Amino Acids (v.48, #1)

ω-Amidase: an underappreciated, but important enzyme in l-glutamine and l-asparagine metabolism; relevance to sulfur and nitrogen metabolism, tumor biology and hyperammonemic diseases by Arthur J. L. Cooper; Yevgeniya I. Shurubor; Thambi Dorai; John T. Pinto; Elena P. Isakova; Yulia I. Deryabina; Travis T. Denton; Boris F. Krasnikov (1-20).
In mammals, two major routes exist for the metabolic conversion of l-glutamine to α-ketoglutarate. The most widely studied pathway involves the hydrolysis of l-glutamine to l-glutamate catalyzed by glutaminases, followed by the conversion of l-glutamate to α-ketoglutarate by the action of an l-glutamate-linked aminotransferase or via the glutamate dehydrogenase reaction. However, another major pathway exists in mammals for the conversion of l-glutamine to α-ketoglutarate (the glutaminase II pathway) in which l-glutamine is first transaminated to α-ketoglutaramate (KGM) followed by hydrolysis of KGM to α-ketoglutarate and ammonia catalyzed by an amidase known as ω-amidase. In mammals, the glutaminase II pathway is present in both cytosolic and mitochondrial compartments and is most prominent in liver and kidney. Similarly, two routes exist for the conversion of l-asparagine to oxaloacetate. In the most extensively studied pathway, l-asparagine is hydrolyzed to l-aspartate by the action of asparaginase, followed by transamination of l-aspartate to oxaloacetate. However, another pathway also exists for the conversion of l-asparagine to oxaloacetate (the asparaginase II pathway). In this pathway, l-asparagine is first transaminated to α-ketosuccinamate (KSM), followed by hydrolysis of KSM to oxaloacetate by the action of ω-amidase. One advantage of both the glutaminase II and the asparaginase II pathways is that they are irreversible, and thus are important in anaplerosis by shuttling 5-C (α-ketoglutarate) and 4-C (oxaloacetate) units into the TCA cycle. In this review, we briefly mention the importance of the glutaminase II and asparaginase II pathways in microorganisms and plants. However, the major emphasis of the review is related to the importance of these pathways (especially the common enzyme component of both pathways—ω-amidase) in nitrogen and sulfur metabolism in mammals and as a source of anaplerotic carbon moieties in rapidly dividing cells. The review also discusses a potential dichotomous function of ω-amidase as having a role in tumor progression. Finally, the possible role of KGM as a biomarker for hyperammonemic diseases is discussed.
Keywords: ω-Amidase; Asparagine transaminase; α-Ketoglutaramate; α-Ketosuccinamate; Glutamine transaminase; Kynurenine aminotransferase; Nitrilase-like protein (Nit2)

Low-protein diets affect ileal amino acid digestibility and gene expression of digestive enzymes in growing and finishing pigs by Liuqin He; Li Wu; Zhiqi Xu; Tiejun Li; Kang Yao; Zhijie Cui; Yulong Yin; Guoyao Wu (21-30).
The objective of this study was to evaluate effects of dietary crude protein (CP) intake on ileal amino acid digestibilities and expression of genes for digestive enzymes in growing and finishing pigs. In Experiment 1, 18 growing pigs (average initial BW = 36.5 kg) were assigned randomly into one of three treatments (n = 6/treatment group) representing normal (18 % CP), low (15 % CP), and very low (12 % CP) protein intake. In Experiment 2, 18 finishing pigs (average initial BW = 62.3 kg) were allotted randomly into one of three treatments (n = 6/treatment group), representing normal (16 % CP), low (13 % CP) and very low (10 % CP) protein intake. In both experiments, diets with low and very low CP were supplemented with crystalline amino acids to achieve equal content of standardized ileal digestible Lys, Met, Thr, and Trp, and were provided to pigs ad libitum. Daily feed intake, BW, and feed/gain ratios were determined. At the end of each experiment, all pigs were slaughtered to collect pancreas, small-intestine samples, and terminal ileal chymes. Samples were used for determining expression of genes for digestive enzymes and ileal amino acid digestibilities. Growing pigs fed the 12 % CP and 15 % CP diets had lower final body weight (P < 0.01) and ADG (P < 0.0001) when compared with pigs fed the 18 % dietary CP diet. Growing pigs fed with the 12 % CP diet showed higher digestibilities for CP (P < 0.05), DM (P < 0.05), Lys (P < 0.0001), Met (P < 0.01), Cys (P < 0.01), Thr (P < 0.01), Trp (P < 0.05), Val (P < 0.05), Phe (P < 0.05), Ala (P < 0.05), Cys (P < 0.01), and Gly (P < 0.05) than those fed the 18 % CP diet. Finishing pigs fed the 16 % CP diet had a higher (P < 0.01) final body weight than those fed the 10 % CP diet. mRNA levels for digestive enzymes (trypsinogen, chymotrypsin B, and dipeptidases-II and III) differed among the three groups of pigs (P < 0.05), and no difference was noted in the genes expression between control group and lower CP group. These results indicated that a reduction of dietary CP by a six-percentage value limited the growth performance of growing–finishing pigs and that a low-protein diet supplemented with deficient amino acids could reduce the excretion of nitrogen into the environment without affecting weight gain.
Keywords: Low nitrogen; Crude protein; Amino acid; Digestive enzyme; Pig

Isopeptidase activity of human transglutaminase 2: disconnection from transamidation and characterization by kinetic parameters by Róbert Király; Kiruphagaran Thangaraju; Zsófia Nagy; Russell Collighan; Zoltán Nemes; Martin Griffin; László Fésüs (31-40).
Transglutaminase 2 (TG2) is a multifunctional protein with diverse catalytic activities and biological roles. Its best studied function is the Ca2+-dependent transamidase activity leading to formation of γ-glutamyl-ε-lysine isopeptide crosslinks between proteins and γ-glutamyl-amine derivatives. TG2 has a poorly studied isopeptidase activity cleaving these bonds. We have developed and characterised TG2 mutants which are significantly deficient in transamidase activity while have normal or increased isopeptidase activity (W332F) and vice versa (W278F). The W332F mutation led to significant changes of both the K m and the V max kinetic parameters of the isopeptidase reaction of TG2 while its calcium and GTP sensitivity was similar to the wild-type enzyme. The W278F mutation resulted in six times elevated amine incorporating transamidase activity demonstrating the regulatory significance of W278 and W332 in TG2 and that mutations can change opposed activities located at the same active site. The further application of our results in cellular systems may help to understand TG2-driven physiological and pathological processes better and lead to novel therapeutic approaches where an increased amount of crosslinked proteins correlates with the manifestation of degenerative disorders.
Keywords: Human transglutaminase 2; Isopeptidase activity; γ-Glutamyl-hydrolase; Transamidation; Regulation of activities; Moonlighting enzyme

