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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.42, #5)

The potential usefulness of taurine on diabetes mellitus and its complications by Takashi Ito; Stephen W. Schaffer; Junichi Azuma (pp. 1529-1539).

Taurine (2-aminoethanesulfonic acid) is a free amino acid found ubiquitously in millimolar concentrations in all mammalian tissues. Taurine exerts a variety of biological actions, including antioxidation, modulation of ion movement, osmoregulation, modulation of neurotransmitters, and conjugation of bile acids, which may maintain physiological homeostasis. Recently, data is accumulating that show the effectiveness of taurine against diabetes mellitus, insulin resistance and its complications, including retinopathy, nephropathy, neuropathy, atherosclerosis and cardiomyopathy, independent of hypoglycemic effect in several animal models. The useful effects appear due to the multiple actions of taurine on cellular functions. This review summarizes the beneficial effects of taurine supplementation on diabetes mellitus and the molecular mechanisms underlying its effectiveness.

Keywords: Taurine; Diabetic mellitus; Diabetic complications; Obesity; Insulin resistance

Development and application of site-specific proteomic approach for study protein S-nitrosylation by Miao Liu; James E. Talmadge; Shi-Jian Ding (pp. 1541-1551).

Protein S-nitrosylation is the covalent redox-related modification of cysteine sulfhydryl groups with nitric oxide, creating a regulatory impact similar to phosphorylation. Recent studies have reported a growing number of proteins to be S-nitrosylated in vivo resulting in altered functions. These studies support S-nitrosylation as a critical regulatory mechanism, fine-tuning protein activities within diverse cellular processes and biochemical pathways. In addition, S-nitrosylation appears to have key roles in the etiology of a broad range of human diseases. In this review, we discuss recent advances in proteomic approaches for the enrichment, identification, and quantitation of cysteine S-nitrosylated proteins and peptides. These advances have provided analytical tools with the power to interpret the impact of S-nitrosylation at the system level, providing a new platform for drug discovery and the identification of diagnostic markers for human diseases.

Keywords: S-nitrosylation; Proteomics; Identification; Quantification

Nutritional and medicinal aspects of d-amino acids by Mendel Friedman; Carol E. Levin (pp. 1553-1582).

This paper reviews and interprets a method for determining the nutritional value of d-amino acids, d-peptides, and amino acid derivatives using a growth assay in mice fed a synthetic all-amino acid diet. A large number of experiments were carried out in which a molar equivalent of the test compound replaced a nutritionally essential amino acid such as l-lysine (l-Lys), l-methionine (l-Met), l-phenylalanine (l-Phe), and l-tryptophan (l-Trp) as well as the semi-essential amino acids l-cysteine (l-Cys) and l-tyrosine (l-Tyr). The results show wide-ranging variations in the biological utilization of test substances. The method is generally applicable to the determination of the biological utilization and safety of any amino acid derivative as a potential nutritional source of the corresponding l-amino acid. Because the organism is forced to use the d-amino acid or amino acid derivative as the sole source of the essential or semi-essential amino acid being replaced, and because a free amino acid diet allows better control of composition, the use of all-amino-acid diets for such determinations may be preferable to protein-based diets. Also covered are brief summaries of the widely scattered literature on dietary and pharmacological aspects of 27 individual d-amino acids, d-peptides, and isomeric amino acid derivatives and suggested research needs in each of these areas. The described results provide a valuable record and resource for further progress on the multifaceted aspects of d-amino acids in food and biological samples.

Keywords: d-Amino acids; d-Peptides; Amino acid derivatives; Nutritional evaluation; Bioavailability; Toxicity

Operator- and software-related post-experimental variability and source of error in 2-DE analysis by Renato Millioni; Lucia Puricelli; Stefano Sbrignadello; Elisabetta Iori; Ellen Murphy; Paolo Tessari (pp. 1583-1590).

In the field of proteomics, several approaches have been developed for separating proteins and analyzing their differential relative abundance. One of the oldest, yet still widely used, is 2-DE. Despite the continuous advance of new methods, which are less demanding from a technical standpoint, 2-DE is still compelling and has a lot of potential for improvement. The overall variability which affects 2-DE includes biological, experimental, and post-experimental (software-related) variance. It is important to highlight how much of the total variability of this technique is due to post-experimental variability, which, so far, has been largely neglected. In this short review, we have focused on this topic and explained that post-experimental variability and source of error can be further divided into those which are software-dependent and those which are operator-dependent. We discuss these issues in detail, offering suggestions for reducing errors that may affect the quality of results, summarizing the advantages and drawbacks of each approach.

Keywords: 2-DE; Post-experimental variability; Pixel-based method; Spot-based method

Neurogenic differentiation of amniotic fluid stem cells by M. Rosner; M. Mikula; A. Preitschopf; M. Feichtinger; K. Schipany; M. Hengstschläger (pp. 1591-1596).

In 2003, human amniotic fluid has been shown to contain stem cells expressing Oct-4, a marker for pluripotency. This finding initiated a rapidly growing and very promising new stem cell research field. Since then, amniotic fluid stem (AFS) cells have been demonstrated to harbour the potential to differentiate into any of the three germ layers and to form three-dimensional aggregates, so-called embryoid bodies, known as the principal step in the differentiation of pluripotent stem cells. Marker selection and minimal dilution approaches allow the establishment of monoclonal AFS cell lineages with high proliferation potential. AFS cells have a lower risk for tumour development and do not raise the ethical issues of embryonic stem cells. Compared to induced pluripotent stem cells, AFS cells do not need exogenic treatment to induce pluripotency, are chromosomal stable and do not harbour the epigenetic memory and accumulated somatic mutations of specific differentiated source cells. Compared to adult stem cells, AFS can be grown in larger quantities and show higher differentiation potential. Accordingly, in the recent past, AFS became increasingly accepted as an optimal tool for basic research and probably also for specific cell-based therapies. Here, we review the current knowledge on the neurogenic differentiation potential of AFS cells.

Keywords: Amniotic fluid; Differentiation; Neurogenic; Neuron; Pluripotent; Stem cell

Metabolism of select amino acids in bacteria from the pig small intestine by Zhao-Lai Dai; Xi-Long Li; Peng-Bin Xi; Jing Zhang; Guoyao Wu; Wei-Yun Zhu (pp. 1597-1608).

