Amino Acids (v.47, #8)

Syntheses of some α-cyclic tripeptides as potential inhibitors for HMG-CoA Reductase by Subrata Chakraborty; Shih-Hung Lin; David Shiuan; Dar-Fu Tai (1495-1505).
α-Cyclic tripeptides (CtPs) are the most rigid members of the cyclic peptide family. However, due to their synthetic difficulty, biological activity has remained undisclosed. The incorporation of side-chain-protected natural amino acids into functional CtPs was performed to explore the potential biological functions. Several novel CtPs that consist of protected serine (S(Bn)) and/or glutamate (E(OBn)) were prepared from corresponding linear tripeptides by chemical synthesis. There is a strong possibility for CtPs that contain 3 phenyl groups to correlate with atorvastatin structure. The binding effects in human HMG-CoA reductase (hHMGR) activities were first evaluated by molecular docking. High docking scores were received with these CtPs for enzyme. Therefore, enzymatic assays were carried out and the compound cyclo(S(Bn))3 was indeed able to moderately inhibit hHMGR (IC50 = 110 μM).
Keywords: Cyclic tripeptide; Turn-inducing unit; Cyclization; Amino acids; Molecular docking; HMG-CoA reductase

Air oxidation method employed for the disulfide bond formation of natural and synthetic peptides by Enrica Calce; Rosa Maria Vitale; Andrea Scaloni; Pietro Amodeo; Stefania De Luca (1507-1515).
Among the available protocols, chemically driven approaches to oxidize cysteine may not be required for molecules that, under the native-like conditions, naturally fold in conformations ensuring an effective pairing of the right disulfide bridge pattern. In this contest, we successfully prepared the distinctin, a natural heterodimeric peptide, and some synthetic cyclic peptides that are inhibitors of the CXCR4 receptor. In the first case, the air oxidation reaction allowed to connect two peptide chains via disulfide bridge, while in the second case allowed the cyclization of rationally designed peptides by an intramolecular disulfide bridge. Computational approaches helped to either drive de-novo design or suggest structural modifications and optimal oxidization protocols for disulfide-containing molecules. They are able to both predict and to rationalize the propensity of molecules to spontaneously fold in suitable conformations to achieve the right disulfide bridges.
Keywords: Disulfide bridges; Peptide folding; Oxidation methods; Native-like oxidation conditions

Dietary l-leucine supplementation enhances intestinal development in suckling piglets by Yuli Sun; Zhenlong Wu; Wei Li; Chen Zhang; Kaiji Sun; Yun Ji; Bin Wang; Ning Jiao; Beibei He; Weiwei Wang; Zhaolai Dai; Guoyao Wu (1517-1525).
l-Leucine is a signaling amino acid in animal metabolism. It is unknown whether supplementing l-leucine to breast-fed neonates may enhance their small-intestinal development. This hypothesis was tested with a piglet model. Seven-day-old sow-reared pigs with an average birth weight of 1.45 kg were assigned randomly to the control or leucine group (n = 30/group). Piglets in the leucine group were orally administrated with 1.4 g l-leucine/kg body weight, whereas piglets in the control group received isonitrogenous l-alanine, twice daily for 14 days. The supplemental l-leucine amounted to 200 % of l-leucine intake from sow’s milk by 7-day-old pigs. At the end of the 2-week experiment, tissue samples were collected for determining intestinal morphology, expression of genes for intestinal leucine transporters (real-time RT-PCR and western blot analysis), and plasma metabolites and hormones. l-leucine administration increased (P < 0.05) villus height in the duodenum, an elevated ratio of villus height to crypt depth in the duodenum and ileum, plasma concentrations of leucine, glutamine and asparagine, as well as body-weight gains. mRNA levels for l-leucine transporters (SLC6A14, SLC6A19 and SLC7A9) and the abundance of the ATB0,+ protein were increased (P < 0.05) but those for SLC7A7 mRNA and the LAT2 protein were decreased (P < 0.05) in the jejunum of leucine-supplemented piglets, compared with the control. Plasma concentrations of ammonia, urea, triglycerides, and growth-related hormones did not differ between the control and leucine groups. Collectively, these results indicate that l-leucine supplementation improves intestinal development and whole-body growth in suckling piglets with a normal birth weight.
Keywords: Swine; Gene expression; Milk; Nutrition; Small intestine

