, kidney, pancreas, stomach, and white and brown adipose tissues, thereby causing changes in blood pressure, blood glucose, appetite, lipolysis, and thermogenesis. CAR-mediated changes in neurotransmission and physiological functions were eliminated by histamine H1 or H3 receptor antagonists (diphenhydramine or thioperamide) and bilateral lesions of the hypothalamic suprachiasmatic nucleus (SCN), a master circadian clock. Moreover, a carnosine-degrading enzyme (carnosinase 2) was shown to be localized to histamine neurons in the hypothalamic tuberomammillary nucleus (TMN). Thus, CAR released from skeletal muscle during exercise may be transported into TMN-histamine neurons and hydrolyzed. The resulting l-histidine may subsequently be converted into histamine, which could be responsible for the effects of CAR on neurotransmission and physiological function. Thus, CAR appears to influence hypoglycemic, hypotensive, and lipolytic activity through regulation of autonomic nerves and with the involvement of the SCN and histamine. These findings are reviewed and discussed in the context of other recent reports, including those on carnosine synthetases, carnosinases, and carnosine transport.

Keywords: Sympathetic nerve; Parasympathetic nerve; Suprachiasmatic nerve; Adrenal gland; Pancreas; Carnosinase

Transforming dietary peptides in promising lead compounds: the case of bioavailable carnosine analogs by Giulio Vistoli; Marina Carini; Giancarlo Aldini (pp. 111-126).

The ability of carnosine to prevent advanced glycoxidation end products (AGEs) and advanced lipoxidation end products (ALEs) formation, on the one hand, and the convincing evidence that these compounds act as pathogenetic factors, on the other hand, strongly support carnosine as a promising therapeutic agent for oxidative-based diseases. The mechanism/s by which carnosine inhibits AGEs and ALEs is still under investigation but an emerging hypothesis is that carnosine acts by deactivating the AGEs and ALEs precursors and in particular the reactive carbonyl species (RCS) generated by both lipid and sugar oxidation. The ability of carnosine to inhibit AGEs and ALEs formation and the corresponding biological effects has been demonstrated in several in vitro studies and in some animal models. However, such effects are in line of principle, limited in humans, due to the effect of serum carnosinase (absent in rodents), which catalyzes the carnosine hydrolysis to its constitutive amino acids. Such a limitation has prompted a great interest in the design of carnosine derivatives, which maintaining (or improving) the reactivity with RCS, are more resistant to carnosinase. The present paper intends to critically review the most recent studies oriented to obtaining carnosine derivatives, optimized in terms of reactivity with RCS, selectivity (no reaction with physiological aldehydes) and the pharmacokinetic profile (mainly through an enhanced resistance to carnosinase hydrolysis). The review also includes a brief description of AGEs and ALEs as drug targets and the evidence so far reported regarding the ability of carnosine as inhibitor of AGEs and ALEs formation and the proposed reaction mechanisms.

Keywords: Carnosine and derivatives thereof; Advanced glycoxidation end products (AGEs); Advanced lipoxidation end products (ALEs); Reactive carbonyl species; Carbonyl quenching

Carnosine enhances diabetic wound healing in the db/db mouse model of type 2 diabetes by Ishrath Ansurudeen; Vivekananda Gupta Sunkari; Jacob Grünler; Verena Peters; Claus Peter Schmitt; Sergiu-Bogdan Catrina; Kerstin Brismar; Elisabete Alcantara Forsberg (pp. 127-134).

Diabetes mellitus (DM) is a progressive disorder with severe late complications. Normal wound healing involves a series of complex and well-orchestrated molecular events dictated by multiple factors. In diabetes, wound healing is grossly impaired due to defective, and dysregulated cellular and molecular events at all phases of wound healing resulting in chronic wounds that fail to heal. Carnosine, a dipeptide of alanine and histidine and an endogenous antioxidant is documented to accelerate healing of wounds and ulcers. However, not much is known about its role in wound healing in diabetes. Therefore, we studied the effect of carnosine in wound healing in db/db mice, a mice model of Type 2 DM. Six millimeter circular wounds were made in db/db mice and analyzed for wound healing every other day. Carnosine (100 mg/kg) was injected (I.P.) every day and also applied locally. Treatment with carnosine enhanced wound healing significantly, and wound tissue analysis showed increased expression of growth factors and cytokines genes involved in wound healing. In vitro studies with human dermal fibroblasts and microvascular-endothelial cells showed that carnosine increases cell viability in presence of high glucose. These effects, in addition to its known role as an antioxidant and a precursor for histamine synthesis, provide evidence for a possible therapeutic use of carnosine in diabetic wound healing.

Keywords: Carnosine; Diabetic wound healing; db/db mice; Human dermal fibroblast; Human dermal microvascular endothelial cells

Carnosine and cancer: a perspective by Frank Gaunitz; Alan R. Hipkiss (pp. 135-142).

The application of carnosine in medicine has been discussed since several years, but many claims of therapeutic effects have not been substantiated by rigorous experimental examination. In the present perspective, a possible use of carnosine as an anti-neoplastic therapeutic, especially for the treatment of malignant brain tumours such as glioblastoma is discussed. Possible mechanisms by which carnosine may perform its anti-tumourigenic effects are outlined and its expected bioavailability and possible negative and positive side effects are considered. Finally, alternative strategies are examined such as treatment with other dipeptides or β-alanine.

Different conformational forms of serum carnosinase detected by a newly developed sandwich ELISA for the measurements of carnosinase concentrations by Katja Adelmann; Dirk Frey; Eva Riedl; Hannes Koeppel; Frederick Pfister; Verena Peters; Claus P. Schmitt; Paula Sternik; Stephanie Hofmann; Hans Walter Zentgraf; Gerjan Navis; Jacob van den Born; Stephan J. L. Bakker; Bernhard K. Krämer; Benito A. Yard; Sibylle J. Hauske (pp. 143-151).

Serum carnosinase (CN-1) measurements are at present mainly performed by assessing enzyme activity. This method is time-consuming, not well suited for large series of samples and can be discordant to measurements of CN-1 protein concentrations. To overcome these limitations, we developed sandwich ELISA assays using different anti-CN-1 antibodies, i.e., ATLAS (polyclonal IgG) and RYSK173 (monoclonal IgG1). With the ATLAS-based assay, similar amounts of CN-1 were detected in serum and both EDTA and heparin plasma. The RYSKS173-based assay detected CN-1 in serum in all individuals at significantly lower concentrations compared to the ATLAS-based assay (range: 0.1–1.8 vs. 1–50 μg/ml, RYSK- vs. ATLAS-based, P < 0.01). CN-1 detection with the RYSK-based assay was increased in EDTA plasma, albeit at significantly lower concentrations compared to ATLAS. In heparin plasma, CN-1 was also poorly detected with the RYSK-based assay. Addition of DTT to serum increased the detection of CN-1 in the RYSK-based assay almost to the levels found in the ATLAS-based assay. Both ELISA assays were highly reproducible (R: 0.99, P < 0.01 and R: 0.93, P < 0.01, for the RYSK- and ATLAS-based assays, respectively). Results of the ATLAS-based assay showed a positive correlation with CN-1 activity (R: 0.62, P < 0.01), while this was not the case for the RYSK-based assay. However, there was a negative correlation between CN-1 activity and the proportion of CN-1 detected in the RYSK-based assay, i.e., CN-1 detected with the RYSK-based assay/CN-1 detected with the ATLAS-based assay × 100% (Spearman–Rang correlation coefficient: −0.6, P < 0.01), suggesting that the RYSK-based assay most likely detects a CN-1 conformation with low CN-1 activity. RYSK173 and ATLAS antibodies reacted similarly in Western blot, irrespective of PNGase treatment. Binding of RYSK173 in serum was not due to differential N-glycosylation as demonstrated by mutant CN-1 cDNA constructs. In conclusion, our study demonstrates a good correlation between enzyme activity and CN-1 protein concentration in ELISA and suggests the presence of different CN-1 conformations in serum. The relevance of these different conformations is still elusive and needs to be addressed in further studies.