This study investigated the metabolism of select amino acids (AA) in bacterial strains (Streptococcus sp., Escherichia coli and Klebsiella sp.) and mixed bacterial cultures derived from the jejunum and ileum of pigs. Cells were incubated at 37°C for 3 h in anaerobic media containing 0.5–5 mM select AA plus [U-¹⁴C]-labeled tracers to determine their decarboxylation and incorporation into bacterial protein. Results showed that all types of bacteria rapidly utilized glutamine, lysine, arginine and threonine. However, rates of the utilization of AA by pure cultures of E. coli and Klebsiella sp. were greater than those for mixed bacterial cultures or Streptococcus sp. The oxidation of lysine, threonine and arginine accounted for 10% of their utilization in these pure bacterial cultures, but values were either higher or lower in mixed bacterial cultures depending on AA, bacterial species and the gut segment (e.g., 15% for lysine in jejunal and ileal mixed bacteria; 5.5 and 0.3% for threonine in jejunal mixed bacteria and ileal mixed bacteria, respectively; and 20% for arginine in ileal mixed bacteria). Percentages of AA used for bacterial protein synthesis were 50–70% for leucine, 25% for threonine, proline and methionine, 15% for lysine and arginine and 10% for glutamine. These results indicate diverse metabolism of AA in small-intestinal bacteria in a species- and gut compartment-dependent manner. This diversity may contribute to AA homeostasis in the gut. The findings have important implications for both animal and human nutrition, as well as their health and well-beings.

Keywords: Small intestine; Amino acid metabolism; Bacteria; Metabolic fate; Swine

Theanine is a candidate amino acid for pharmacological stabilization of mast cells by N. H. Kim; H. J. Jeong; H. M. Kim (pp. 1609-1618).

The increasing occurrences of allergic disorders may be attributed to exposure to environmental factors that contribute to the pathogenesis of allergy. The health benefits of green tea have been widely reported but are largely unsubstantiated. Theanine is the major amino acid present in green tea. In this study, we investigated the role of theanine in both IgE- and non- IgE-induced allergic response. Theanine inhibited compound 48/80-induced systemic anaphylactic shock and ear swelling responses. IgE-mediated passive cutaneous anaphylaxis was inhibited by the oral administration or pharmaceutical acupuncture of theanine. Histamine release from mast cells was decreased with the treatment of theanine. Theanine also repressed phorbol 12-myristate 13-acetate and calcium ionophore A23187-induced TNF-α, IL-1β, IL-6, and IL-8 secretion by suppressing NF-κB activation. Furthermore, theanine suppressed the activation of caspase-1 and the expression of receptor interacting protein-2. The current study demonstrates for the first time that theanine might possess mast cell-stabilizing capabilities.

Keywords: Caspase-1; Histamine; Nuclear factor-κB; Receptor interacting protein-2; Theanine

Predicting subcellular location of apoptosis proteins with pseudo amino acid composition: approach from amino acid substitution matrix and auto covariance transformation by Xiaoqing Yu; Xiaoqi Zheng; Taigang Liu; Yongchao Dou; Jun Wang (pp. 1619-1625).

Apoptosis proteins are very important for understanding the mechanism of programmed cell death. Obtaining information on subcellular location of apoptosis proteins is very helpful to understand the apoptosis mechanism. In this paper, based on amino acid substitution matrix and auto covariance transformation, we introduce a new sequence-based model, which not only quantitatively describes the differences between amino acids, but also partially incorporates the sequence-order information. This method is applied to predict the apoptosis proteins’ subcellular location of two widely used datasets by the support vector machine classifier. The results obtained by jackknife test are quite promising, indicating that the proposed method might serve as a potential and efficient prediction model for apoptosis protein subcellular location prediction.

Keywords: Apoptosis proteins; Subcellular location; Substitution matrix; Auto covariance transformation; Support vector machine

Effect of Irbesartan treatment on plasma and urinary markers of protein damage in patients with type 2 diabetes and microalbuminuria by Naila Rabbani; Antonysunil Adaikalakoteswari; Kasper Rossing; Peter Rossing; Lise Tarnow; Hans-Henrik Parving; Paul J. Thornalley (pp. 1627-1639).

The aim of this study was to assess the effect of the angiotensin II receptor blocker Irbesartan on protein damage by glycation, oxidation and nitration in patients with type 2 diabetes and microalbuminuria. In a double-masked randomised crossover trial of 52 hypertensive type 2 diabetic patients, antihypertensive treatment was replaced with bendroflumethiazide. After 2-months wash-out, patients were treated randomly with Irbesartan 300, 600, and 900 mg o.d., each dose for 2 months in a three-way crossover study. Glycation, oxidation and nitration adduct residues in plasma protein and related urinary free adducts were determined by stable isotopic dilution analysis liquid chromatography–tandem mass spectrometry. Treatment with Irbesartan decreased urinary excretion of advanced glycation endproducts (AGEs)—methylglyoxal- and glyoxal-derived hydroimidazolones, MG-H1 and G-H1. Urinary AGEs were decreased by 30–32%. In plasma protein, treatment with Irbesartan increased content of glycation adducts N _ε-fructosyl-lysine, AGEs N _ε-carboxymethyl-lysine, N _ε-carboxyethyl-lysine and pentosidine, and also increased content of oxidation markers N-formylkynurenine and dityrosine. This was attributed to decreased clearance of plasma protein modified by N _ε-fructosyl-lysine and oxidative markers through the glomerular filter tightened by Irbesartan treatment. Treatment of patients with type 2 diabetes with Irbesartan decreased urinary excretion of MG-H1, G-H1 and 3-NT, which may result from decreased exposure to these AGEs. This is likely achieved by blocking angiotensin II signalling and related down-regulation of glyoxalase 1 and may contribute to health benefits of Irbesartan therapy.

Keywords: Diabetic nephropathy; Microalbuminuria; Angiotensin receptor blocker; Glycation; Oxidative stress; Nitrotyrosine; Methylglyoxal; Glycosylation gap

Purification and identification of antioxidant peptides from the skin protein hydrolysate of two marine fishes, horse mackerel (Magalaspis cordyla) and croaker (Otolithes ruber) by N. S. Sampath Kumar; R. A. Nazeer; R. Jaiganesh (pp. 1641-1649).

In the current study, two peptides with antioxidant properties were purified from skin protein hydrolysates of horse mackerel (Magalaspis cordyla) and croaker (Otolithes ruber) by consecutive chromatographic fractionations including ion exchange chromatography and gel filtration chromatography. By electron spray ionization double mass spectrometry (ESI-MS/MS), the sequence of the peptide from the skin protein hydrolysate of horse mackerel was identified to be Asn-His-Arg-Tyr-Asp-Arg (856 Da) and that of croaker to be Gly-Asn-Arg-Gly-Phe-Ala-Cys-Arg-His-Ala (1101.5 Da). The antioxidant activity of these peptides was tested by electron spin resonance (ESR) spectrometry using 1-diphenyl-2-picryl hydrazyl (DPPH^·) and hydroxyl (OH^·) radical scavenging assays. Both peptides exhibited higher activity against polyunsaturated fatty acid (PUFA) peroxidation than the natural antioxidant α-tocopherol. These results suggest that the two peptides isolated from the skin protein hydrolysates of horse mackerel and croaker are potent antioxidants and may be effectively used as food additives and as pharmaceutical agents.