The role of leucine and its metabolites in protein and energy metabolism by Yehui Duan; Fengna Li; Yinghui Li; Yulong Tang; Xiangfeng Kong; Zemeng Feng; Tracy G. Anthony; Malcolm Watford; Yongqing Hou; Guoyao Wu; Yulong Yin (41-51).
Leucine (Leu) is a nutritionally essential branched-chain amino acid (BCAA) in animal nutrition. It is usually one of the most abundant amino acids in high-quality protein foods. Leu increases protein synthesis through activation of the mammalian target of rapamycin (mTOR) signaling pathway in skeletal muscle, adipose tissue and placental cells. Leu promotes energy metabolism (glucose uptake, mitochondrial biogenesis, and fatty acid oxidation) to provide energy for protein synthesis, while inhibiting protein degradation. Approximately 80 % of Leu is normally used for protein synthesis, while the remainder is converted to α-ketoisocaproate (α-KIC) and β-hydroxy-β-methylbutyrate (HMB) in skeletal muscle. Therefore, it has been hypothesized that some of the functions of Leu are modulated by its metabolites. Both α-KIC and HMB have recently received considerable attention as nutritional supplements used to increase protein synthesis, inhibit protein degradation, and regulate energy homeostasis in a variety of in vitro and in vivo models. Leu and its metabolites hold great promise to enhance the growth and health of animals (including humans, birds and fish).
Keywords: Leucine; α-Ketoisocaproate; β-Hydroxy-β-methylbutyrate; Protein metabolism; Energy homeostasis; Pigs

Dietary supplementation with l-glutamate and l-aspartate alleviates oxidative stress in weaned piglets challenged with hydrogen peroxide by Jielin Duan; Jie Yin; Wenkai Ren; Ting Liu; Zhijie Cui; Xingguo Huang; Li Wu; Sung Woo Kim; Gang Liu; Xi Wu; Guoyao Wu; Tiejun Li; Yulong Yin (53-64).
This study was to evaluate the protective roles of l-glutamate (Glu) and l-aspartate (Asp) in weaned piglets challenged with H2O2. Forty weaned piglets were assigned randomly into one of five groups (8 piglets/group): (1) control group (NC) in which pigs were fed a corn- and soybean meal-based diet and received intraperitoneal administration of saline; (2) H2O2 group (PC) in which pigs were fed the basal diet and received intraperitoneal administration of 10 % H2O2 (1 ml/kg body weight once on days 8 and repeated on day 11); (3) PC + Glu group (PG) in which pigs were fed the basal diet supplemented with 2.0 % Glu before intraperitoneal administration of 10 % H2O2; (4) PC + Asp group (PA) in which pigs were fed the basal diet supplemented with 1.0 % Asp before intraperitoneal administration of 10 % H2O2; (5) PC + Glu + Asp group (PGA) in which pigs were fed the basal diet supplemented with 2.0 % Glu plus 1.0 % Asp before intraperitoneal administration of 10 % H2O2. Measured parameters included daily feed intake (DFI), average daily gain (ADG), feed conversion rate (FCR), and serum anti-oxidative enzyme activities (catalase, superoxide dismutase, glutathione peroxidase-1), serum malondialdehyde and H2O2 concentrations, serum amino acid (AA) profiles, and intestinal expression of AA transporters. Dietary supplementation with Glu, Asp or their combination attenuated the decreases in DFI, ADG and feed efficiency, the increase in oxidative stress, the alterations of serum AA concentrations, and the changed expression of intestinal AA transporters in H2O2-challenged piglets. Thus, dietary supplementation with Glu or Asp alleviates growth suppression and oxidative stress, while restoring serum the amino acid pool in H2O2-challenged piglets.
Keywords: l-Glutamate; l-Aspartate; Oxidative stress; Hydrogen peroxide; Performance; Pigs

18F-Labeled wild-type annexin V: comparison of random and site-selective radiolabeling methods by Amanda Perreault; James C. Knight; Monica Wang; Jenilee Way; Frank Wuest (65-74).
Early stage apoptosis is characterized by the externalization of phosphatidylserine (PS) from the inner leaflet of the plasma membrane to the outer periphery. Consequently, PS represents an excellent target for non-invasive imaging of apoptosis by positron emission tomography. Annexin V is a 36 kDa protein which binds with high affinity to PS. Radiolabeling of wild-type annexin V with fluorine-18 (18F) can be accomplished via random acylation of 23 amine groups (22 lysine residues and one N-terminal amine) with [18F]SFB or site-specific alkylation reaction on cysteine residue at position 315 with maleimide-containing prosthetic groups like [18F]FBEM. The effect upon random and site-directed 18F labeling of annexin V was studied with EL4 mouse lymphoma cells. Both, randomly and site-selectively radiolabeled annexin V demonstrated comparable binding to apoptotic EL4 cells. This finding suggests that the 18F radiolabeling method has no significant effect on the ability of 18F-labeled wild-type annexin V to bind PS in apoptotic cells.
Keywords: Wild-type annexin V; Fluorine-18; Apoptosis; [18F]SFB; [18F]FBEM