Keywords: Carnosinase; ELISA; Conformation; Antibodies; Plasma

Carnosine derivatives: new multifunctional drug-like molecules by Francesco Bellia; Graziella Vecchio; Enrico Rizzarelli (pp. 153-163).

Carnosine (β-alanyl-l-histidine) is an endogenous dipeptide widely and abundantly distributed in the muscle and nervous tissues of several animal species. Many functions have been proposed for this compound because of its antioxidant and metal ion-chelator properties. Many potential therapeutic properties have been recognized especially related to the antioxidant activity, but the therapeutic uses are strongly limited by the mechanism governing its homeostasis. This fact has been the main reason for developing the synthesis of carnosine derivatives with interesting potentiality, but until now there have been very few applications. These derivatives could represent the future drugs for many pathologies related to oxidative stress and metal ion dyshomeostasis.

Keywords: Carnosine; Dipeptide; Derivatisation; Carnosinase

Carnosine and its (S)-Trolox™ derivative protect animals against oxidative stress by S. Stvolinsky; K. Toropova; M. Gordeeva; V. Kazey; T. Sato; K. Meguro; A. Boldyrev (pp. 165-170).

The novel synthetic derivative of carnosine, (S)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl-β-alanyl-l-histidine (S-Trolox™-Carnosine, STC) increases the resistance of rats to experimental acute hypobaric hypoxia (AHH) thus protecting brain from the oxidative damage. This effect is accompanied by better preservation of the acquired skills in Morris water maze possibly by increasing efficiency of the brain antioxidant system. In addition, STC caused an increase in life span of both male and female fruit fly Drosophila melanogaster whereas carnosine increased life span only in male fruit flies. The results indicate that development of the drug based on STC could be beneficial in neurology and gerontology.

Keywords: Carnosine; Trolox™; Acute hypobaric hypoxia; Oxidative stress; Brain; Antioxidant defense

Insulin resistance and the metabolism of branched-chain amino acids in humans by María M. Adeva; Jesús Calviño; Gema Souto; Cristóbal Donapetry (pp. 171-181).

Peripheral resistance to insulin action is the major mechanism causing the metabolic syndrome and eventually type 2 diabetes mellitus. The metabolic derangement associated with insulin resistance is extensive and not restricted to carbohydrates. The branched-chain amino acids (BCAAs) are particularly responsive to the inhibitory insulin action on amino acid release by skeletal muscle and their metabolism is profoundly altered in conditions featuring insulin resistance, insulin deficiency, or both. Obesity, the metabolic syndrome and diabetes mellitus display a gradual increase in the plasma concentration of BCAAs, from the obesity-related low-grade insulin-resistant state to the severe deficiency of insulin action in diabetes ketoacidosis. Obesity-associated hyperinsulinemia succeeds in maintaining near-normal or slightly elevated plasma concentration of BCAAs, despite the insulin-resistant state. The low circulating levels of insulin and/or the deeper insulin resistance occurring in diabetes mellitus are associated with more marked elevation in the plasma concentration of BCAAs. In diabetes ketoacidosis, the increase in plasma BCAAs is striking, returning to normal when adequate metabolic control is achieved. The metabolism of BCAAs is also disturbed in other situations typically featuring insulin resistance, including kidney and liver dysfunction. However, notwithstanding the insulin-resistant state, the plasma level of BCAAs in these conditions is lower than in healthy subjects, suggesting that these organs are involved in maintaining BCAAs blood concentration. The pathogenesis of the decreased BCAAs plasma level in kidney and liver dysfunction is unclear, but a decreased afflux of these amino acids into the blood stream has been observed.

Keywords: Insulin resistance; Branched-chain amino acids; Leucine; Isoleucine; Valine

Protective immunity provided by a new modified SERA protein peptide: its immunogenetic characteristics and correlation with 3D structure by Adriana Bermúdez; Armando Moreno-Vranich; Manuel E. Patarroyo (pp. 183-194).

The serine repeat antigen (SERA) protein is a leading candidate molecule for inclusion as a component in a multi-antigen, multi-stage, minimal subunit-based, chemically synthesised anti-malarial vaccine. Peptides having high red blood cell binding affinity (known as HABPs) have been identified in this protein. The 6733 HABP was located in the C-terminal portion of the 47-kDa fragment while HABP 6754 was located in the C-terminal region of the 56-kDa fragment. These conserved HABPs failed to induce an immune response. Critical red blood cell binding residues and/or their neighbours (assessed by glycine-analogue scanning) were replaced by others having the same mass, volume and surface but different polarity, rendering some of them highly immunogenic when assessed by antibody production against the parasite or its proteins and protection-inducers against experimental challenge with a highly infectious Aotus monkey-adapted Plasmodium falciparum strain. This manuscript presents some modified HABPs as vaccine candidate components for enriching our tailor-made anti-malarial vaccine repertoire, as well as their 3D structure obtained by ¹H-NMR displaying a short-structured region, differently from the native ones having random structures.

Keywords: SERA 5; NMR; Structure; Malaria vaccine

Helix formation and capping energetics of arginine analogs with varying side chain length by Richard P. Cheng; Yi-Jen Weng; Wei-Ren Wang; Marc J. Koyack; Yuta Suzuki; Cheng-Hsun Wu; Po-An Yang; Hao-Chun Hsu; Hsiou-Ting Kuo; Prashant Girinath; Chun-Jen Fang (pp. 195-206).

Arginine (Arg) has been used for recognizing negatively charged biological molecules, cell penetration, and oligosaccharide mass signal enhancement. The versatility of Arg has inspired the need to develop Arg analogs and to research the structural effects of incorporating Arg analogs. Accordingly, we investigated the effect of Arg side chain length on helix formation by studying 12 Ala-based peptides containing the Arg analogs (S)-2-amino-6-guanidino-hexanoic acid (Agh), (S)-2-amino-4-guanidinobutyric acid (Agb), and (S)-2-amino-3-guanidinopropionic acid (Agp). Solid phase guanidinylation with orthogonal protection strategies was necessary to synthesize Agb- and Agp-containing peptides using Fmoc-based chemistry. The fraction helix for the peptides was determined by circular dichroism spectroscopy, and used to derive the statistical mechanical parameters and energetics for N-capping, C-capping, and helix propagation (propensity). All four Arg analogs were unfavorable for N-capping. The C-cap parameter followed the trend Agp < Agb < Arg < Agh, showing more favorable C-cap energetics with increasing side chain length. In contrast, helix propensity followed the trend Agp < Agb < Arg > Agh, highlighting the uniqueness of the Arg side chain length in helix formation. Molecular mechanics calculations and a survey on protein structures were consistent with the experimental results. Furthermore, calculations and survey both showed that the g– conformation for the χ₁ dihedral was present for the first two residues at the N-terminus of helices, but not favored in the center or C-terminus of helices due to sterics. These results should serve as the foundation for developing Arg-related bioactive compounds and technologies.

Keywords: Arginine; Side chain length; Peptide; Helix

Design, conformational studies and analysis of structure–function relationships of PTH (1–11) analogues: the essential role of Val in position 2 by A. Caporale; L. Gesiot; M. Sturlese; A. Wittelsberger; S. Mammi; E. Peggion (pp. 207-218).

The N-terminal 1–34 segment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it elicits all the biological responses characteristic of the native intact PTH. Recent studies reported potent helical analogues of the PTH (1–11) with helicity-enhancing substitutions. This work describes the synthesis, biological activity, and conformational studies of analogues obtained from the most active non-natural PTH (1–11) peptide H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH₂; specifically, the replacement of Val in position 2 with d-Val, l-(αMe)-Val and N-isopropyl-Gly was studied. The synthesized analogues were characterized functionally by in-cell assays and their structures were determined by CD and NMR spectroscopy. To clarify the relationship between the structure and activity, the structural data were used to generate a pharmacophoric model, obtained overlapping all the analogues. This model underlines the fundamental functional role of the side chain of Val² and, at the same time, reveals that the introduction of conformationally constrained C^α-tetrasubstituted α-amino acids in the peptides increases their helical content, but does not necessarily ensure significant biological activity.