Keywords: Horse mackerel; Croaker; Antioxidant peptide; In vitro digestion; Lipid peroxidation

A rapid and robust assay for the determination of the amino acid hypusine as a possible biomarker for a high-throughput screening of antimalarials and for the diagnosis and therapy of different diseases by Annette Kaiser; Alex R. Khomutov; Alina Simonian; Enzo Agostinelli (pp. 1651-1659).

Eukaryotic initiation factor 5A (eIF5A) has recently been identified as a biomarker of prognostic significance and therapeutic potential for the treatment in hepatocellular carcinoma. This prompted us to establish a rapid and robust assay to determine deoxyhypusine and hypusine formed with the purified enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) from Plasmodium to develop a rapid screening assay for antimalarial drugs. The peptide hydrolysate obtained from hypusinylated eIF5A was analyzed by ultra performance liquid chromatography (UPLC) with retention times for deoxyhypusine of 7.44 min and for hypusine of 7.30 min, respectively. The limit of detection for both compounds was 0.144 ng/μl. Determination of the specific activity of Plasmodium DOHH resulted in a twofold higher specific activity than its human counterpart. Following the iron-complexing strategy of the ferrous iron which is present in the active site of Plasmodium DOHH, a series of iron chelating compounds was tested. 2,2′-Dipyridyl and mimosine abolished DOHH activity completely while 4-oxo-piperidine-carboxylates i.e. the nitrophenylether JK8-2 and EHW 437, the oxime ether of the piperidine aldehyde, showed no inhibition although they were highly active in in vitro cultures of Plasmodium and in vivo in a rodent mouse model. The method allows a high-throughput screening (HPTS) of antimalarial drugs and the evaluation of eIF5A as a biomarker.

Keywords: Ultra performance liquid chromatography; Hypusine; Plasmodium ; Analytics of a biomarker

Drosophila as a platform to predict the pathogenicity of novel aminoacyl-tRNA synthetase mutations in CMT by Ricardo Leitão-Gonçalves; Biljana Ermanoska; An Jacobs; Els De Vriendt; Vincent Timmerman; James R. Lupski; Patrick Callaerts; Albena Jordanova (pp. 1661-1668).

Charcot-Marie-Tooth disease (CMT) is the major form of inherited peripheral neuropathy in humans. CMT is clinically and genetically heterogeneous and four aminoacyl-tRNA synthetases have been implicated in disease etiology. Mutations in the YARS gene encoding a tyrosyl-tRNA synthetase (TyrRS) lead to Dominant Intermediate CMT type C (DI-CMTC). Three dominant YARS mutations were so far associated with DI-CMTC. To further expand the spectrum of CMT causing genetic defects in this tRNA synthetase, we performed DNA sequencing of YARS coding regions in a cohort of 181 patients with various types of peripheral neuropathy. We identified a novel K265N substitution that in contrast to all previously described mutations is located at the anticodon recognition domain of the enzyme. Further genetic analysis revealed that this variant represents a benign substitution. Using our recently developed DI-CMTC Drosophila model, we tested in vivo the pathogenicity of this new YARS variant. We demonstrated that the developmental and behavioral defects induced by all DI-CMTC causing mutations were not present upon ubiquitous or panneuronal TyrRS K265N expression. Thus, in line with our genetic studies, functional analysis confirmed that the K265N substitution does not induce toxicity signs in Drosophila. The consistency observed throughout this work underscores the robustness of our DI-CMTC animal model and identifies Drosophila as a valid read-out platform to ascertain the pathogenicity of novel mutations to be identified in the future.

Keywords: CMT; Drosophila ; YARS; Disease model; Mutation

Modulation of mercury-induced mitochondria-dependent apoptosis by glycine in hepatocytes by Pabitra Bikash Pal; Sankhadeep Pal; Joydeep Das; Parames C. Sil (pp. 1669-1683).

Mercury (Hg) is one of the universal environmental pollutants and is responsible for various organ pathophysiology including oxidative stress-induced hepatic disorders. In the present study, we aimed to explore the protective role of glycine in Hg-induced cytotoxicity and cell death in murine hepatocytes. Exposure of mercury (20 μM), in the form HgCl₂ for 1 h, significantly enhanced the ALT and ALP leakage, increased reactive oxygen species production, reduced cell viability and distorted the antioxidant status of hepatocytes. Flow cytometric analyses shows that Hg-induced apoptotic death in hepatocytes. Mechanism of this pathophysiology involves reduced mitochondrial membrane potential, variations in Bcl-2/Bad proteins, activation of caspases and cleavage of PARP protein. In addition, Hg distinctly increased NF-κB phosphorylation in association with IKKα phosphorylation and IκBα degradation. Concurrent treatment with glycine (45 mM), however, reduced Hg-induced oxidative stress, attenuated the changes in NF-κB phosphorylation and protects hepatocytes from Hg-induced apoptotic death. Hg also distinctly increased the phosphorylation of p38, JNK and ERK mitogen-activated protein kinase (MAPKs). Glycine treatment suppressed these apoptotic events, signifying its protective role in Hg-induced hepatocyte apoptosis as referred by reduction of p38, JNK and ERK MAPK signaling pathways. Results suggest that glycine can modulate Hg-induced oxidative stress and apoptosis in hepatocytes probably because of its antioxidant activity and functioning via mitochondria-dependent pathways and could be a beneficial agent in oxidative stress-mediated liver diseases.

Keywords: Mercury; Oxidative stress; Reactive oxygen species; NF-κB; MAPKinases; Apoptosis; Glycine; Antioxidant

Diastereoselective Michael reaction of chiral nickel(II) glycinate with nitroalkenes for asymmetric synthesis of β-substituted α,γ-diaminobutyric acid derivatives in water by Jiang Wang; Xun Ji; Jianmei Shi; Haifeng Sun; Hualiang Jiang; Hong Liu (pp. 1685-1694).

We have developed the first operationally simple and environmentally benign protocol for the aqueous asymmetric Michael addition reaction of chiral nickel(II) glycinate with nitroalkenes. The reactions proceeded smoothly in the presence of TBAB (tetrabutyl ammonium bromide) in neat water at room temperature and provided good yields of β-substituted α,γ-diaminobutyric acid derivatives with excellent diastereoselectivities.