Co-dependence of genotype and dietary protein intake to affect expression on amino acid/peptide transporters in porcine skeletal muscle by Y. Liu; X. Kong; F. Li; B. Tan; Y. Li; Y. Duan; Y. Yin; J. He; C. Hu; F. Blachier; Guoyao Wu (75-90).
A total of 96 barrows (48 pure-bred Bama mini-pigs representing fatty genotype, and 48 Landrace pigs representing lean genotype) were randomly assigned to either a low- or adequate-protein treatment diet. The experimental period commenced at 5 weeks of age and extended to the finishing period. After euthanasia, blood and skeletal muscle samples were collected from pigs at the nursery, growing, and finishing phases. Our results indicate that the concentrations of free AAs in the plasma and muscle decreased as the age of the pigs increased. In addition, a strain × growth phase interaction (P < 0.05) was observed for the free AA pool in the plasma and muscle. The low-protein diet upregulated (P < 0.05) the mRNA levels for T1R1/T1R3 involved in glutamate binding, but downregulated (P < 0.05) the mRNA levels for PAT1, PAT2, and ASCT2, which transport neutral AAs into muscles. Bama mini-pigs had higher (P < 0.05) mRNA levels for LAT1, SNAT2, and EAAC1, but a lower (P < 0.05) mRNA level for PepT1, compared with Landrace pigs. Collectively, our findings indicate that adequate provision of dietary protein plays an important role in regulating profiles of free AA pools and expression of key AA/peptide transporters/transceptors in a genotype- and tissue-specific manner.
Keywords: Bama mini-pig; Dietary protein; Skeletal muscle; AA transporter; AA receptor

SPECT imaging of interleukin-6 receptor in ovarian tumor xenografts with a novel radiotracer of 99mTc-HYNIC-Aca-LSLITRL by Fei Li; Zhenzhong Zhang; Teng Cheng; Rui Wei; Yun Dai; Mengqin Lv; Danfeng Luo; Xiaohua Zhu; Ding Ma; Ling Xi; Qingjian Dong; Xiangyi Ma (91-101).
Growing evidences have shown that the IL-6/IL-6R signal pathway promotes the tumor growth, angiogenesis, invasion and migration in various cancers, especially for epithelial ovarian cancer. Hence, including anti-IL-6 antibody (Siltuximab) and anti-IL-6R antibody (Tocilizumab), more and more therapeutic drugs targeting IL-6/IL-6R pathway were developed to block their activity. The molecular imaging of IL-6R is a significant factor for predicting tumor response to IL-6/IL-6R targeted drugs. However, few probes targeting IL-6R were designed and used for the specific detection. The purpose of this study was to develop and evaluate a novel radiotracer, 99mTc-HYNIC-Aca-LSLITRL, for SPECT imaging of interleukin-6 receptor. The expression of IL-6R was determined by western blot, immunofluorescence and immunohistochemistry. HYNIC-Aca-LSLITRL and HYNIC-Aca-TLQASIL were synthesized, and then were labeled with 99mTc. The stability and the cell-binding assay were performed. Ovarian tumor xenografts were established and subjected to SPECT imaging after injection of these two radiopharmaceuticals with or without excess primary peptides. The biodistribution of these two radiotracers was performed in nude mice bearing C13K tumors. 99mTc-HYNIC-Aca-LSLITRL and 99mTc-HYNIC-Aca-TLQASIL were obtained in >95 % labeling yield with favorably stability. In vitro studies demonstrated that the interleukin-6 receptor was overexpressed in ovarian cancer C13K cells. The SPECT imaging of interleukin-6 receptor and biodistribution studies showed that 99mTc-HYNIC-Aca-LSLITRL had higher tumor uptake and significantly lower kidney accumulation compared to 99mTc-HYNIC-Aca-TLQASIL. 99mTc-HYNIC-Aca-LSLITRL could be a promising agent for SPECT imaging of interleukin-6 receptor of ovarian cancer especially for those anti-IL-6R drugs under clinical trials, such as tocilizumab.
Keywords: Interleukin-6 receptor; Ovarian cancer; 99mTc-HYNIC-Aca-LSLITRL; 99mTc-HYNIC-Aca-TLQASIL; Molecular imaging

Reduced cortical neurotransmitter receptor complex levels in fetal Down syndrome brain by Soheil Keihan Falsafi; Mara Dierssen; Maryam Ghafari; Arnold Pollak; Gert Lubec (103-116).
In this study, cortical receptor complex levels were determined in fetal Down syndrome (DS, trisomy 21) brain. Frontal cortices were obtained from individuals with DS (19th–22nd week of gestation) and controls. Membrane proteins were extracted, assayed on blue native gels and immunoblotted with brain receptor antibodies. Levels of a D1R-containing complex were markedly decreased in male and female cortices of DS individuals. Females with DS had significant reductions of nicotinic acetylcholine receptors α4 and α7, NMDA receptor GluN1 and AMPA receptor GluA1- and GluA3-containing receptor complexes. Levels of other brain receptor complexes (5-hydroxytryptamine 1A, GluA2 and GluR4 receptor-containing complexes) were comparable between the groups of females. Levels of GluA2- and GluA3-containing complexes were significantly increased in males. Decreased levels of D1R complexes in both sexes, along with the significant reduction of α4, α7-containing receptor complexes observed in females, may explain the brain deficits and impaired cognition observed in DS.
Keywords: Fetal Down syndrome; Dopamine receptor; Brain receptors; Receptor complexes

The epithelial-to-mesenchymal transition (EMT) plays a vital role in carcinogenesis, invasion, and metastasis of many epithelial tumors including oral squamous cell carcinoma (OSCC), a common malignancy of the head and neck. However, the functional role of the actin-sequestering protein thymosin β4 (Tβ4) in the EMT in OSCCs remains unclear. Thus, we investigated whether overexpression of Tβ4 could induce in vitro tumorigenesis such as cell proliferation and anchorage independency and an EMT-like phenotype in OSCCs. Also, we examined whether it affects invasiveness and cell motility-associated signaling molecules. Tβ4-overexpressing OSCCs, SCC-15_Tβ4 and SCC-25_Tβ4, enhanced cell proliferation and colony formation. In addition, we observed that Tβ4 overexpression induced an EMT-like phenotype, accompanied by a decrease in expression of the epithelial cell marker E-cadherin and an increase in expression of mesenchymal cell markers vimentin and N-cadherin. Also, the expression level of Twist1, an EMT-inducing transcription factor, was significantly enhanced in SCC-15_Tβ4 and SCC-25_Tβ4 cells. Tβ4 overexpression augmented in vitro invasion and MMP-2 activity and enhanced the phosphorylation of paxillin and cortactin and expression of LIMK1. Taken together, these results suggest that Tβ4 overexpression could be one of the causes of tumorigenesis and progression in OSCCs. Further investigation on the Tβ4 molecule would encourage the development of specific targets for cancer treatment.
Keywords: Thymosin β4; Oral squamous cell carcinoma; Epithelial-to-mesenchymal transition; Proliferation; Invasion