Keywords: PTH; NMR analysis; Pharmacophoric model; αMeVal; Conformational analogues; Structure/activity analysis

Cyclic RGD peptides interfere with binding of the Helicobacter pylori protein CagL to integrins α_Vβ₃ and α₅β₁ by Jens Conradi; Sylwia Huber; Katharina Gaus; Felix Mertink; Soledad Royo Gracia; Ulf Strijowski; Steffen Backert; Norbert Sewald (pp. 219-232).

The human pathogen Helicobacter pylori that may cause different gastric diseases exploits integrins for infection of gastric cells. The H. pylori protein CagL present on the outer region of the type IV secretion pilus contains an RGD sequence (-Arg-Gly-Asp-) that enables binding to cells presenting integrins α₅β₁ and α_Vβ₃. This interaction can be inhibited with conformationally designed cyclic RGD peptides derived from the CagL epitope -Ala-Leu-Arg-Gly-Asp-Leu-Ala-. The inhibition of the CagL–α_Vβ₃ interaction by different RGD peptides strongly suggests the importance of the RGD motif for CagL binding. CagL point mutants (RAD, RGA) show decreased affinity to integrin α_Vβ₃. Furthermore, structure–activity relationship studies with cyclic RGD peptides in a spatial screening approach show the distinct influence of the three-dimensional arrangement of RGD motif on the ability to interfere with this interaction. Resulting from these studies, similar structural requirements for the CagL epitope as previously suggested for other ligands of integrin α_Vβ₃ are proposed.

Keywords: CagL; Integrins; α_Vβ₃ ; RGD peptides; SAR studies

Regulatory role for l-arginine in the utilization of amino acids by pig small-intestinal bacteria by Zhao-Lai Dai; Xi-Long Li; Peng-Bin Xi; Jing Zhang; Guoyao Wu; Wei-Yun Zhu (pp. 233-244).

We recently reported that bacteria from the pig small intestine rapidly utilize and metabolize amino acids (AA). This study investigated the effect of l-arginine on the utilization of AA by pure bacterial strains (Streptococcus sp., Escherichia coli and Klebsiella sp.) and mixed bacterial cultures derived from the pig small intestine. Bacteria were incubated at 37°C for 3 h in anaerobic AA media containing 0–5 mmol/L of arginine to determine the effect of arginine on the bacterial utilization of AA. Amino acids in the medium plus cell extracts were analyzed by high-performance liquid chromatography. Results indicated concentration-dependent increases in the bacterial utilization of arginine and altered fluxes of arginine into ornithine and citrulline in the bacteria. Net glutamine utilization increased in pure bacterial strains with increased concentrations of arginine. With the addition of arginine, net utilization of threonine, glycine, phenylalanine and branched-chain AA increased (P < 0.05) in Streptococcus sp. and Klebsiella sp., but decreased in E. coli. Net utilization of lysine, threonine, isoleucine, leucine, glycine and alanine by jejunal or ileal mixed bacteria decreased (P < 0.05) with the addition of arginine. Complete utilization of asparagine, aspartate and serine were observed in pig small-intestinal bacteria after 3 h of incubation. Overall, the addition of arginine affected the metabolism of the arginine-family of AA and the serine- and aspartate-family of AA in small-intestinal bacteria and reduced the utilization of most AA in ileal mixed bacteria. These novel findings indicate that arginine exerts its beneficial effects on swine nutrition partially by regulating AA utilization and metabolism in the small-intestinal microbiota.

Keywords: Amino acids; Intestinal bacteria; Nutrition; Small intestine; Swine

Glycine and its N-methylated analogues cause pH-dependent membrane damage to enterotoxigenic Escherichia coli by D. Vanhauteghem; G. P. J. Janssens; A. Lauwaerts; S. Sys; F. Boyen; I. D. Kalmar; E. Meyer (pp. 245-253).

The current study first investigates the emulsifying potential of glycine and its N-methylated derivatives N-methylglycine (sarcosine), N,N-dimethylglycine (DMG) and N,N,N-trimethylglycine (betaine) under varying pH conditions. Subsequently, the effect of these test compounds on the membrane integrity of enterotoxigenic Escherichia coli (ETEC) was evaluated. Oil in water emulsions containing each compound show that DMG is a more potent enhancer of emulsification than glycine, sarcosine and betaine under the conditions tested. Flow cytometry was used to investigate whether the emulsifying potential is associated with an effect on ETEC membrane integrity. The bacteria were exposed to each of the test compounds under varying pH conditions and membrane integrity was assessed using the LIVE/DEAD BacLight kit. Results show a membrane deteriorating effect caused by glycine, sarcosine and DMG, but not by betaine. This effect is pH- and time-dependent and has an apparent threshold at pH 9.0. Conventional plate counts confirmed concomitant changes in culturability of the membrane comprised bacteria.

Keywords: Glycine; Methylamine; Flow cytometry; Membrane integrity; Emulsification; Alkaline stress

Site-directed mutagenesis and feedback-resistant N-acetyl-L-glutamate kinase (NAGK) increase Corynebacterium crenatum L-arginine production by Meijuan Xu; Zhiming Rao; Wenfang Dou; Juan Yang; Jian Jin; Zhenghong Xu (pp. 255-266).

N-Acetyl-l-glutamate kinase (EC 2.7.2.8) is first committed in the specific l-arginine pathway of Corynebacterium sp. A limited increase of l-arginine production for the argB overexpression in the engineering C. creantum SYPA-CCB strain indicated that l-arginine feedback inhibition plays an influence on the l-arginine production. In this study, we have performed site-directed mutagenesis of the key enzyme (NAGK) and the three mutations (E19R, H26E and H268D) exhibited the increase of I _0.5 ^R efficiently. Thereby, the multi-mutated NAGK_M3 (including E19R/H26E/H268D) was generated and its I _0.5 ^R of l-arginine of the mutant was increased remarkably, whereas the NAGK enzyme activities did not declined. To get a feedback-resistant and robust l-arginine producer, the engineered strains SYPA-CCB_M3 were constructed. Introducing the argB_M3 gene enabled the NAGK enzyme activity insensitive to the intracellular arginine concentrations resulted in an enhanced arginine biosynthesis flux and decreased formation of by-products. The l-arginine synthesis was largely enhanced due to the overexpression of the argB_M3, which is resistant to feedback resistant by l-arginine. Thus l-arginine production could reach 45.6 g/l, about 41.7% higher compared with the initial strain. This is an example of up-modulation of the flux through the l-arginine metabolic pathway by deregulating the key enzyme of the pathway.

Keywords: N-Acetyl-l-glutamate kinase (NAGK); Feedback inhibition; Site-directed mutagenesis; Corynebacterium crenatum ; l-Arginine production

Effect of double mutations K214/A–E215/Q of FRATide on GSK3β: insights from molecular dynamics simulation and normal mode analysis by Shao-Yong Lu; Yong-Jun Jiang; Jian-Wei Zou; Tian-Xing Wu (pp. 267-277).

Glycogen synthase kinase 3β (GSK3β) is a multifunctional serine/threonine protein kinase that is involved in several biological processes including insulin and Wnt signaling pathways. The Wnt signaling via FRAT-mediated displacement of axin inhibits GSK3β activity toward non-primed substrates without affecting its activity toward primed substrates. Herein, molecular dynamics simulation, molecular mechanics generalized Born/surface area (MM_GBSA) calculation, and normal mode analysis are performed to explore the structural influence of the double mutations K214/A–E215/Q of FRATide on the GSK3β–FRATide complex. The results reveal that the priming phosphate-binding site, the primed substrate-binding site, the alignment of the critical active site residues in the ATP-binding site, as well as the periodic open–closed conformational change of the ATP-binding site, which are critical for the catalytic activity of GSK3β, are negligibly influenced in the mutated system compared with the wild-type (WT) system. This indicates that FRATide does not inhibit the GSK3β activity toward primed substrates. Additionally, MM_GBSA calculation indicates that the less energy-favorable GSK3β–FRATide complex is observed in the mutant than in the WT complex.