Keywords: Michael reaction; Nickel(II) glycinate; Asymmetric synthesis; Diaminobutyric acids

Effects of creatine supplementation on muscle wasting and glucose homeostasis in rats treated with dexamethasone by Humberto Nicastro; Bruno Gualano; Wilson Max Almeida Monteiro de Moraes; Vitor de Salles Painelli; Claudia Ribeiro da Luz; Andre dos Santos Costa; Fabiana de Salvi Guimarães; Alessandra Medeiros; Patricia Chakur Brum; Antonio Herbert Lancha Jr (pp. 1695-1701).

We aimed to investigate the possible role of creatine (CR) supplementation in counteracting dexamethasone-induced muscle wasting and insulin resistance in rats. Also, we examined whether CR intake would modulate molecular pathways involved in muscle remodeling and insulin signaling. Animals were randomly divided into four groups: (1) dexamethasone (DEX); (2) control pair-fed (CON-PF); (3) dexamethasone plus CR (DEX-CR); and (4) CR pair-fed (CR-PF). Dexamethasone (5 mg/kg/day) and CR (5 g/kg/day) were given via drinking water for 7 days. Plantaris and extensor digitorum longus (EDL) muscles were removed for analysis. Plantaris and EDL muscle mass were significantly reduced in the DEX-CR and DEX groups when compared with the CON-PF and CR-PF groups (P < 0.05). Dexamethasone significantly decreased phospho-Ser⁴⁷³-Akt protein levels compared to the CON-PF group (P < 0.05) and CR supplementation aggravated this response (P < 0.001). Serum glucose was significantly increased in the DEX group when compared with the CON-PF group (DEX 7.8 ± 0.6 vs. CON-PF 5.2 ± 0.5 mmol/l; P < 0.05). CR supplementation significantly exacerbated hyperglycemia in the dexamethasone-treated animals (DEX-CR 15.1 ± 2.4 mmol/l; P < 0.05 vs. others). Dexamethasone reduced GLUT-4 translocation when compared with the CON-PF and CR-PF (P < 0.05) groups and this response was aggravated by CR supplementation (P < 0.05 vs. others). In conclusion, supplementation with CR resulted in increased insulin resistance and did not attenuate muscle wasting in rats treated with dexamethasone. Given the contrast with the results of human studies that have shown benefits of CR supplementation on muscle atrophy and insulin sensitivity, we suggest caution when extrapolating this animal data to human subjects.

Keywords: Glucocorticoid; Atrophy; Glucose homeostasis; GLUT-4

Predicting sub-cellular localization of tRNA synthetases from their primary structures by Bharat Panwar; G. P. S. Raghava (pp. 1703-1713).

Since endo-symbiotic events occur, all genes of mitochondrial aminoacyl tRNA synthetase (AARS) were lost or transferred from ancestral mitochondrial genome into the nucleus. The canonical pattern is that both cytosolic and mitochondrial AARSs coexist in the nuclear genome. In the present scenario all mitochondrial AARSs are nucleus-encoded, synthesized on cytosolic ribosomes and post-translationally imported from the cytosol into the mitochondria in eukaryotic cell. The site-based discrimination between similar types of enzymes is very challenging because they have almost same physico-chemical properties. It is very important to predict the sub-cellular location of AARSs, to understand the mitochondrial protein synthesis. We have analyzed and optimized the distinguishable patterns between cytosolic and mitochondrial AARSs. Firstly, support vector machines (SVM)-based modules have been developed using amino acid and dipeptide compositions and achieved Mathews correlation coefficient (MCC) of 0.82 and 0.73, respectively. Secondly, we have developed SVM modules using position-specific scoring matrix and achieved the maximum MCC of 0.78. Thirdly, we developed SVM modules using N-terminal, intermediate residues, C-terminal and split amino acid composition (SAAC) and achieved MCC of 0.82, 0.70, 0.39 and 0.86, respectively. Finally, a SVM module was developed using selected attributes of split amino acid composition (SA-SAAC) approach and achieved MCC of 0.92 with an accuracy of 96.00%. All modules were trained and tested on a non-redundant data set and evaluated using fivefold cross-validation technique. On the independent data sets, SA-SAAC based prediction model achieved MCC of 0.95 with an accuracy of 97.77%. The web-server ‘MARSpred’ based on above study is available at http://www.imtech.res.in/raghava/marspred/ .

Keywords: Mitochondrial tRNA synthetase; Support vector machine; Prediction; MARSpred

Oostatic peptides containing d-amino acids: synthesis, oostatic activity, degradation, accumulation in ovaries and NMR study by Jan Hlaváček; Richard Tykva; Josef Holík; Blanka Bennettová; Miloš Buděšínský; Věra Vlasáková; Bohuslav Černý; Jiřina Slaninová (pp. 1715-1725).

Analogs of the H-Tyr-Asp-Pro-Ala-Pro-OH pentapeptide with d-amino acid residues either in differing or in all of the positions of the sequences were prepared and their oostatic potency was compared with that of the parent pentapeptide. The d-amino acid residue containing analogs exhibited an equal or even higher oostatic effect in the flesh fly Neobellieria bullata than the parent peptide. Contrary to the rapid incorporation of radioactivity from the labeled H-Tyr-Asp-[³H]Pro-Ala-Pro-OH pentapeptide into the ovaries of N. bullata in vitro, the radioactivity incorporation from the labeled pentapeptides with either d-aspartic acid or d-alanine was significantly delayed. As compared to the parent pentapeptide, also the degradation of both the d-amino acid-containing analogs mentioned above proceeded at a significantly lower rate. The decreased intake of radioactivity, the lower degradation and finally also the high oostatic effect may be ascribed to the decreased enzymatic degradation of the peptide bonds neighboring the d-amino acid residues in the corresponding peptides. The introduction of the non-coded d-amino acids thus enhances the oostatic effect in N. bullata owing to the prolonged half-life of the corresponding pentapeptides, which can thus affect more ovarian cells.

Keywords: d-amino acids; Oostatic peptide synthesis; ³H labeling; Oostatic activity in Neobellieria bullata ; ³H incorporation; Peptide degradation; NMR study

Synthesis of camptothecin–amino acid carbamate linkers by Marcus A. Etienne; Mikhail Kostochka; Joseph A. Fuselier; David H. Coy (pp. 1727-1733).