Characterization of the diversity of mycosporine-like amino acids in lichens from high altitude region of Himalaya by Vertika Shukla; Rupender Kumari; Davendra K. Patel; Dalip K. Upreti (129-136).
Lichens are tolerant to a number of environmental variables including high-intensity solar radiations, which is mainly due to the presence of chemical substances in the thallus. Especially, cyanobacterial lichens synthesize a unique class of chemical substances known as mycosporine-like amino acids (MAAs) the primary characteristic of which is strong ultraviolet (UV) absorption between 300 and 360 nm. In view of its UV-protecting potential, the applicability of mass spectral fragmentation using electrospray ionization tandem mass spectrometric analysis for the characterization of MAAs in lichen samples was explored. MAA compounds were characterized in four cyanobacteria-containing lichen species belonging to genus Peltigera, Stereocaulon and Lobaria. Among them, Peltigera and Lobaria are true cyanobacteria containing lichens (cyanolichens) while Stereocaulon is a tripartite lichen, as it contains both green algae (in the thallus) and cyanobacteria (in the cephalodia), collected from higher altitudes of Himalaya (Tungnath-Chopta in Garhwal Himalaya, 3432 m) from an exposed locality experiencing high light intensity. Mass spectral data of distinctive fragmentation pattern revealed that all the four species have good diversity of MAA compounds, especially Lobaria retigera was found to be enriched with highest diversity of oxo and imino MAAs. Overall, different numbers of oxo and imino MAA compounds were detected in the remaining lichen species. Good diversity of imino MAAs has ecological significance which is required to be investigated further. Moreover, the impressive diversity characterized in each lichen species suggests that lichens should be thoroughly studied for their MAAs contents.
Keywords: Cyanobacterial lichens; MAAs diversity; UV radiance; Photo protection

Homeostatic effect of p-chloro-diphenyl diselenide on glucose metabolism and mitochondrial function alterations induced by monosodium glutamate administration to rats by Caroline B. Quines; Suzan G. Rosa; Pietro M. Chagas; Juliana T. da Rocha; Fernando Dobrachinski; Nélson R. Carvalho; Félix A. Soares; Sônia C. Almeida da Luz; Cristina W. Nogueira (137-148).
The metabolic syndrome is a group of metabolic alterations considered a worldwide public health problem. Organic selenium compounds have been reported to have many different pharmacological actions, such as anti-hypercholesterolemic and anti-hyperglycemic. The aim of this study was to evaluate the effect of p-chloro-diphenyl diselenide (p-ClPhSe)2, an organic selenium compound, in a model of obesity induced by monosodium glutamate (MSG) administration in rats. The rats were treated during the first ten postnatal days with MSG and received (p-ClPhSe)2 (10 mg/kg, intragastrically) from 45th to 51th postnatal day. Glucose, lipid and lactate levels were determined in plasma of rats. Glycogen levels and activities of tyrosine aminotransferase, hexokinase, citrate synthase and glucose-6-phosphatase (G-6-Pase) were determined in livers of rats. Renal G-6-Pase activity was also determined. The purine content [Adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate] and mitochondrial functionality in the liver were also investigated. p-(ClPhSe)2 did not alter the reduction in growth performance and in the body weight caused by MSG but reduced epididymal fat deposition of rats. p-(ClPhSe)2 restored glycemia, triglycerides, cholesterol and lactate levels as well as the glucose metabolism altered in rats treated with MSG. p-(ClPhSe)2 restored hepatic mitochondrial dysfunction and the decrease in citrate synthase activity and ATP and ADP levels caused by MSG in rats. In summary, (p-ClPhSe)2 had homeostatic effects on glucose metabolism and mitochondrial function alterations induced by MSG administration to rats.
Keywords: Metabolic syndrome; Obesity; Monosodium glutamate; Organoselenium; Rats

High intake of dietary cysteine is extremely toxic to animals and the underlying mechanism remains largely unknown. This study was conducted to test the hypothesis that excessive l-cysteine induces cell death by activating endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) signaling in intestinal porcine epithelial cells. Jejunal enterocytes were cultured in the presence of 0–10 mmol/L l-cysteine. Cell viability, morphologic alterations, mRNA levels for genes involved in ER stress, protein abundances for glucose-regulated protein 78, C/EBP homologous protein (CHOP), alpha subunit of eukaryotic initiation factor-2 (eIF2α), extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal protein kinase (JNK1/2) were determined. The results showed that l-cysteine (5–10 mmol/L) reduced cell viability (P < 0.05) and led to vacuole-like cell death in intestinal porcine epithelial cells. These adverse effects of L-cysteine  were not affected by the autophagy inhibitor 3-methyladenine. The protein abundances for CHOP, phosphorylated (p)-eIF2α, p-JNK1/2, p-p38 MAPK, and the spliced form of XBP-1 mRNA were enhanced (P < 0.05), whereas those for p-ERK1/2 were reduced (P < 0.05). Collectively,  excessive l-cysteine induces vacuole-like cell death via the  activation of ER stress and MAPK signaling in small intestinal epithelial cells. These signaling pathways may be potential targets for developing effective strategies to prevent the toxicity of dietary cysteine.
Keywords: l-Cysteine; Intestinal epithelial cells; Endoplasmic reticulum stress; Mitogen-activated protein kinase; Cell death