Synthesis and antiproliferative activity of glutamic acid-based dipeptides by Gastón Silveira-Dorta; Víctor S. Martín; José M. Padrón (1527-1532).
A small and focused library of 22 dipeptides derived from N,N-dibenzylglutamic acid α- and γ-benzyl esters was prepared in a straightforward manner. The evaluation of the antiproliferative activity in the human solid tumor cell lines HBL-100 (breast), HeLa (cervix), SW1573 (non-small cell lung), T-47D (breast), and WiDr (colon) provided γ-glutamyl methionine (GI50 = 6.0–41 μM) and α-glutamyl proline (GI50 = 7.5–18 μM) as lead compounds. In particular, glutamyl serine and glutamyl proline dipeptides were more active in the resistant cancer cell line WiDr than the conventional anticancer drugs cisplatin and etoposide. Glutamyl tryptophan dipeptides did not affect cell growth of HBL-100, while in T-47D cells, proliferation was inhibited. This result might be attributed to the inhibition of the ATB0,+ transporter.
Keywords: Antitumor agents; Dipeptide; Glutamic acid; Structure–activity relationships

Taurine supplementation ameliorates glucose homeostasis, prevents insulin and glucagon hypersecretion, and controls β, α, and δ-cell masses in genetic obese mice by Junia C. Santos-Silva; Rosane Aparecida Ribeiro; Jean F. Vettorazzi; Esperanza Irles; Sarah Rickli; Patrícia C. Borck; Patricia M. Porciuncula; Ivan Quesada; Angel Nadal; Antonio C. Boschero; Everardo M. Carneiro (1533-1548).
Taurine (Tau) regulates β-cell function and glucose homeostasis under normal and diabetic conditions. Here, we assessed the effects of Tau supplementation upon glucose homeostasis and the morphophysiology of endocrine pancreas, in leptin-deficient obese (ob) mice. From weaning until 90-day-old, C57Bl/6 and ob mice received, or not, 5 % Tau in drinking water (C, CT, ob and obT). Obese mice were hyperglycemic, glucose intolerant, insulin resistant, and exhibited higher hepatic glucose output. Tau supplementation did not prevent obesity, but ameliorated glucose homeostasis in obT. Islets from ob mice presented a higher glucose-induced intracellular Ca2+ influx, NAD(P)H production and insulin release. Furthermore, α-cells from ob islets displayed a higher oscillatory Ca2+ profile at low glucose concentrations, in association with glucagon hypersecretion. In Tau-supplemented ob mice, insulin and glucagon secretion was attenuated, while Ca2+ influx tended to be normalized in β-cells and Ca2+ oscillations were increased in α-cells. Tau normalized the inhibitory action of somatostatin (SST) upon insulin release in the obT group. In these islets, expression of the glucagon, GLUT-2 and TRPM5 genes was also restored. Tau also enhanced MafA, Ngn3 and NeuroD mRNA levels in obT islets. Morphometric analysis demonstrated that the hypertrophy of ob islets tends to be normalized by Tau with reductions in islet and β-cell masses, but enhanced δ-cell mass in obT. Our results indicate that Tau improves glucose homeostasis, regulating β-, α-, and δ-cell morphophysiology in ob mice, indicating that Tau may be a potential therapeutic tool for the preservation of endocrine pancreatic function in obesity and diabetes.
Keywords: Glucagon secretion; Insulin secretion; Obesity; Somatostatin; Taurine supplementation