A more convenient and facile approach for the synthesis and production of camptothecin–amino acids carbamate linkers, that can be used in the synthesis of bioconjugate peptides JF-10-81, JF-10-71, and other peptide analogs designed to target somatostatin receptors has been described.

Keywords: Camptothecin; Somatostatin; Bioconjugates; Amino acids; Linkers

Anti-inflammatory mechanism of taurine against ischemic stroke is related to down-regulation of PARP and NF-κB by Ming Sun; Yumei Zhao; Yi Gu; Chao Xu (pp. 1735-1747).

Taurine is reported to reduce tissue damage induced by inflammation and to protect the brain against experimental stroke. The objective of this study was to investigate whether taurine reduced ischemic brain damage through suppressing inflammation related to poly (ADP-ribose) polymerase (PARP) and nuclear factor-kappaB (NF-κB) in a rat model of stroke. Rats received 2 h ischemia by intraluminal filament and were then reperfused. Taurine (50 mg/kg) was administered intravenously 1 h after ischemia. Treatment with taurine markedly reduced neurological deficits, lessened brain swelling, attenuated cell death, and decreased the infarct volume 72 h after ischemia. Our data showed the up-regulation of PARP and NF-κB p65 in cytosolic fractions in the core and nuclear fractions in the penumbra and core, and the increases in the nuclear poly (ADP-ribose) levels and the decreases in the intracellular NAD⁺ levels in the penumbra and core at 22 h of reperfusion; these changes were reversed by taurine. Moreover, taurine significantly reduced the levels of tumor necrosis factor-α, interleukin-1β, inducible nitric oxide synthase, and intracellular adhesion molecule-1, lessened the activities of myeloperoxidase and attenuated the infiltration of neutrophils in the penumbra and core at 22 h of reperfusion. These data demonstrate that suppressing the inflammatory reaction related to PARP and NF-κB-driven expression of inflammatory mediators may be one mechanism of taurine against ischemic stroke.

Keywords: Experimental stroke; Taurine; PARP; NF-κB; Inflammation

Using predicted shape string to enhance the accuracy of γ-turn prediction by Yaojuan Zhu; Tonghua Li; Dapeng Li; Yun Zhang; Wenwei Xiong; Jiangming Sun; Zehui Tang; Guanyan Chen (pp. 1749-1755).

Numerous methods for predicting γ-turns in proteins have been developed. However, the results they generally provided are not very good, with a Matthews correlation coefficient (MCC) ≤0.18. Here, an attempt has been made to develop a method to improve the accuracy of γ-turn prediction. First, we employ the geometric mean metric as optimal criterion to evaluate the performance of support vector machine for the highly imbalanced γ-turn dataset. This metric tries to maximize both the sensitivity and the specificity while keeping them balanced. Second, a predictor to generate protein shape string by structure alignment against the protein structure database has been designed and the predicted shape string is introduced as new variable for γ-turn prediction. Based on this perception, we have developed a new method for γ-turn prediction. After training and testing the benchmark dataset of 320 non-homologous protein chains using a fivefold cross-validation technique, the present method achieves excellent performance. The overall prediction accuracy Q _total can achieve 92.2% and the MCC is 0.38, which outperform the existing γ-turn prediction methods. Our results indicate that the protein shape string is useful for predicting protein tight turns and it is reasonable to use the dihedral angle information as a variable for machine learning to predict protein folding. The dataset used in this work and the software to generate predicted shape string from structure database can be obtained from anonymous ftp site ftp://cheminfo.tongji.edu.cn/GammaTurnPrediction/ freely.

Keywords: Imbalanced data; Shape string; Support vector machine (SVM); γ-Turn prediction

Substrate specificity of a peptidyl-aminoacyl-l/d-isomerase from frog skin by Alexander Jilek; Christa Mollay; Karl Lohner; Günther Kreil (pp. 1757-1764).

In the skin of fire-bellied toads (Bombina species), an aminoacyl-l/d-isomerase activity is present which catalyses the post-translational isomerization of the l- to the d-form of the second residue of its substrate peptides. Previously, this new type of enzyme was studied in some detail and genes potentially coding for similar polypeptides were found to exist in several vertebrate species including man. Here, we present our studies to the substrate specificity of this isomerase using fluorescence-labeled variants of the natural substrate bombinin H with different amino acids at positions 1, 2 or 3. Surprisingly, this enzyme has a rather low selectivity for residues at position 2 where the change of chirality at the alpha-carbon takes place. In contrast, a hydrophobic amino acid at position 1 and a small one at position 3 of the substrate are essential. Interestingly, some peptides containing a Phe at position 3 also were substrates. Furthermore, we investigated the role of the amino-terminus for substrate recognition. In view of the rather broad specificity of the frog isomerase, we made a databank search for potential substrates of such an enzyme. Indeed, numerous peptides of amphibia and mammals were found which fulfill the requirements determined in this study. Expression of isomerases with similar characteristics in other species can therefore be expected to catalyze the formation of peptides containing d-amino acids.

Keywords: d-amino acid; Chirality; Post-translational modification; Peptide biosynthesis; Amphibian skin

Asymmetric dimethylarginine in children with homocystinuria or phenylketonuria by Nele Kanzelmeyer; Dimitrios Tsikas; Kristine Chobanyan-Jürgens; Bibiana Beckmann; Bernhard Vaske; Sabine Illsinger; Anibh M. Das; Thomas Lücke (pp. 1765-1772).

Plasma concentration of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis from l-arginine and a cardiovascular risk factor, was found to be elevated in plasma of homocysteinemic adults. Enhanced cardiovascular risk due to homocystinuria and impaired renal function has been found in patients with phenylketonuria (PKU) on protein-restricted diet. However, it is still unknown whether ADMA synthesis is also elevated in children with homocystinuria due to cystathionine beta-synthase deficiency (classical homocystinuria), and whether ADMA may play a role in phenylketonuria in childhood. In the present study, we investigated the status of the l-arginine/NO pathway in six young patients with homocystinuria, in 52 young phenylketonuria patients on natural protein-restricted diet, and in age- and gender-matched healthy children serving as controls. ADMA in plasma and urine was determined by GC–MS/MS. The NO metabolites nitrate and nitrite in plasma and urine, and urinary dimethylamine (DMA), the dimethylarginine dimethylaminohydrolase (DDAH) metabolite of ADMA, were measured by GC–MS. Unlike urine ADMA excretion, plasma ADMA concentration in patients with homocystinuria was significantly higher than in controls (660 ± 158 vs. 475 ± 77 nM, P = 0.035). DMA excretion rate was considerably higher in children with homocystinuria as compared to controls (62.2 ± 24.5 vs. 6.5 ± 2.9 μmol/mmol creatinine, P = 0.068), indicating enhanced DDAH activity in this disease. In contrast and unexpectedly, phenylketonuria patients had significantly lower ADMA plasma concentrations compared to controls (512 ± 136 vs. 585 ± 125 nM, P = 0.009). Phenylketonuria patients and controls had similar l-arginine/ADMA molar ratios in plasma. Urinary nitrite excretion was significantly higher in phenylketonuria as compared to healthy controls (1.7 ± 1.7 vs. 0.7 ± 1.2 μmol/mmol creatinine, P = 0.003). Our study shows that the l-arginine/NO pathway is differently altered in children with phenylketonuria and homocystinuria. Analogous to hyperhomocysteinemic adults, elevated ADMA plasma concentrations could be a cardiovascular risk factor in children with homocystinuria. In phenylketonuria, the l-arginine/NO pathway seems not be altered. Delineation of the role of ADMA in childhood phenylketonuria and homocystinuria demands further investigation.