Inclusion of a pH-responsive amino acid-based amphiphile in methotrexate-loaded chitosan nanoparticles as a delivery strategy in cancer therapy by Daniele Rubert Nogueira; Laís E. Scheeren; Letícia B. Macedo; Ana Isa P. Marcolino; M. Pilar Vinardell; Montserrat Mitjans; M. Rosa Infante; Ammad A. Farooqi; Clarice M. B. Rolim (157-168).
The encapsulation of antitumor drugs in nanosized systems with pH-sensitive behavior is a promising approach that may enhance the success of chemotherapy in many cancers. The nanocarrier dependence on pH might trigger an efficient delivery of the encapsulated drug both in the acidic extracellular environment of tumors and, especially, in the intracellular compartments through disruption of endosomal membrane. In this context, here we reported the preparation of chitosan-based nanoparticles encapsulating methotrexate as a model drug (MTX-CS-NPs), which comprises the incorporation of an amino acid-based amphiphile with pH-responsive properties (77KS) on the ionotropic complexation process. The presence of 77KS clearly gives a pH-sensitive behavior to NPs, which allowed accelerated release of MTX with decreasing pH as well as pH-dependent membrane-lytic activity. This latter performance demonstrates the potential of these NPs to facilitate cytosolic delivery of endocytosed materials. Outstandingly, the cytotoxicity of MTX-loaded CS-NPs was higher than free drug to MCF-7 tumor cells and, to a lesser extent, to HeLa cells. Based on the overall results, MTX-CS-NPs modified with the pH-sensitive surfactant 77KS could be potentially useful as a carrier system for intracellular drug delivery and, thus, a promising targeting anticancer chemotherapeutic agent.
Keywords: Chitosan nanoparticles; In vitro antitumor activity; Lysine-based surfactant methotrexate; pH-sensitivity

Detection of COL III in parchment by amino acid analysis by Dorte V. P. Sommer; René Larsen (169-181).
Cultural heritage parchments made from the reticular dermis of animals have been subject to studies of deterioration and conservation by amino acid analysis. The reticular dermis contains a varying mixture of collagen I and III (COL I and III). When dealing with the results of the amino acid analyses, till now the COL III content has not been taken into account. Based on the available amino acid sequences, we present a method for determining the amount of COL III in the reticular dermis of new and historical parchments calculated from the ratio of Ile/Val. We find COL III contents between 7 and 32 % in new parchments and between 0.2 and 40 % in the historical parchments. This is consistent with results in the literature. The varying content of COL III has a significant influence on the uncertainty of the amino acid analysis. Although we have not found a simple correlation between the COL III content and the degree of deterioration, our results show that this question must be taken into consideration in future studies of the chemical and physical deterioration of parchment measured by amino acid analysis and other analytical methods.
Keywords: Amino acid analysis; Collagen I; Collagen III; Cultural heritage; Oxidation; Parchment

Changes in urinary amino acids excretion in relationship with muscle activity markers over a professional cycling stage race: in search of fatigue markers by Roberto Corsetti; Alessandra Barassi; Silvia Perego; Veronica Sansoni; Alessandra Rossi; Clara Anna Linda Damele; Gianlodovico Melzi D’Eril; Giuseppe Banfi; Giovanni Lombardi (183-192).
The aim of this study was to identify the relationship between metabolic effort, muscular damage/activity indices, and urinary amino acids profile over the course of a strenuous prolonged endurance activity, as a cycling stage race is, in order to identify possible fatigue markers. Nine professional cyclists belonging to a single team, competing in the Giro d’Italia cycling stage race, were anthropometrically characterized and sampled for blood and urine the day before the race started, and on days 12 and 23 of the race. Diet was kept the same over the race, and power output and energy expenditure were recorded. Sera were assayed for muscle markers (lactate dehydrogenase, aspartate aminotransferase, and creatine kinase activities, and blood urea nitrogen), and creatinine, all corrected for plasma volume changes. Urines were profiled for amino acid concentrations, normalized on creatinine excretion. Renal function, in terms of glomerular filtration rate, was monitored by MDRD equation corrected on body surface area. Creatine kinase activity and blood urea were increased during the race as did serum creatinine while kidney function remained stable. Among the amino acids, taurine, glycine, cysteine, leucine, carnosine, 1-methyl histidine, and 3-methyl histidine showed a net decreased, while homocysteine was increased. Taurine and the dipeptide carnosine (β-alanyl-l-histidine) were significantly correlated with the muscle activity markers and the indices of effort. In conclusion, the metabolic profile is modified strikingly due to the effort. Urinary taurine and carnosine seem useful tools to evaluate the muscle damage and possibly the fatigue status on a long-term basis.
Keywords: Urinary amino acids; Fatigue; Cycling stage race; Muscular activity; Endurance

The structures of buried water molecules were studied in an ensemble of high-quality and non-redundant protein crystal structures. Buried water molecules were clustered and classified in lake-like clusters, which are completely isolated from the bulk solvent, and bay-like clusters, which are in contact with the bulk solvent through a surface water molecule. Buried water molecules are extremely common: lake-like clusters are found in 89 % of the protein crystal structures and bay-like clusters in 93 %. Clusters with only one water molecule are much more common than larger clusters. Both cluster types incline to be surrounded by loop residues, and to a minor extent by residues in extended secondary structure. Helical residues on the contrary do not tend to surround clusters of buried water molecules. One buried water molecule is found every 30–50 amino acid residues, depending on the secondary structures that are more abundant in the protein. Both main- and side-chain atoms are in contact with buried waters; they form four hydrogen bonds with the first water and 1–1.5 additional hydrogen bond for each additional water in the cluster. Consequently, buried water molecules appear to be firmly packed and rigid like the protein atoms. In this regard, it is remarkable to observe that prolines often surround water molecules buried in the protein interior. Interestingly, clusters of buried water molecules tend to be just beneath the protein surface. Moreover, water molecules tend to form a one-dimensional wire rather than more compact arrangements. This agrees with recent evidence of the mechanisms of solvent exchange between internal cavities and bulk solvent.
Keywords: Water molecule; Protein core; Protein hydration; Protein structure