Taurine increases testicular function in aged rats by inhibiting oxidative stress and apoptosis by Jiancheng Yang; Xiaomeng Zong; Gaofeng Wu; Shumei Lin; Ying Feng; Jianmin Hu (1549-1558).
In males, the decline of androgen synthesis, spermatogenesis and sexual function are the main phenotypes of aging, which may be attributed to testicular dysfunction. Taurine can act as an antioxidant, a testosterone secretion stimulator, a sperm membrane stabilizer and motility factor, and an anti-apoptotic agent. Recent observational studies suggested that taurine may play an important role in spermatogenesis, but to date whether taurine has anti-aging effects on testes remains unknown. We found that in aged rats testicular SDH and G6PDH activities, marker enzymes of testes, serum testosterone, testicular 3β-HSD and 17β-HSD mRNA expression levels were significantly increased by taurine treatment. Taurine administration also markedly raised the sperm count, viability and motility, decreased the sperm abnormality. Our data suggested that taurine can postpone testicular function deterioration in aged rats. Importantly, we observed obvious elevation of testicular antioxidant enzymes (SOD, GSH, GSH-Px) activities, and remarkable reduction of ROS and MDA by taurine administration, indicating taurine can decrease testicular oxidative stress and lipid peroxidation in aged rats. Finally, we found taurine effectively reduced testicular DNA fragmentation, increased testicular Bcl-2 protein expression, and decreased cytochrome c, Bax, Fas, FasL and caspase-3 expression, suggesting taurine can prohibit aged testicular apoptosis by mitochondrial dependent and independent signal pathway. In summary, our results indicated that taurine can suppress testicular function deterioration by increasing antioxidant ability and inhibiting apoptosis.
Keywords: Taurine; Increases testicular function; Oxidative stress; Apoptosis; Aged rats

l-Glycyl-l-glutamine provides the isolated and perfused young and middle-aged rat heart protection against ischaemia–reperfusion injury by Amer Almashhadany; Othman A. Alghamdi; Thomas Van der Touw; Graham L. Jones; Nicola King (1559-1565).
The amino acids glycine and glutamine have been implicated in myocardial protection of the much studied young adult heart. This study aimed to determine whether such protection could be enhanced using the dipeptide, l-glycyl-l-glutamine (gly-gln) in both young hearts and in middle-aged hearts representative of a more clinically relevant age group. Hearts from 8-week-old and 36-week-old rats were perfused in the Langendorff mode for 20 min, before 40 min global normothermic ischaemia and 30 min reperfusion. Where present, 0.5, 2, or 5 mM gly-gln was added 10 min into baseline perfusion, was present throughout ischaemia and was washed out after 10 min reperfusion. Reperfusion damage was assessed from the release of lactate dehydrogenase. Metabolic fitness was assessed from the time to ischaemic contracture and the accumulation of lactate and thiobarbituric acid reactive substances during ischaemia. The presence of 5 mM gly-gln significantly improved the post-ischaemic rate pressure product (RPP) and decreased reperfusion damage in both the 8 (RPP in control on reperfusion 5527 ± 957  vs. 10,320 ± 795 mmHg beat min−1 in 5 mM gly-gln, n = 6 ± SE, p < 0.05) and 36-week-old (RPP in control on reperfusion 1964.33 ± 876.3 vs. 4008 ± 675 mmHg beat min−1, n = 6 ± SE, p < 0.01) hearts. Five mM gly-gln also increased the time to ischaemic contracture and was able to protect against the rise in lactate that occurred in the controls during ischaemia. These results suggest that gly-gln has good potential as a combatant against ischaemia–reperfusion injury in both the young adult and middle-aged populations.
Keywords: l-Glycyl-l-glutamine; Middle-aged; Ischaemia–reperfusion injury; Myocardial protection

The N-terminal cytoplasmic domain of neuregulin 1 type III is intrinsically disordered by Maryna Chukhlieb; Arne Raasakka; Salla Ruskamo; Petri Kursula (1567-1577).
Axonally expressed neuregulin 1 (NRG1) type III is a transmembrane protein involved in various neurodevelopmental processes, including myelination and Schwann cell migration. NRG1 type III has one transmembrane domain and a C-terminal extracellular segment, which contains an epidermal growth factor homology domain. Little is known, however, about the intracellular N terminus of NRG1 type III, and the structure–function relationships of this cytoplasmic domain have remained uncharacterized. In the current study, we carried out the first structural and functional studies on the NRG1 type III cytoplasmic domain. Based on sequence analyses, the domain is predicted to be largely disordered, while a strictly conserved region close to the transmembrane segment may contain helical structure and bind metal ions. As shown by synchrotron radiation circular dichroism spectroscopy, the recombinant NRG1 type III cytoplasmic domain was disordered in solution, but it was able to fold partially into a helical structure, especially when both metals and membrane-mimicking compounds were present. NRG1 cytoplasmic tail binding to metals was further confirmed by calorimetry. These results suggest that the juxtamembrane segment of the NRG1 type III cytoplasmic domain may fold onto the membrane surface upon metal binding. Using synchrotron small-angle X-ray scattering, we further proved that the NRG1 cytoplasmic domain is intrinsically disordered, highly elongated, and behaves like a random polymer. Our work provides the first biochemical and biophysical data on the previously unexplored cytoplasmic domain of NRG1 type III, which will help elucidate the detailed structure–function relationships of this domain.
Keywords: Myelin; Neuregulin; Intrinsically disordered proteins; Metal binding; Membrane; Protein structure