Keywords: ADMA; l-Arginine; Dimethylamine; Dimethylarginine dimethylaminohydrolase; Homocysteine; Nitric oxide

Wavelet transform analysis of NMR structure ensembles to reveal internal fluctuations of enzymes by Mei Hu; Yizhou Li; Gang Yang; Gongbing Li; Menglong Li; Zhining Wen (pp. 1773-1781).

Internal motions and flexibility are essential for biological functions in proteins. To assess the internal fluctuations and conformational flexibility of proteins, reliable computational methods are needed. In this study, wavelet transformation was used to filter out the noise and facilitate investigating the internal positional fluctuations of enzymes within nuclear magnetic resonance (NMR) structure ensembles. Moreover, potential active sites were identified by combining with positional fluctuation score, sequence conservation, and solvent accessible surface area. Among the total 107 catalytic residues in 44 examined enzymes, 69 residues were identified correctly. Our results suggest that wavelet transform analysis of structure ensemble is applicable to extract essential fluctuation information of proteins; furthermore, analysis of positional fluctuations is helpful for the identification of catalytic residues.

Keywords: Structures alignment; Mean square fluctuation; Potential active sites

l-Carnitine induces recovery of liver lipid metabolism in cancer cachexia by Renata Silvério; Alessandro Laviano; Filippo Rossi Fanelli; Marília Seelaender (pp. 1783-1792).

Cancer cachexia causes metabolic alterations with a marked effect on hepatic lipid metabolism. l-Carnitine modulates lipid metabolism and its supplementation has been proposed as a therapeutic strategy in many diseases. In the present study, the effects of l-carnitine supplementation on gene expression and on liver lipid metabolism-related proteins was investigated in cachectic tumour-bearing rats. Wistar rats were assigned to receive 1 g/kg of l-carnitine or saline. After 14 days, supplemented and control animals were assigned to a control (N), control supplemented with l-carnitine (CN), tumour-bearing Walker 256 carcinosarcoma (TB) and tumour-bearing supplemented with l-carnitine (CTB) group. The mRNA expression of carnitine palmitoyltransferase I and II (CPT I and II), microsomal triglyceride transfer protein (MTP), liver fatty acid-binding protein (L-FABP), fatty acid translocase (FAT/CD36), peroxisome proliferator-activated receptor-alpha (PPAR-alpha) and organic cation transporter 2 (OCTN2) was assessed, and the maximal activity of CPT I and II in the liver measured, along with plasma and liver triacylglycerol content. The gene expression of MTP, and CPT I catalytic activity were reduced in TB, who also showed increased liver (150%) and plasma (3.3-fold) triacylglycerol content. l-Carnitine supplementation was able to restore these parameters back to control values (p < 0.05). These data show that l-carnitine preserves hepatic lipid metabolism in tumour-bearing animals, suggesting its supplementation to be of potential interest in cachexia.

Keywords: l-Carnitine; Cachexia; Liver; Lipid metabolism; Steatosis

TG2 transamidating activity acts as a reostat controlling the interplay between apoptosis and autophagy by Federica Rossin; Manuela D’Eletto; Douglas Macdonald; Maria Grazia Farrace; Mauro Piacentini (pp. 1793-1802).

Tissue transglutaminase (TG2) activity has been implicated in inflammatory disease processes such as Celiac disease, infectious diseases, cancer, and neurodegenerative diseases, such as Huntington’s disease. Furthermore, four distinct biochemical activities have been described for TG2 including protein crosslinking via transamidation, GTPase, kinase and protein disulfide isomerase activities. Although the enzyme plays a complex role in the regulation of cell death and autophagy, the molecular mechanisms and the putative biochemical activity involved in each is unclear. Therefore, the goal of the present study was to determine how TG2 modulates autophagy and/or apoptosis and which of its biochemical activities is involved in those processes. To address this question, immortalized embryonic fibroblasts obtained from TG2 knock-out mice were reconstituted with either wild-type TG2 or TG2 lacking its transamidating activity and these were subjected to different treatments to induce autophagy or apoptosis. We found that knock out of the endogenous TG2 resulted in a significant exacerbation of caspase 3 activity and PARP cleavage in MEF cells subjected to apoptotic stimuli. Interestingly, the same cells showed the accumulation of LC3 II isoform following autophagy induction. These findings strongly suggest that TG2 transamidating activity plays a protective role in the response of MEF cells to death stimuli, because the expression of the wild-type TG2, but not its transamidation inactive C277S mutant, resulted in a suppression of caspase 3 as well as PARP cleavage upon apoptosis induction. Additionally, the same mutant was unable to catalyze the final steps in autophagosome formation during autophagy. Our findings clearly indicate that the TG2 transamidating activity is the primary biochemical function involved in the physiological regulation of both apoptosis and autophagy. These data also indicate that TG2 is a key regulator of cross-talk between autophagy and apoptosis.

Keywords: Transglutaminase 2; Apoptosis; Autophagy; Transamidating activity

Effects of red bull energy drink on repeated sprint performance in women athletes by Todd A. Astorino; Angela J. Matera; Jency Basinger; Mindy Evans; Taylor Schurman; Rodney Marquez (pp. 1803-1808).