Keywords: GSK3β; Wnt signaling; MD simulation; MM_GBSA; Normal mode analysis

Synchrotron vacuum ultraviolet (VUV) photo-induced fragmentation of cyclic dipeptides radical cations by Liyun Zhang; Liangyuan Jia; Lidong Zhang; Huijun Guo; Zhongyue Zhou; Junjie Weng; Fei Qi (pp. 279-287).

Cyclic dipeptides, due to their chemical properties and various bioactivities, are very attractive for medicinal chemistry. Fragmentations of three simple cyclic dipeptides including cyclo(Gly–Gly), cyclo(Ala–Ala) and cyclo(Gly–Val) in the gas-phase are determined with synchrotron vacuum ultraviolet (VUV) photoionization mass spectrometry (VUV PIMS) and theoretical calculations. Cyclo(Gly-Gly) and cyclo(Ala-Ala) show the similar fragmentation pathways. The primary decomposition reactions of cyclo(Gly-Gly) and cyclo(Ala-Ala) radical cations are found to be HNCO loss and CO elimination. The appearance energies (AEs) of fragment ions [CH₂NHCOCH₂]^+• and [CH₃CHNHCOCHCH₃]^+• are measured to be 10.21 and 9.66 ± 0.05 eV, respectively, which are formed from cyclo(Gly-Gly) and cyclo(Ala-Ala) radical cations with HNCO elimination. Due to the stabilization of the radical cation of cyclo(Gly-Val) with isopropyl side group, the dominant fragment ion m/z 114 assigned as [C₄H₆N₂O₂]^+• is produced by γ-H migration and i cleavage to lose propylene. The ionization energies (IEs) of three cyclic dipeptides decrease in the order cyclo(Gly-Gly) (9.33 ± 0.05 eV) > cyclo(Ala-Ala) (9.21 ± 0.05 eV) > cyclo(Gly-Val) (9.09 ± 0.05 eV) from measurements of photoionization efficiency spectra. It implies that IEs of cyclic dipeptides are affected by substituent groups and symmetrical characterization of molecular structures. These observations of the chemical properties of cyclic dipeptides radical ion (M^+•) may be important for understanding gas-phase molecular reactivity of 2,5-diketopiperazines and guiding diketopiperazine-based drug design.

Keywords: Cyclic dipeptide; VUV photoionization; Mass spectrometry; Fragmentation

Variation of the intercalating proline in artificial peptides mimicking the DNA binding and bending IHF protein by S. Scholz; E. K. Liebler; B. Eickmann; H.-J. Fritz; U. Diederichsen (pp. 289-298).

The integration host factor (IHF) is a protein which sequence specifically induces a bend of double-stranded DNA by more than 160°. Based on IHF as lead structure, a peptide mimic was introduced resembling the positively charged body of the protein by a lysine dendrimer and the minor groove recognition loop by a cyclopeptide. The proline located close to the tip of the recognition loop intercalates between the base pair plane. It was modified in order to evaluate the influence of the side chain residue with respect to size (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), aromaticity (phenylalanine), conformation of the five-membered ring [(4R)-fluoroproline, (4S)-fluoroproline, 3,4-dehydroproline], and the peptide backbone conformation (α-methylproline) on binding dsDNA and bending the double strand. Binding and bending studies were carried out by fluorescence resonance energy transfer experiments and gel electrophoresis using DNA sequences prepared by PCR with the IHF binding site in central or terminal position. Whereas aromatic residues and α-methylproline were not tolerated as proline substitute, incorporation of (4S)-fluoroproline and 3,4-dehydroproline provided enhanced binding.

Keywords: Amino acids; DNA binding and bending; IHF protein; Conformation; Peptides

Using the same organocatalyst for asymmetric synthesis of both enantiomers of glutamic acid-derived Ni(II) complexes via 1,4-additions of achiral glycine and dehydroalanine Schiff base Ni(II) complexes by Yuri N. Belokon; Zalina T. Gugkaeva; Karine V. Hakobyan; Victor I. Maleev; Margarita A. Moskalenko; Victor N. Khrustalev; Ashot S. Saghyan; Alan T. Tsaloev; Kiryl K. Babievsky (pp. 299-308).

(S)- and (R)-BIMBOL were efficient PT catalysts of asymmetric Michael addition of prochiral Ni–PBP–Gly (1) to acrylic esters and malonic esters to Ni–PBP–Δ-Ala (2) correspondingly. The salient feature of the catalysis is opposite configurations of Glu prepared via the two paths with BIMBOL of the same configuration and a perspective novel catalytic procedure for the synthesis of Gla derivatives.

Keywords: γ-Carboxyglutamic acid; Glutamic acid; Asymmetric organocatalysis; Michael addition

Bi-functional peptides with both trypsin-inhibitory and antimicrobial activities are frequent defensive molecules in Ranidae amphibian skins by Xiuwen Yan; Huan Liu; Xuening Yang; Qiaolin Che; Rui Liu; Hailong Yang; Xiuhong Liu; Dewen You; Aili Wang; Jianxu Li; Ren Lai (pp. 309-316).

Amphibian skins act as the first line against noxious aggression by microorganisms, parasites, and predators. Anti-microorganism activity is an important task of amphibian skins. A large amount of gene-encoded antimicrobial peptides (AMPs) has been identified from amphibian skins. Only a few of small protease inhibitors have been found in amphibian skins. From skin secretions of 5 species (Odorrana livida, Hylarana nigrovittata, Limnonectes kuhlii, Odorrana grahami, and Amolops loloensis) of Ranidae frogs, 16 small serine protease inhibitor peptides have been purified and characterized. They have lengths of 17–20 amino acid residues (aa). All of them are encoded by precursors with length of 65–70 aa. These small peptides show strong trypsin-inhibitory abilities. Some of them can exert antimicrobial activities. They share the conserved GCWTKSXXPKPC fragment in their primary structures, suggesting they belong to the same families of peptide. Signal peptides of precursors encoding these serine protease inhibitors share obvious sequence similarity with those of precursors encoding AMPs from Ranidae frogs. The current results suggest that these small serine protease inhibitors are the common defensive compounds in frog skin of Ranidae as amphibian skin AMPs.

Keywords: Amphibian Skin; Antimicrobial peptide; Innate immunity; Protease inhibitor; Serine

Homocysteine metabolism in peripheral blood mononuclear cells: evidence for cystathionine beta-synthase activity in resting state by Monika Katko; Erzsebet Zavaczki; Viktoria Jeney; Gyorgy Paragh; Jozsef Balla; Zsuzsa Varga (pp. 317-326).

Activated peripheral blood mononuclear cells (PBMC) release homocysteine and possess cystathionine β-synthase (CBS) activity; however, it was thought that there is no CBS in resting state. Previously, we found that nickel decreased intracellular homocysteine concentration in un-stimulated (e.g. resting) PBMC, suggesting that resting PBMC might also have active homocysteine metabolism. Here, we demonstrated that un-stimulated PBMC synthesize (incorporate l-[methyl-¹⁴C]methionine to DNA, lipids and proteins), release (increase extracellular homocysteine), and metabolize homocysteine. Intracellular homocysteine concentration varied with incubation time, depending on extracellular concentrations of methionine, homocysteine, and glutathione. Methionine synthase activity was constant and independent of thiol concentrations. In Western blot, CBS protein was clearly identified in freshly isolated PBMC. CBS protein level and activity increased with incubation time, upon stimulation, and similar to intracellular homocysteine, depending on intra- and extracellular homocysteine and glutathione concentrations. According to our knowledge, this is the first evidence that certifies homocysteine metabolism and regulatory role of CBS activity to keep balanced intracellular homocysteine level in resting PBMC. Homocysteine, released by PBMC, in turn can modulate its functions contributing to the development of hyperhomocysteinemia-induced diseases.