To date, there have been few reports analyzing the amino acid requirement for growth of hyperthermophilic archaea. We here found that the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 requires Thr, Leu, Val, Phe, Tyr, Trp, His and Arg in the medium for growth, and shows slow growth in medium lacking Met or Ile. This largely corresponds to the presence, or absence, of genes related to amino acid biosynthesis in its genome, though there are exceptions. The amino acid requirements were dramatically lost by addition of d-isomers of Met, Leu, Val, allo-Ile, Phe, Tyr, Trp and Arg. Tracer analysis using 14C-labeled d-Trp showed that d-Trp in the medium was used as a protein component in the cells, suggesting the presence of d-amino acid metabolic enzymes. Pyridoxal 5′-phosphate (PLP)-dependent racemase activity toward Met, Leu and Phe was detected in crude extract of P. horikoshii and was enhanced in cells grown in the medium supplemented with d-amino acids, especially d-allo-Ile. The gene encoding the racemase was narrowed down to one open reading frame on the basis of enzyme purification from P. horikoshii cells, and the recombinant enzyme exhibited PLP-dependent racemase activity toward several amino acids, including Met, Leu and Phe, but not Pro, Asp or Glu. This is the first report showing the presence in a hyperthermophilic archaeon of a PLP-dependent amino acid racemase with broad substrate specificity that is likely responsible for utilization of d-amino acids for growth.
Keywords: Hyperthermophilic archaea; Pyrococcus horikoshii ; Amino acid requirement; d-Amino acid utilization; Amino acid racemase

In the present study, a new strategy based on chemical analysis and chemometrics methods was proposed for the comprehensive analysis and profiling of underivatized free amino acids (FAAs) and small peptides among various Luo-Han-Guo (LHG) samples. Firstly, the ultrasound-assisted extraction (UAE) parameters were optimized using Plackett–Burman (PB) screening and Box–Behnken designs (BBD), and the following optimal UAE conditions were obtained: ultrasound power of 280 W, extraction time of 43 min, and the solid–liquid ratio of 302 mL/g. Secondly, a rapid and sensitive analytical method was developed for simultaneous quantification of 24 FAAs and 3 active small peptides in LHG at trace levels using hydrophilic interaction ultra-performance liquid chromatography coupled with triple–quadrupole linear ion-trap tandem mass spectrometry (HILIC-UHPLC-QTRAP®/MS2). The analytical method was validated by matrix effects, linearity, LODs, LOQs, precision, repeatability, stability, and recovery. Thirdly, the proposed optimal UAE conditions and analytical methods were applied to measurement of LHG samples. It was shown that LHG was rich in essential amino acids, which were beneficial nutrient substances for human health. Finally, based on the contents of the 27 analytes, the chemometrics methods of unsupervised principal component analysis (PCA) and supervised counter propagation artificial neural network (CP-ANN) were applied to differentiate and classify the 40 batches of LHG samples from different cultivated forms, regions, and varieties. As a result, these samples were mainly clustered into three clusters, which illustrated the cultivating disparity among the samples. In summary, the presented strategy had potential for the investigation of edible plants and agricultural products containing FAAs and small peptides.
Keywords: Siraitiae Fructus; Free amino acids; Peptides; HILIC-UHPLC-QTRAP®/MS2 ; Chemometrics; Artificial neural network

Keywords: Superoxide; Superoxide dismutase; Methoxyestradiol; Inhibition; Method