Energy drinks are frequently consumed by athletes prior to competition to improve performance. This study examined the effect of Red Bull™ on repeated sprint performance in women athletes. Fifteen collegiate soccer players participated, with mean age, height, and body mass equal to 19.5 ± 1.1 year, 168.4 ± 5.8 cm, and 63.4 ± 6.1 kg, respectively. After performing a familiarization trial, subjects performed three sets of eight bouts of the modified t test after ingestion of 255 mL of placebo or Red Bull 1 h pre-exercise in a randomized, placebo-controlled crossover design. Throughout testing, sprint time, heart rate (HR), and rating of perceived exertion (RPE) were continuously obtained. Repeated measures analysis of variance was used to examine differences in variables between drink conditions. Across athletes, t test time ranged from 10.4 to 12.7 s. Mean sprint time was similar (p > 0.05) between Red Bull (11.31 ± 0.61 s) and placebo (11.35 ± 0.61 s). HR and RPE increased (p < 0.05) during the bouts, but there was no effect (p > 0.05) of Red Bull on either variable versus placebo. Findings indicate that 255 mL of Red Bull containing 1.3 mg/kg of caffeine and 1 g of taurine does not alter repeated sprint performance, RPE, or HR in women athletes versus placebo. One serving of this energy drink provides no ergogenic benefit for women athletes engaging in sprint-based exercise.

Keywords: Caffeine; Ergogenic; Sprint performance; Fatigue; Women

GPCR-MPredictor: multi-level prediction of G protein-coupled receptors using genetic ensemble by Muhammad Naveed; Asif Ullah Khan (pp. 1809-1823).

G protein-coupled receptors (GPCRs) are transmembrane proteins, which transduce signals from extracellular ligands to intracellular G protein. Automatic classification of GPCRs can provide important information for the development of novel drugs in pharmaceutical industry. In this paper, we propose an evolutionary approach, GPCR-MPredictor, which combines individual classifiers for predicting GPCRs. GPCR-MPredictor is a web predictor that can efficiently predict GPCRs at five levels. The first level determines whether a protein sequence is a GPCR or a non-GPCR. If the predicted sequence is a GPCR, then it is further classified into family, subfamily, sub-subfamily, and subtype levels. In this work, our aim is to analyze the discriminative power of different feature extraction and classification strategies in case of GPCRs prediction and then to use an evolutionary ensemble approach for enhanced prediction performance. Features are extracted using amino acid composition, pseudo amino acid composition, and dipeptide composition of protein sequences. Different classification approaches, such as k-nearest neighbor (KNN), support vector machine (SVM), probabilistic neural networks (PNN), J48, Adaboost, and Naives Bayes, have been used to classify GPCRs. The proposed hierarchical GA-based ensemble classifier exploits the prediction results of SVM, KNN, PNN, and J48 at each level. The GA-based ensemble yields an accuracy of 99.75, 92.45, 87.80, 83.57, and 96.17% at the five levels, on the first dataset. We further perform predictions on a dataset consisting of 8,000 GPCRs at the family, subfamily, and sub-subfamily level, and on two other datasets of 365 and 167 GPCRs at the second and fourth levels, respectively. In comparison with the existing methods, the results demonstrate the effectiveness of our proposed GPCR-MPredictor in classifying GPCRs families. It is accessible at http://111.68.99.218/gpcr-mpredictor/ .

Keywords: GPCRs; Support vector machine; Amino acid; Pseudo amino acid; Dipeptide compositions; GA-based ensemble; GPCR-MPredictor

Erratum to: GPCR-MPredictor: multi-level prediction of G protein-coupled receptors using genetic ensemble by Muhammad Naveed; Asifullah Khan (pp. 1825-1825).

Interactions of melatonin with mammalian mitochondria. Reducer of energy capacity and amplifier of permeability transition by P. Martinis; L. Zago; M. Maritati; V. Battaglia; S. Grancara; V. Rizzoli; E. Agostinelli; M. Bragadin; A. Toninello (pp. 1827-1837).

Melatonin, a metabolic product of the amino acid tryptophan, induces a dose-dependent energy drop correlated with a decrease in the oxidative phosphorylation process in isolated rat liver mitochondria. This effect involves a gradual decrease in the respiratory control index and significant alterations in the state 4/state 3 transition of membrane potential (ΔΨ). Melatonin, alone, does not affect the insulating properties of the inner membrane but, in the presence of supraphysiological Ca²⁺, induces a ΔΨ drop and colloid-osmotic mitochondrial swelling. These events are sensitive to cyclosporin A and the inhibitors of Ca²⁺ transport, indicative of the induction or amplification of the mitochondrial permeability transition. This phenomenon is triggered by oxidative stress induced by melatonin and Ca²⁺, with the generation of hydrogen peroxide and the consequent oxidation of sulfydryl groups, glutathione and pyridine nucleotides. In addition, melatonin, again in the presence of Ca²⁺, can also induce substantial release of cytochrome C and AIF (apoptosis-inducing factor), thus revealing its potential as a pro-apoptotic agent.

Keywords: Melatonin; Mitochondria; Permeability transition; Ca²⁺ ; Oxidative stress; Pro-apoptotic factors

Taurine protects rat testes against doxorubicin-induced oxidative stress as well as p53, Fas and caspase 12-mediated apoptosis by Joydeep Das; Jyotirmoy Ghosh; Prasenjit Manna; Parames C. Sil (pp. 1839-1855).

The protective effect of taurine against doxorubicin-induced testicular oxidative stress and apoptosis was investigated in rats. Male rats 8 weeks of age were treated with doxorubicin alone (3 mg/kg, i.p. every other day for 3 doses), taurine alone (150 mg/kg, i.p. every other day for 3 doses) or taurine plus doxorubicin (each dose given 1 day post-taurine). After 28 days, rat testes were collected and analysed. Rats treated with doxorubicin alone displayed reduced body and testicular weights, decreased sperm counts, increased the extent of testicular toxicity (as evident from the decreased activity of testicular marker enzyme, SDH) and oxidative stress (reduced GSH, increased GSSG and MDA levels), decreased antioxidant (SOD, CAT, GST, GPx, GR) and membrane-bound (Na⁺–K⁺ and Ca²⁺ ATPases) enzyme activities as well as plasma testosterone. Reverse transcriptase-PCR analysis revealed that doxorubicin induced a marked decrease in the expression of key enzymes for testicular androgenesis (3β-HSD, 17β-HSD) and testicular steroidogenic acute regulatory (StAR) protein. Western blot analysis showed that doxorubicin administration markedly increased the levels of caspase-9, 3, -8, -12, Fas, Bid and disturbed the Bcl-2 family protein balance. These results suggest that doxorubicin can trigger intrinsic, extrinsic and endoplasmic reticulum-associated apoptotic pathways in testicular pathophysiology. Doxorubicin also triggered activation of JNK, p38MAP kinases and p53. However, taurine could effectively prevent nearly all of these doxorubicin-induced testicular abnormalities, thereby proving to be an effective cytoprotectant.