Polyvinyl alcohol nanofiber formulation of the designer antimicrobial peptide APO sterilizes Acinetobacter baumannii-infected skin wounds in mice by Istvan Sebe; Eszter Ostorhazi; Aron Fekete; Krisztian N. Kovacs; Romana Zelko; Ilona Kovalszky; Wenyi Li; John D. Wade; Dora Szabo; Laszlo Otvos Jr. (203-211).
Native and designer cationic antimicrobial peptides are increasingly acknowledged as host defense molecules rather than true antimicrobials. Due to their ability to activate the innate immune system, these structures are used to treat uninfected and bacterially-infected wounds, including those harboring Acinetobacter baumannii. Previously we documented that when administered intramuscularly or topically in liquid formulations, the proline-rich host defense peptide dimer A3-APO accelerates uninfected wound re-epithelization and eliminates systemic and local A. baumannii, methicillin-resistant Staphylococcus aureus and other pathogen load from infected lesions better than conventional antibiotics. In the current study we sought to produce and characterize a novel delivery system, suitable for immediate and convenient application in non-hospital environments. The APO monomer was incorporated into polyvinyl alcohol nanofibers and the complex was polymerized into a solid patch dressing. Mice were subjected to skin abrasion where the wounds were either left uninfected or were inoculated with a near lethal dose of multidrug resistant A. baumannii strain. Analyzed after 3 days, APO monomer-containing patches improved wound appearance significantly better than polymer patches without antibiotics. When compared to colistin, the APO patches accelerated wound healing, and statistically significantly reduced wound size and wound bacterial load. The in vivo antimicrobial effect was more extensive than after intramuscular administration of the peptide drug, by using only one tenth of the active pharmaceutical ingredient. These data suggest that the APO monomer-impregnated nanofiber dressing can be developed as an economical first-line treatment option to skin injuries in general and battlefield burn and blast injuries in particular.
Keywords: Antibacterial peptide; Cutaneous infection; Nanoformulation; Topical treatment; Wound healing

Metabolomic study of urinary polyamines in rat exposed to 915 MHz radiofrequency identification signal by Man-Jeong Paik; Hye Sun Kim; Yun-Sil Lee; Hyung Do Choi; Jeong-Ki Pack; Nam Kim; Young Hwan Ahn (213-217).
Metabolomic analysis of urinary polyamines (PAs) from rat exposed to 915 MHz radiofrequency identification (RFID) signal for 8 h/day for 2 weeks was performed by gas chromatography–mass spectrometry as N-ethoxycarbonyl/N-pentafluoropropionyl derivatives. Large alterations in nine PA levels including four aliphatic and five acetylated PAs were monitored in sham-exposed and RFID-exposed groups. Total PA and urinary levels of N 1-acetylputrescine, N 1-acetylcadaverine, putrescine, cadaverine, N 1-acetylspermidine, N 8-acetylspermidine, spermidine and spermine were reduced, whereas N 1-acetylspermine was significantly increased after sham and RFID exposure compared with those before exposure. Their levels were normalized to the corresponding group means before exposure and then plotted into star symbol patterns. N 1-Acetylspermine after RFID exposure was 54 % higher compared to the level before RFID exposure, while it was elevated by only 17 % in the sham group. The results suggest that 915 MHz RFID exposure may induce metabolic disturbance of PA. It may also elevate spermidine/spermine acetyltransferase (SSAT) activity. Thus, the present metabolic profiling combined with star pattern recognition method might be useful for understanding the complexity of biochemical events after exposure to RFID signal.
Keywords: Metabolomic analysis; Urinary polyamines; Radiofrequency identification; Gas chromatography-mass spectrometry; Star pattern recognition

An ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-qTOF-MS) method using hydrophilic interaction liquid chromatography was developed and validated for simultaneous quantification of 18 free amino acids in urine with a total acquisition time including the column re-equilibration of less than 18 min per sample. This method involves simple sample preparation steps which consisted of 15 times dilution with acetonitrile to give a final composition of 25 % aqueous and 75 % acetonitrile without the need of any derivatization. The dynamic range for our calibration curve is approximately two orders of magnitude (120-fold from the lowest calibration curve point) with good linearity (r 2 ≥ 0.995 for all amino acids). Good separation of all amino acids as well as good intra- and inter-day accuracy (<15 %) and precision (<15 %) were observed using three quality control samples at a concentration of low, medium and high range of the calibration curve. The limits of detection (LOD) and lower limit of quantification of our method were ranging from approximately 1–300 nM and 0.01–0.5 µM, respectively. The stability of amino acids in the prepared urine samples was found to be stable for 72 h at 4 °C, after one freeze thaw cycle and for up to 4 weeks at −80 °C. We have applied this method to quantify the content of 18 free amino acids in 646 urine samples from a dietary intervention study. We were able to quantify all 18 free amino acids in these urine samples, if they were present at a level above the LOD. We found our method to be reproducible (accuracy and precision were typically <10 % for QCL, QCM and QCH) and the relatively high sample throughput nature of this method potentially makes it a suitable alternative for the analysis of urine samples in clinical setting.
Keywords: Free amino acids; Human urine; Absolute quantification; HILIC-UPLC-qTOF-MS

Ubr11 in the fission yeast Schizosaccharomyces pombe is an evolutionarily conserved ubiquitin ligase functioning in the Arg/N-end rule pathway, which promotes degradation of substrate proteins via the proteasome. Ubr11 recognizes the N-degron sequence in substrates. The primary N-degron contains a destabilization-inducing N-terminal amino acid, which is either a basic (type 1) or bulky hydrophobic (type 2) residue. Dipeptides are known to inhibit proteolytic degradation via the Arg/N-end rule pathway. Here, I examined the potency of some amino acid- or dipeptide-related molecules in their inhibition of Ubr11/N-end rule-mediated degradation. An amide form of l-arginine and l-tryptophan had weak inhibitory activity for type 1 and type 2 substrates, respectively, although the unmodified amino acid monomer and its carboxymethylated ester were ineffective. Among the naturally occurring dipeptides tested, Lys-Leu and Tyr-Leu showed potent inhibitory activity, but their effect was transient, especially at submillimolar concentrations. l-arginine-β-naphthylamide (Arg-βNA) showed stronger activity than several dipeptides for type 1 substrates, but all Lys-Leu, Tyr-Leu, and Arg-βNA caused growth retardation. The inhibitory activity of the l-phenylalanine carbobenzoxy-hydrazide for type 2 substrates was not very strong, but it prolonged the action of Tyr-Leu at low concentrations and, importantly, did not interfere with cell growth. Apart from their utility, these dipeptidomimetics provide a clue for understanding the determinants of recognition by Ubr ubiquitin ligase and further designing novel inhibitors of the Arg/N-end rule pathway.
Keywords: N-End rule pathway; Ubiquitin ligase; Ubr protein; Dipeptidomimetic; S. pombe