Keywords: PBMC; Cystathionine β-synthase; Intracellular homocysteine; Extracellular homocysteine; Methionine; Glutathione

Different environmental temperatures affect amino acid metabolism in the eurytherm teleost Senegalese sole (Solea senegalensis Kaup, 1858) as indicated by changes in plasma metabolites by Benjamín Costas; Cláudia Aragão; Ignacio Ruiz-Jarabo; Luis Vargas-Chacoff; Francisco J. Arjona; Juan M. Mancera; Maria T. Dinis; Luís E. C. Conceição (pp. 327-335).

Senegalese sole (Solea senegalensis) is a eurytherm teleost that under natural conditions can be exposed to annual water temperature fluctuations between 12 and 26°C. This study assessed the effects of temperature on sole metabolic status, in particular in what concerns plasma free amino acid changes during thermal acclimation. Senegalese sole maintained at 18°C were acclimated to either cold (12°C) or warm (26°C) environmental temperatures for 21 days. Fish maintained at 18°C served as control. Plasma concentrations of cortisol, glucose, lactate, triglycerides, proteins, and free amino acids were assessed. Cold acclimation influenced interrenal responses of sole by increasing cortisol release. Moreover, plasma glucose and lactate concentrations increased linearly with temperature, presumably reflecting a higher metabolic activity of sole acclimated to 26°C. Acclimation temperature affected more drastically plasma concentrations of dispensable than that of indispensable amino acids, and different acclimation temperatures induced different responses. Asparagine, glutamine and ornithine seem to be of particular importance for ammonia detoxification mechanisms, synthesis of triglycerides that may be used during homeoviscous adaptation and, to a lesser extent, as energetic substrates in specimens acclimated to 12°C. When sole is acclimated to 26°C taurine, glutamate, GABA and glycine increased, which may suggest important roles as antioxidant defences, in osmoregulatory processes and/or for energetic purposes at this thermal regimen. In conclusion, acclimation to different environmental temperatures induces several metabolic changes in Senegalese sole, suggesting that amino acids may be important for thermal acclimation.

Keywords: Acclimation; Dispensable amino acids; Homeoviscous adaptation; Solea senegalensis ; Temperature

Optimization of enzymatic hydrolysis conditions for the production of antioxidant peptides from muscles of Nemipterus japonicus and Exocoetus volitans using response surface methodology by Shabeena Yousuf Naqash; R. A. Nazeer (pp. 337-345).

In the present study, protein of muscles of commercially important marine fishes Nemipterus japonicus and Exocoetus volitans were extracted by trypsin and their hydrolysis conditions viz., temperature, time, and enzyme to substrate concentration on degree of hydrolysis were studied by response surface methodology. The optimum values for N. japonicus was found as temperature, 30°C, hydrolysis time of 100 min an enzyme/substrate concentration of 1.59% whereas, for E. volitans muscle protein, optimum hydrolysis conditions were temperature, 30°C, hydrolysis time of 115 min and enzyme/substrate concentration of 1.67%. Furthermore, amino acid sequence of antioxidant peptides derived after chromatographic purification was identified by ESI–MS/MS. The analysis of peptides showed sequences as Glu-Ser-Asp-Arg-Pro (620.3 Da) and Gly-Trp-Met-Gly-Cys-Trp (747.3) for N. japonicus and E. volitans muscle, respectively. The peptides contained important antioxidant amino acids and acted as good antioxidant peptides to scavenge free radicals.

Keywords: Nemipterus japonicus ; Exocoetus volitans ; Antioxidant peptides; Amino acids

Erythrocyte glutathione transferase: a potential new biomarker in chronic kidney diseases which correlates with plasma homocysteine by Mariarita Dessì; Annalisa Noce; Kutayba F. Dawood; Francesco Galli; Massimo Taccone-Gallucci; Raffaele Fabrini; Alessio Bocedi; Renato Massoud; Giorgio Fucci; Anna Pastore; Simone Manca di Villahermosa; Viviana Zingaretti; Giorgio Federici; Giorgio Ricci (pp. 347-354).

The erythrocyte glutathione S-transferase (e-GST) is a member of a superfamily of inducible enzymes involved in cell detoxification that shows an increased expression in chronic kidney disease (CKD) patients. We propose a new automated analysis procedure for e-GST activity that has been validated in 72 CKD patients and 62 maintenance hemodialysis patients (MHD). Regression analysis was carried out to assess association between e-GST activity data, main clinical variables, and plasma homocysteine (Hcy), a modified sulfur amino acid known as potential risk factor for cardiovascular disease that is increased above normal levels in more than 90% of the uremic patients. An increased e-GST activity was confirmed in MHD patients (N = 62; 10.2 ± 0.4 U/gHb) compared with healthy subjects (N = 80; 5.8 ± 0.4 U/gHb), and as an original finding, a significant increase of e-GST activity was observed in pre-dialysis CKD patients with a positive correlation with disease severity weighted according to the four stages of “Kidney Disease Outcomes Quality Initiative” classification (7.4 ± 0.5, 8 ± 1, 9.5 ± 0.6, 12 ± 1 U/gHb, respectively). No correlation was found between e-GST activity and hemoglobin, transferrin, blood iron and the markers of systemic inflammation and renal function such as alpha-1 acid glycoprotein and high-sensitive C-Reactive Protein, beta-2 microglobulin and the index of malnutrition-inflammation PINI, while a significant correlation was observed for the first time between plasma Hcy and e-GST activity (r ² = 0.64, P < 0.0001) in MHD patients. Hcy, however, was not identified as an inhibitor of e-GST enzyme. The results in this study suggest the potential for automated e-GST analysis as a valuable tool to further explore phase II-related uremic toxicity in CKD and MHD patients.

Keywords: Chronic kidney disease; Erythrocyte glutathione transferase; Hyperhomocysteinemia; Maintenance hemodialysis

Increased concentrations of both NMDA receptor co-agonists d-serine and glycine in global ischemia: a potential novel treatment target for perinatal asphyxia by Sabine A. Fuchs; Cacha M. P. C. D. Peeters-Scholte; Martina M. J. de Barse; Martin W. Roeleveld; Leo W. J. Klomp; Ruud Berger; Tom J. de Koning (pp. 355-363).

Worldwide, perinatal asphyxia is an important cause of morbidity and mortality among term-born children. Overactivation of the N-methyl-d-aspartate receptor (NMDAr) plays a central role in the pathogenesis of cerebral hypoxia–ischemia, but the role of both endogenous NMDAr co-agonists d-serine and glycine remains largely elusive. We investigated d-serine and glycine concentration changes in rat glioma cells, subjected to oxygen and glucose deprivation (OGD) and CSF from piglets exposed to hypoxia–ischemia by occlusion of both carotid arteries and hypoxia. We illustrated these findings with analyses of cerebrospinal fluid (CSF) from human newborns affected by perinatal asphyxia. Extracellular concentrations of glycine and d-serine were markedly increased in rat glioma cells exposed to OGD, presumably through increased synthesis from l-serine. Upon reperfusion glycine concentrations normalized and d-serine concentrations were significantly lowered. The in vivo studies corroborated the finding of initially elevated and then normalizing concentrations of glycine and decreased d-serine concentrations upon reperfusion These significant increases of both endogenous NMDAr co-agonists in combination with elevated glutamate concentrations, as induced by global cerebral ischemia, are bound to lead to massive NMDAr activation, excitotoxicity and neuronal damage. Influencing these NMDAr co-agonist concentrations provides an interesting treatment target for this common, devastating and currently poorly treatable condition.