Discovery and microassay of a nitrite-dependent carbonic anhydrase activity by stable-isotope dilution gas chromatography–mass spectrometry by Maximilian Zinke; Erik Hanff; Anke Böhmer; Claudiu T. Supuran; Dimitrios Tsikas (245-255).
The intrinsic activity of carbonic anhydrase (CA) is the hydration of CO2 to carbonic acid and its dehydration to CO2. CA may also function as esterase and phosphatase. Recently, we demonstrated that renal CA is mainly responsible for the reabsorption of nitrite (NO2 ) which is the most abundant reservoir of the biologically highly potent nitric oxide (NO). By means of a stable-isotope dilution GC–MS method, we discovered a novel CA activity which strictly depends upon nitrite. We found that bovine erythrocytic CAII (beCAII) catalyses the incorporation of 18O from H 2 18 O into nitrite at pH 7.4. After derivatization with pentafluorobenzyl bromide, gas chromatographic separation and mass spectrometric analysis, we detected ions at m/z 48 for singly 18O-labelled nitrite (16O=N–18O/18O=N–16O) and at m/z 50 for doubly 18O-labelled nitrite (18O=N–18O) in addition to m/z 46 for unlabelled nitrite. Using 15N-labelled nitrite (15NO2 , m/z 47) as an internal standard and selected-ion monitoring of m/z 46, m/z 48, m/z 50 and m/z 47, we developed a GC–MS microassay for the quantitative determination of the nitrite-dependent beCAII activity. The CA inhibitors acetazolamide and FC5 207A did not alter beCAII-catalysed formation of singly and doubly 18O-labelled nitrite. Cysteine and the experimental CA inhibitor DIDS (a diisothiocyanate) increased several fold the beCAII-catalysed formation of the 18O-labelled nitrite species. Cysteine, acetazolamide, FC5 207A, and DIDS by themselves had no effect on the incorporation of 18O from H 2 18 O into nitrite. We conclude that erythrocytic CA possesses a nitrite-dependent activity which can only be detected when nitrite is used as the substrate and the reaction is performed in buffers of neutral pH values prepared in H 2 18 O. This novel CA activity, i.e., the nitrous acid anhydrase activity, represents a bioactivation of nitrite and may have both beneficial (via S-nitrosylation and subsequent NO release) and possibly adverse (via C- and N-nitrosylation) effects in living organisms.
Keywords: Carbonic anhydrase; GC–MS; H 2 18 O; Labelling; Nitric oxide; Nitrite

Leucine supplementation of a chronically restricted protein and energy diet enhances mTOR pathway activation but not muscle protein synthesis in neonatal pigs by Rodrigo Manjarín; Daniel A. Columbus; Agus Suryawan; Hanh V. Nguyen; Adriana D. Hernandez-García; Nguyet-Minh Hoang; Marta L. Fiorotto; Teresa Davis (257-267).
Suboptimal nutrient intake represents a limiting factor for growth and long-term survival of low-birth weight infants. The objective of this study was to determine if in neonates who can consume only 70 % of their protein and energy requirements for 8 days, enteral leucine supplementation will upregulate the mammalian target of rapamycin (mTOR) pathway in skeletal muscle, leading to an increase in protein synthesis and muscle anabolism. Nineteen 4-day-old piglets were fed by gastric tube 1 of 3 diets, containing (kg body weight−1·day−1) 16 g protein and 190 kcal (CON), 10.9 g protein and 132 kcal (R), or 10.8 g protein + 0.2 % leucine and 136 kcal (RL) at 4-h intervals for 8 days. On day 8, plasma AA and insulin levels were measured during 6 post-feeding intervals, and muscle protein synthesis rate and mTOR signaling proteins were determined at 120 min post-feeding. At 120 min, leucine was highest in RL (P < 0.001), whereas insulin, isoleucine and valine were lower in RL and R compared to CON (P < 0.001). Compared to RL and R, the CON diet increased (P < 0.01) body weight, protein synthesis, phosphorylation of S6 kinase (p-S6K1) and 4E-binding protein (p-4EBP1), and activation of eukaryotic initiation factor 4 complex (eIF4E·eIF4G). RL increased (P ≤ 0.01) p-S6K1, p-4EBP1 and eIF4E·eIF4G compared to R. In conclusion, when protein and energy intakes are restricted for 8 days, leucine supplementation increases muscle mTOR activation, but does not improve body weight gain or enhance skeletal muscle protein synthesis in neonatal pigs.
Keywords: Leucine; Premature infant; Muscle; Synthesis

Optimization of oncocin for antibacterial activity using a SPOT synthesis approach: extending the pathogen spectrum to Staphylococcus aureus by Daniel Knappe; Serge Ruden; Stefanie Langanke; Tarun Tikkoo; Jennifer Ritzer; Ralf Mikut; Lisandra L. Martin; Ralf Hoffmann; Kai Hilpert (269-280).
The identification of lead molecules against multidrug-resistant bacteria ensuing the development of novel antimicrobial drugs is an urgent task. Proline-rich antimicrobial peptides are highly active in vitro and in vivo, but only against a few Gram-negative human pathogens, with rather weak activities against Pseudomonas aeruginosa and Staphylococcus aureus. This reduced level of efficacy could be related to inadequate uptake mechanisms or structural differences of the intracellular target proteins, i.e., the 70S ribosome or chaperone DnaK. Here we synthesized peptide arrays on cellulose membranes using cleavable linkers to release the free individual peptides for further antimicrobial tests. Thus, a library of singly substituted oncocin analogs was produced by replacing each residue by all other 19 canonical amino acids yielding a set of 361 individual peptides to be evaluated against a luminescent P. aeruginosa strain. Thirteen substitutions appeared promising and their improved antibacterial activities were confirmed for different bacteria after larger scale synthesis of these analogs. By combining two favorable substitutions into one peptide, we finally obtained an oncocin analog that was ten times more active against P. aeruginosa and even 100-fold more active against S. aureus than the original oncocin, providing minimal inhibitory concentrations of 4–8 and 0.5 µg/mL, respectively.
Keywords: Oncocin; Proline-rich antimicrobial peptide; Pseudomonas aeruginosa ; Quartz crystal microbalance; SPOT synthesis

Maternal and fetal tryptophan metabolism in gestating rats: effects of intrauterine growth restriction by Mitsue Sano; Véronique Ferchaud-Roucher; Bertrand Kaeffer; Guillaume Poupeau; Blandine Castellano; Dominique Darmaun (281-290).
l-Tryptophan (l-Trp) is a precursor for serotonin (5-HT) and nicotinamide adenine dinucleotide (NAD) synthesis. Both 5-HT and NAD may impact energy metabolism during gestation given that recent studies have demonstrated that increased 5-HT production is crucial for increasing maternal insulin secretion, and that sirtuin, an NAD+-dependent protein deacetylase, regulates endocrine signaling. Infants born with intrauterine growth restriction (IUGR) are at a higher risk of metabolic disease once they reach adulthood. IUGR is associated with altered maternal–fetal amino acid transfer. Whether IUGR affects l-Trp metabolism in mother and fetus has not been fully elucidated. Recently, we developed an analytical method using stable isotope-labeled l-Trp to explore the metabolism of l-Trp and its main metabolites, l-kynurenine (l-Kyn), 5-HT and quinolinic acid (QA). In this study, dams submitted to dietary protein restriction throughout gestation received intravenous infusions of stable isotope-labeled 15N2-l-Trp to determine whether l-Trp metabolism is affected by IUGR. Samples were obtained from maternal, fetal and umbilical vein plasma, as well as the amniotic fluid (AF), placenta and liver of the mother and the fetus after isotope infusion. We observed evidence for active l-Trp transfer from mother to fetus, as well as de novo synthesis of 5-HT in the fetus. Plasma 5-HT was decreased in undernourished mothers. In IUGR fetuses, maternal–fetal l-Trp transfer remained unaffected, but conversion to QA was impaired, implying that NAD production also decreased. Whether such alterations in tryptophan metabolism during gestation have adverse consequences and contribute to the increased risk of metabolic disease in IUGR remains to be explored.
Keywords: IUGR; Gestation; Fetus; Tryptophan metabolism; Serotonin; NAD; Stable isotope

Inhibition efficiencies of three amino acids [tryptophan (B), tyrosine (c), and serine (A)] have been studied as green corrosion inhibitors on corrosion of carbon steel using density functional theory (DFT) method in gas and aqueous phases. Quantum chemical parameters such as EHOMO (highest occupied molecular orbital energy), ELUMO (lowest unoccupied molecular orbital energy), hardness (η), polarizability ( $$ alpha $$ α ), total negative charges on atoms (TNC), molecular volume (MV) and total energy (TE) have been calculated at the B3LYP level of theory with 6-311++G** basis set. Consistent with experimental data, theoretical results showed that the order of inhibition efficiency is tryptophan (B) > tyrosine (C) > serine (A). In order to determine the possible sites of nucleophilic and electrophilic attacks, local reactivity has been evaluated through Fukui indices.
Keywords: Corrosion; Inhibitory efficiency; Amino acid; DFT; Steel

The anti-inflammatory action of the analgesic kyotorphin neuropeptide derivatives: insights of a lipid-mediated mechanism by Katia Conceição; Pedro R. Magalhães; Sara R. R. Campos; Marco M. Domingues; Vasanthakumar G. Ramu; Matthias Michalek; Philippe Bertani; António M. Baptista; Montserrat Heras; Eduard R. Bardaji; Burkhard Bechinger; Mônica Lopes Ferreira; Miguel A. R. B. Castanho (307-318).
Recently, a designed class of efficient analgesic drugs derived from an endogenous neuropeptide, kyotorphin (KTP, Tyr-Arg) combining C-terminal amidation (KTP-NH2) and N-terminal conjugation to ibuprofen (Ib), IbKTP-NH2, was developed. The Ib moiety is an enhancer of KTP-NH2 analgesic action. In the present study, we have tested the hypothesis that KTP-NH2 is an enhancer of the Ib anti-inflammatory action. Moreover, the impact of the IbKTP-NH2 conjugation on microcirculation was also evaluated by a unified approach based on intravital microscopy in the murine cremasteric muscle. Our data show that KTP-NH2 and conjugates do not cause damage on microcirculatory environment and efficiently decrease the number of leukocyte rolling induced by lipopolysaccharide (LPS). Isothermal titration calorimetry showed that the drugs bind to LPS directly thus contributing to LPS aggregation and subsequent elimination. In a parallel study, molecular dynamics simulations and NMR data showed that the IbKTP-NH2 tandem adopts a preferential “stretched” conformation in lipid bilayers and micelles, with the simulations indicating that the Ib moiety is anchored in the hydrophobic core, which explains the improved partition of IbKTP-NH2 to membranes and the permeability of lipid bilayers to this conjugate relative to KTP-NH2. The ability to bind glycolipids concomitant to the anchoring in the lipid membranes through the Ib residue explains the analgesic potency of IbKTP-NH2 given the enriched glycocalyx of the blood–brain barrier cells. Accumulation of IbKTP-NH2 in the membrane favors both direct permeation and local interaction with putative receptors as the location of the KTP-NH2 residue of IbKTP-NH2 and free KTP-NH2 in lipid membranes is the same.
Keywords: Kyotorphin; Kyotorphin amide; Ibuprofen; Analgesia; Microcirculation

Retraction Note to: Structure-based development and optimization of therapy antibody drugs against TNFα by Wenyan Fu; Xiaoze Wang; Weili Yang; Hiroaki Takeda; Shi Hu; Zhiyong Lou; Jian Zhao; Augus N. Bethune; Yajun Guo (319-319).