Keywords: d-Serine; Glutamate; NMDA receptor; Perinatal asphyxia; Ischemia

Binding activity, structure, and immunogenicity of synthetic peptides derived from Plasmodium falciparum CelTOS and TRSP proteins by Hernando Curtidor; Gabriela Arévalo-Pinzón; Adriana Bermudez; Dayana Calderon; Magnolia Vanegas; Liliana C. Patiño; Manuel A. Patarroyo; Manuel E. Patarroyo (pp. 365-378).

Several sporozoite proteins have been associated with Plasmodium falciparum cell traversal and hepatocyte invasion, including the cell-traversal protein for ookinetes and sporozoites (CelTOS), and thrombospondin-related sporozoite protein (TRSP). CelTOS and TRSP amino acid sequences have been finely mapped to identify regions specifically binding to HeLa and HepG2 cells, respectively. Three high-activity binding peptides (HABPs) were found in CelTOS and one HABP was found in TRSP, all of them having high α-helical structure content. These HABPs’ specific binding was sensitive to HeLa and HepG2 cells’ pre-treatment with heparinase I and chondroitinase ABC. Despite their similarity at three-dimensional (3D) structural level, TRSP and TRAP HABPs located in the TSR domain did not compete for the same binding sites. CelTOS and TRSP HABPs were used as a template for designing modified sequences to then be assessed in the Aotus monkey experimental model. Antibodies directed against these modified HABPs were able to recognize both the native parasite protein by immunofluorescence assay and the recombinant protein (expressed in Escherichia coli) by Western blot and ELISA assays. The results suggested that these modified HABPs could be promising targets in designing a fully effective, antimalarial vaccine.

Keywords: Plasmodium falciparum ; Sporozoite; CelTOS; TRSP; Peptide; Vaccine

Proteomic characterization of Kunitz trypsin inhibitor variants, Tia and Tib, in soybean [Glycine max (L.) Merrill] by K. J. Lee; J.-B. Kim; B.-K. Ha; S. H. Kim; S.-Y. Kang; B.-M. Lee; D. S. Kim (pp. 379-388).

The soybean Kunitz trypsin inhibitor (KTi) has several polymorphic variants. Of these, Tia and Tib, which differ by nine amino acids, are the two main types. In this study, differences in KTi proteome between Tia and Tib were investigated using three soybean cultivars and three mutant lines. Two cultivars, Baekwoon (BW) and Paldal (PD), and one mutant line, SW115-24, were Tia type, whereas one soybean cultivar, Suwon115 (SW115), and two mutant lines, BW-7-2 and PD-5-10, were Tib type. Protein from the six soybean lines was extracted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), non-denaturing polyacrylamide gel electrophoresis (non-denaturing PAGE), and two-dimensional polyacrylamide gel electrophoresis (2-DE). By SDS-PAGE, there was no difference between soybean cultivars and mutant lines, except for SW115-24. Western blot analysis revealed that, in comparison with Tia, Tib type accumulated relatively low amounts of KTi. By non-denaturing PAGE, the three soybean lines of Tib type were characterized by slower mobility than the three soybean lines of Tia type. Zymography detected eight distinct zones of trypsin inhibitory activity among which Tia and Tib lacked the fifth and sixth zone, respectively. By two-dimensional native polyacrylamide gel electrophoresis (2-DN), the spots related to trypsin inhibitory activity showed different mobilities, whereas only one KTi (21.5 kDa) spot was resolved by 2-DE. By two-dimensional zymography (2-DZ), Tib showed a broader activity zone (pI 4–7) in comparison with Tia (pI 4–5). The results indicate that the genotypes with a different type of KTi present different proteomic profiles and trypsin inhibitory activities.

Keywords: Kunitz trypsin inhibitor; Mutation; Proteome; Soybean

Solution structure of a novel α-conotoxin with a distinctive loop spacing pattern by Bingbing Zhang; Feijuan Huang; Weihong Du (pp. 389-396).

α-Pharmacological conotoxins are among the most selective ligands of nicotinic acetylcholine receptors with typical cysteine frameworks. They are characterized by the intercysteine loop and classified into various subfamilies, such as α3/5 and α4/7 conotoxins. A novel α-conotoxin, Pu14a (DCPPHPVPGMHKCVCLKTC), with a distinct loop spacing pattern between cysteines was reported recently. Pu14a belongs to the Cys framework 14 (–C–C–C–C) family containing four proline residues in the loop 1 region. Similar to another framework 14 conotoxin Lt14a (MCPPLCKPSCTNC-NH₂), Pu14a has C1–C3/C2–C4 disulfide linkage, and can inhibit some subtypes of nicotinic acetylcholine receptors. In this study, the solution structure of Pu14a was investigated using ¹H nuclear magnetic resonance spectroscopy to understand the structure-activity relationship of this conotoxin. 20 converged structures of this conopeptide, with RMSD value of 0.77 Å, were obtained based on distance constraints, dihedral angles and disulfide bond constraints. The three-dimensional structure of Pu14a showed remarkable difference from typical α-conotoxins because of a large intercysteine loop between C2 and C13, as well as a 3₁₀-helix near the C-terminal. Furthermore, four proline residues in Pu14a adopted the trans conformation that may correlate with the large loop configuration and the biological activity of this conopeptide. The distinct structural characteristics of Pu14a will be very useful for studying the structure-activity relationship of α-conotoxins.

Keywords: α-Conotoxin; Pu14a; Solution structure; Proline

Antinociceptive effect of intrathecal administration of hypotaurine in rat models of inflammatory and neuropathic pain by Koji Hara; Motohiro Nakamura; Yasunori Haranishi; Tadanori Terada; Kazunori Kataoka; Takeyoshi Sata (pp. 397-404).

Hypotaurine is an intermediate in taurine biosynthesis from cysteine in astrocytes. Although hypotaurine functions as an antioxidant and organic osmolyte, its physiological role in the central nervous system remains unclear. This study used behavioral assessments to determine whether hypotaurine influenced nociceptive transmission in acute, inflammatory, and neuropathic pain. The tail flick, paw pressure, and formalin tests were performed in male Sprague–Dawley rats to examine the effects of the intrathecal administration of hypotaurine (100, 200, 400, 600 μg) on thermal, mechanical, and chemical nociception. Chronic constriction injury (CCI) to the sciatic nerve was induced in the rats, and the electronic von Frey test and plantar test were performed to assess the effects on neuropathic pain. To determine which neurotransmitter pathway(s) was involved in the action of hypotaurine, in this study, we examined how the antagonists of spinal pain processing receptors altered the effect of 600 μg hypotaurine. To explore whether hypotaurine affected motor performance, the Rotarod test was conducted. Hypotaurine had antinociceptive effects on thermal, mechanical, and chemical nociception in the spinal cord. In CCI rats, hypotaurine alleviated mechanical allodynia and thermal hyperalgesia. These effects were reversed completely by pretreatment with an intrathecal injection of strychnine, a glycine receptor antagonist. Conversely, hypotaurine did not affect motor performance. This study demonstrated that intrathecal hypotaurine suppressed acute, inflammatory, and neuropathic pain. Hypotaurine may regulate nociceptive transmission physiologically by activating glycinergic neurons in the spinal cord, and it is a promising candidate for treating various pain states.

Keywords: Glycinergic neurotransmission; Spinal cord; Taurine; Nociceptive transmission

In vivo targeting of HER2-positive tumor using 2-helix affibody molecules by Gang Ren; Jack M. Webster; Zhe Liu; Rong Zhang; Zheng Miao; Hongguang Liu; Sanjiv S. Gambhir; Faisal A. Syud; Zhen Cheng (pp. 405-413).

Molecular imaging of human epidermal growth factor receptor type 2 (HER2) expression has drawn significant attention because of the unique role of the HER2 gene in diagnosis, therapy and prognosis of human breast cancer. In our previous research, a novel cyclic 2-helix small protein, MUT-DS, was discovered as an anti-HER2 Affibody analog with high affinity through rational protein design and engineering. MUT-DS was then evaluated for positron emission tomography (PET) of HER2-positive tumor by labeling with two radionuclides, ⁶⁸Ga and ¹⁸F, with relatively short half-life (t _1/2 < 2 h). In order to fully study the in vivo behavior of 2-helix small protein and demonstrate that it could be a robust platform for labeling with a variety of radionuclides for different applications, in this study, MUT-DS was further radiolabeled with ⁶⁴Cu or ¹¹¹In and evaluated for in vivo targeting of HER2-positive tumor in mice. Design 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated MUT-DS (DOTA–MUT-DS) was chemically synthesized using solid phase peptide synthesizer and I₂ oxidation. DOTA–MUT-DS was then radiolabeled with ⁶⁴Cu or ¹¹¹In to prepare the HER2 imaging probe (⁶⁴Cu/¹¹¹In-DOTA–MUT-DS). Both biodistribution and microPET imaging of the probe were evaluated in nude mice bearing subcutaneous HER2-positive SKOV3 tumors. DOTA–MUT-DS could be successfully synthesized and radiolabeled with ⁶⁴Cu or ¹¹¹In. Biodistribution study showed that tumor uptake value of ⁶⁴Cu or ¹¹¹In-labeled DOTA–MUT-DS was 4.66 ± 0.38 or 2.17 ± 0.15%ID/g, respectively, in nude mice bearing SKOV3 xenografts (n = 3) at 1 h post-injection (p.i.). Tumor-to-blood and tumor-to-muscle ratios for ⁶⁴Cu-DOTA-MUT-DS were attained to be 3.05 and 3.48 at 1 h p.i., respectively, while for ¹¹¹In-DOTA–MUT-DS, they were 2.04 and 3.19, respectively. Co-injection of the cold Affibody molecule Z_HER2:342 with ⁶⁴Cu-DOTA-MUT-DS specifically reduced the SKOV3 tumor uptake of the probe by 48%. ¹¹¹In-DOTA–MUT-DS displayed lower liver uptake at all the time points investigated and higher tumor to blood ratios at 4 and 20 h p.i., when compared with ⁶⁴Cu-DOTA–MUT-DS. This study demonstrates that the 2-helix protein based probes, ⁶⁴Cu/¹¹¹In DOTA–MUT-DS, are promising molecular probes for imaging HER2-positive tumor. Two-helix small protein scaffold holds great promise as a novel and robust platform for imaging and therapy applications.

Keywords: Affibody; HER2; PET; Molecular imaging; ¹¹¹In; ⁶⁴Cu

New 1,3-amino alcohols derived from enantiopure bridgehead β-aminobicyclo[2.2.2]oct-5-ene-2-carboxylic acids by Christophe André; Monique Calmès; Françoise Escale; Muriel Amblard; Jean Martinez; Olivier Songis (pp. 415-421).

Constrained enantiopure bicyclic β-amino acids derived from the asymmetric Diels–Alder reaction of the (R)-benzyl-4-(3-acryloyloxy-4,4-dimethyl-2-oxopyrrolidin-1-yl)-benzoate and the 1-(benzyloxycarbonylamino)cyclohexadiene provide original templates for the construction of new rigid enantiopure 1,3-amino alcohols.

Keywords: Bicyclic β-amino acid derivatives; Cyclic amino alcohols; Constrained chiral β-amino acids; Reduction

Facile synthesis of hybrid sulfonophosphinodipeptides composing of taurines and 1-aminoalkylphosphinic acids by Fanhua Meng; Fengdan He; Xiuqing Song; Leilei Zhang; Wenxiang Hu; Gang Liu; Jiaxi Xu (pp. 423-429).

Both sulfonopeptides and phosphonopeptides are important analogs of naturally occurring peptides and have been widely used as enzyme inhibitors and haptens for producing catalytic antibodies due to their tetrahedrally structural features. A series of hybrid sulfonophosphinodipeptides composing of taurines and 1-aminoalkylphosphinic acids were first and conveniently synthesized in satisfactory to good yields via a Mannich-type reaction of N-benzyloxycarbonylaminoalkanesulfonamides, aldehydes, and aryldichlorophosphines, and subsequent hydrolysis. The current method provides an efficient and direct synthesis of hybrid sulfonophosphinodipeptides.

Keywords: Aminoalkanesulfonamide; Mannich reaction; Peptide; Phosphinopeptide; Synthesis

Potential benefits of taurine in the prevention of skeletal muscle impairment induced by disuse in the hindlimb-unloaded rat by Sabata Pierno; Antonella Liantonio; Giulia M. Camerino; Michela De Bellis; Maria Cannone; Gianluca Gramegna; Antonia Scaramuzzi; Simonetta Simonetti; Grazia Paola Nicchia; Davide Basco; Maria Svelto; Jean-François Desaphy; Diana Conte Camerino (pp. 431-445).

Hindlimb unloading (HU) in rats induces severe atrophy and a slow-to-fast phenotype transition in postural slow-twitch muscles, as occurs in human disuse conditions, such as spaceflight or bed rest. In rats, a reduction of soleus muscle weight and a decrease of cross-sectional area (CSA) were observed as signs of atrophy. An increased expression of the fast-isoform of myosin heavy chain (MHC) showed the phenotype transition. In parallel the resting cytosolic calcium concentration (restCa) was decreased and the resting chloride conductance (gCl), which regulates muscle excitability, was increased toward the values of the fast-twitch muscles. Here, we investigated the possible role of taurine, which is known to modulate calcium homeostasis and gCl, in the restoration of muscle impairment due to 14-days-HU. We found elevated taurine content and higher expression of the taurine transporter TauT in the soleus muscle as compared to the fast-twitch extensor digitorum longus (EDL) muscle of control rats. Taurine level was reduced in the HU soleus muscle, although, TauT expression was not modified. Taurine oral supplementation (5 g/kg) fully prevented this loss, and preserved resting gCl and restCa together with the slow MHC phenotype. Taurine supplementation did not prevent the HU-induced drop of muscle weight or fiber CSA, but it restored the expression of MURF-1, an atrophy-related gene, suggesting a possible early protective effect of taurine. In conclusion, taurine prevented the HU-induced phenotypic transition of soleus muscle and might attenuate the atrophic process. These findings argue for the beneficial use of taurine in the treatment of disuse-induced muscle dysfunction.

Keywords: Taurine; Slow- and fast-twitch skeletal muscle; Hindlimb unloading; Atrophy; Resting chloride conductance; Resting intracellular calcium

Predicting protein sumoylation sites from sequence features by Shaolei Teng; Hong Luo; Liangjiang Wang (pp. 447-455).

Protein sumoylation is a post-translational modification that plays an important role in a wide range of cellular processes. Small ubiquitin-related modifier (SUMO) can be covalently and reversibly conjugated to the sumoylation sites of target proteins, many of which are implicated in various human genetic disorders. The accurate prediction of protein sumoylation sites may help biomedical researchers to design their experiments and understand the molecular mechanism of protein sumoylation. In this study, a new machine learning approach has been developed for predicting sumoylation sites from protein sequence information. Random forests (RFs) and support vector machines (SVMs) were trained with the data collected from the literature. Domain-specific knowledge in terms of relevant biological features was used for input vector encoding. It was shown that RF classifier performance was affected by the sequence context of sumoylation sites, and 20 residues with the core motif ΨKXE in the middle appeared to provide enough context information for sumoylation site prediction. The RF classifiers were also found to outperform SVM models for predicting protein sumoylation sites from sequence features. The results suggest that the machine learning approach gives rise to more accurate prediction of protein sumoylation sites than the other existing methods. The accurate classifiers have been used to develop a new web server, called seeSUMO ( http://bioinfo.ggc.org/seesumo/ ), for sequence-based prediction of protein sumoylation sites.

Keywords: Protein sumoylation site prediction; Random forests; Support vector machines; Biological features; SeeSUMO

Purification and identification of antioxidant peptides from egg white protein hydrolysate by Chen Chen; Yu-Jie Chi; Ming-Yang Zhao; Lei Lv (pp. 457-466).

Egg white proteins were hydrolysed separately using five different proteases to obtain antioxidant peptides. The antioxidant activity of egg white protein hydrolysates was influenced by the time of hydrolysis and the type of enzyme. Of the various hydrolysates produced, papain hydrolysate obtained by 3-h hydrolysis (PEWPH) displayed the highest DPPH radical scavenging activity. PEWPH could also quench the superoxide anion and hydroxyl radicals, effectively inhibit lipid peroxidation and exhibit reducing power. Then, PEWPH was purified sequentially by ultrafiltration, gel filtration, RP-HPLC and two fractions with relatively strong antioxidant activity were subsequently subjected to LC–MS/MS for peptide sequence identification. The sequences of the two antioxidant peptides were identified to be Tyr-Leu-Gly-Ala-Lys (551.54 Da) and Gly-Gly-Leu-Glu-Pro-Ile-Asn-Phe-Gln (974.55 Da), and they were identified for the first time from food-derived protein hydrolysates. Last, the two purified peptides were synthesized and they showed 7.48- and 6.02-fold higher DPPH radical scavenging activity compared with the crude PEWPH, respectively. These results indicate that PEWPH and/or its isolated peptides may be useful ingredients in food and nutraceutical applications.

Keywords: Egg white protein hydrolysate; Antioxidant activity; Purification; Antioxidant peptide

Efficacy and toxicity of the antimicrobial peptide M33 produced with different counter-ions by Alessandro Pini; Luisa Lozzi; Andrea Bernini; Jlenia Brunetti; Chiara Falciani; Silvia Scali; Stefano Bindi; Tiziana Di Maggio; Gian Maria Rossolini; Neri Niccolai; Luisa Bracci (pp. 467-473).

The tetra-branched peptide M33 (Pini et al. in FASEB J 24:1015–1022, 2010) is under evaluation in animal models for its activity as antimicrobial agent in lung infections and sepsis. The preclinical development of a new drug requires medium-scale manufacture for tests of efficacy, biodistribution, pharmacokinetics and toxicity. In order to produce the most suitable peptide form for these purposes, we evaluated the behaviour of the peptide M33 obtained with different counter-ions. We compared activity and toxicity in vitro and in vivo of the peptide M33 produced as trifluoroacetate salt (TFacetate) and as acetate salt. The two forms did not differ substantially in terms of efficacy in vitro or in vivo but showed different toxicities for human cells and in animals. M33-TFacetate proved to be 5–30% more toxic than M33-acetate for cells derived from normal bronchi and cells carrying ΔF508 mutation in the CFTR gene, the most frequent variant in cystic fibrosis. M33-TFacetate produced manifest signs of in vivo toxicity immediately after administration, whereas M33-acetate only generated mild signs, which disappeared within a few hours. The peptide M33-acetate proved more suitable for the development of a new drug, and was therefore chosen for further characterization.

Keywords: Antimicrobial peptides; Trifluoroacetic acid; Branched peptides; Gram-negative bacteria; Cystic fibrosis; Peptide toxicity

Preparative scale isolation, purification and derivatization of mimosine, a non-proteinogenic amino acid by Kiyoshi Nokihara; Akiyoshi Hirata; Tetsuya Sogon; Takafumi Ohyama (pp. 475-482).

Focusing on drug discovery non-proteinogenic amino acids have often been used as important building blocks for construction of compound libraries in the filed of combinatorial chemistry and chemical biology. Highly homogeneous l-mimosine, α-amino-β-(3-hydoxy-4-oxo-1,4-dihydropyridin-1-yl)-propanoic acid, a non-proteinogenic amino acid, has been successfully isolated and purified on an industrial scale from wild leaves of Leucaena (Leucaena leucocephala de Wit) which is a widely distributed legume in Okinawa, a sub-tropical island in Japan. Optical purity determinations used for quality control have been established through diastereomer formation. Physico-chemical properties and biological properties of purified mimosine have been clarified. Mimosine is sparingly soluble in water and organic solvents but can be dissolved in aqueous alkaline solution. The tyrosinase pathway is of particular interest in the cosmetic field, since mimosine is an analog of tyrosine. Thus the present purified mimosine have been tested in tyrosinase inhibitory assays. The IC₅₀ for tyrosinase inhibitory activity of purified Mim was compared with kojic acid. Mimosine shows significant inhibition of melanin production in murine melanoma cells. The derivatization of mimosine has been investigated with a focus on its use in conventional peptide syntheses to generate mimosyl peptides. N-(9-Fluorenylmethoxycarbonyloxy)-mimosine and resin-bound mimosine for solid-phase syntheses have also been performed. Highly homogeneous Mim is a useful material for the development of functional cosmetics or active pharmaceutical ingredients.

Keywords: Derivatization; Mimosine; Non-proteinogenic amino acid; Leucaena; Chiral determination; Fmoc-mimosine; Mimosyl-resin

Role of conserved active site tryptophan-101 in functional activity and stability of phosphoserine aminotransferase from an enteric human parasite by Vibhor Mishra; Ashutosh Kumar; Vahab Ali; Tomoyoshi Nozaki; Kam Y. J. Zhang; Vinod Bhakuni (pp. 483-491).

Site-directed mutagenesis study was performed to elucidate the role of conserved tryptophan-101 present at the active site of phosphoserine aminotransferase from an enteric human parasite Entamoeba histolytica. Fluorescence resonance energy transfer and molecular dynamic simulation show that the indole ring of Trp101 stacks with the cofactor PLP. Loss of enzymatic activity and PLP polarization values suggest that Trp101 plays a major role in maintaining a defined PLP microenvironment essentially required for optimal enzymatic activity. Studies on W101F, W101H and W101A mutants show that only the indole ring of the conserved Trp101 forms most favorable stacking interaction with the pyridine ring of the cofactor PLP. Protein stability was compromised on substitution of Trp101 with Phe/His/Ala amino acids. A difference in conformational free energy of 1.65 kcal mol⁻¹ was observed between WT-protein and W101A mutant.

Keywords: EhPSAT; Entamoeba; Site-directed mutagenesis; FRET; MD simulation

Coenzyme preference of Streptococcus pyogenes δ¹-pyrroline-5-carboxylate reductase: evidence supporting NADPH as the physiological electron donor by Davide Petrollino; Giuseppe Forlani (pp. 493-497).

The streptococcal enzyme that catalyzes the last step in proline biosynthesis was heterologously expressed and the recombinant protein was purified to electrophoretic homogeneity and characterized thoroughly. As for δ¹-pyrroline-5-carboxylate reductases from other sources, it was able to use either NADH or NADPH as the electron donor in vitro. However, with NADH the activity was markedly inhibited by physiological levels of NADP⁺. Results also strengthen the possibility that an unusual ordered substrate binding occurs, in which the dinucleotide binds last.

Keywords: Electron donor; Proline biosynthesis; Substrate ambiguity; Substrate binding

Extraordinary metabolic stability of peptides containing α-aminoxy acids by Fei Chen; Bin Ma; Zong-Chang Yang; Ge Lin; Dan Yang (pp. 499-503).

The metabolic stability of peptides containing a mixed sequence of α-aminoxy acids and α-amino acids is significantly improved compared to peptides composed of only natural α-amino acids. The introduction of an α-aminoxy acid into peptide chain dramatically improves the stability of the amide bonds immediately before and after it. These peptides containing α-aminoxy acids represent excellent structural scaffold for the design of metabolically stable and biologically active peptides.

Keywords: Metabolism; Peptides; Stability; Aminoxy acid

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.43, #1)