Keywords: Doxorubicin; Testicular oxidative stress; MAPKs; p53; Apoptosis; Taurine; Antioxidant; Cell survival

Formation of 3-nitrotyrosine by riboflavin photosensitized oxidation of tyrosine in the presence of nitrite by Mario Fontana; Carla Blarzino; Laura Pecci (pp. 1857-1865).

The results of the present investigation show the susceptibility of tyrosine to undergo visible light-induced photomodification to 3-nitrotyrosine in the presence of nitrite and riboflavin, as sensitizer. By changing H₂O by D₂O, it could be established that singlet oxygen has a minor role in the reaction. The finding that nitration of tyrosine still occurred to a large extent under anaerobic conditions indicates that the process proceeds mainly through a type I mechanism, which involves the direct interaction of the excited state of riboflavin with tyrosine and nitrite to give tyrosyl radical and nitrogen dioxide radical, respectively. The tyrosyl radicals can either dimerize to yield 3,3′-dityrosine or combine with nitrogen dioxide radical to form 3-nitrotyrosine. The formation of 3-nitrotyrosine was found to increase with the concentration of nitrite added and was accompanied by a decrease in the recovery of 3,3′-dityrosine, suggesting that tyrosine nitration competes with dimerization reaction. The riboflavin photosensitizing reaction in the presence of nitrite was also able to induce nitration of tyrosine residues in proteins as revealed by the spectral changes at 430 nm, a characteristic absorbance of 3-nitrotyrosine, and by immunoreactivity using 3-nitrotyrosine antibodies. Since riboflavin and nitrite are both present endogenously in living organism, it is suggested that this pathway of tyrosine nitration may potentially occur in tissues and organs exposed to sunlight such as skin and eye.

Keywords: Tyrosine nitration; 3-Nitrotyrosine; Nitrite; Riboflavin; Photosensitizers; α-Crystallin; Elastin

Using Markov model to improve word normalization algorithm for biological sequence comparison by Qi Dai; Xiaoqing Liu; Yuhua Yao; Fukun Zhao (pp. 1867-1877).

There are two crucial problems with statistical measures for sequence comparison: overlapping structures and background information of words in biological sequences. Word normalization in improved composition vector method took into account these problems and achieved better performance in evolutionary analysis. The word normalization is desirable, but not sufficient, because it assumes that the four bases A, C, T, and G occur randomly with equal chance. This paper proposed an improved word normalization which uses Markov model to estimate exact k-word distribution according to observed biological sequence and thus has the ability to adjust the background information of the k-word frequencies in biological sequences. The improved word normalization was tested with three experiments and compared with the existing word normalization. The experiment results confirm that the improved word normalization using Markov model to estimate the exact k-word distribution in biological sequences is more efficient.

Keywords: Markov model; Word normalization; Sequence comparison; Classification; Phylogenetic analysis

Low amino acids affect expression of 11β-HSD2 in BeWo cells through leptin-activated JAK-STAT and MAPK pathways by Yueli Shang; Xiaojing Yang; Rui Zhang; Huafeng Zou; Ruqian Zhao (pp. 1879-1887).

Maternal protein restriction diminishes placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) activity and causes fetal growth restriction in mammals. However, it is unknown whether such effect was caused directly by nutrient deficiency, or indirectly through the mediation of maternal hormones. In the present study, a human placental cell line (BeWo) was cultured in F12K as control and F12 as low amino acids (LAA) media for 48 h to investigate the effects of amino acids deficiency on 11β-HSD2 expression and activity. Despite a significant up-regulation of 11β-HSD2 mRNA expression in LAA cells, 11β-HSD2 activity and protein content were decreased by 38 and 54%, respectively (P < 0.05), indicating a mechanism of post-transcriptional regulation. Among 5 miRNAs targeting 11β-HSD2, miR-498 was expressed significantly higher in LAA cells. Leptin concentration was significantly lower (P < 0.01) in LAA medium. The mRNA expression of both isoforms of leptin receptor was significantly higher in LAA cells, although no difference was detected at protein level. To further clarify whether leptin is involved in mediating the effect of LAA on 11β-HSD2 activity, leptin was supplemented to LAA medium, whereas three specific inhibitors of leptin signaling pathways, WP1066 for JAK-STAT, PD98059 for MAPK and LY294002 for PI3K, respectively were added to control medium. Leptin restored the diminished 11β-HSD2 activity in LAA cells, whereas WP1066 (5 nM) and PD98059 (50 nM) significantly decreased 11β-HSD2 activity in control cells. In conclusion, the present results indicate that LAA diminishes 11β-HSD2 expression and activity in BeWo cells through leptin-activated JAK-STAT and MAPK pathways.

Keywords: 11β-HSD2; Leptin; miRNA; Low amino acids; BeWo cells

A simple method for the analysis by MS/MS of underivatized amino acids on dry blood spots from newborn screening by Chunyan Wang; Wenyan Zhang; Fengrui Song; Zhiqiang Liu; Shuying Liu (pp. 1889-1895).

The analysis by electrospray-ionization tandem mass spectrometry of amino acids with butyl esterification and isotopically labeled internal standard is routine in newborn screening laboratories worldwide. In the present study, we established a direct analysis method of higher accuracy that uses a non-deuterated internal standard. The automatic sampler and the pump of an LC apparatus were used to inject sample and mobile phase to MS, but no LC column was needed. The dry blood spot (DBS) material was prepared at levels of low, medium and high concentration; the running time was 1 min. In parallel to the new procedure, we applied the established method to analyze nine amino acids on DBS of healthy newborns and phenylketonuria newborns. The newly proposed method of product ion confirmation scan along with multiple reaction monitoring resulted in a very accurate identification of each amino acid. Our innovative protocol had high sensitivity and specificity in the analysis of cases of suspected metabolic diseases.

Keywords: Newborn screening; Blood spot; Underivatized amino acids; Electrospray-ionization tandem mass spectrometry

Rapid determination of amino acids in biological samples using a monolithic silica column by Yanting Song; Takashi Funatsu; Makoto Tsunoda (pp. 1897-1902).

A high-performance liquid chromatography method in which fluorescence detection is used for the simultaneous determination of 21 amino acids is proposed. Amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and then separated on a monolithic silica column (MonoClad C18-HS, 150 mm × 3 mm i.d.). A mixture of 25 mM citrate buffer containing 25 mM sodium perchlorate (pH 5.5) and acetonitrile was used as the mobile phase. We found that the most significant factor in the separation was temperature, and a linear temperature gradient from 30 to 49°C was used to control the column temperature. The limits of detection and quantification for all amino acids ranged from 3.2 to 57.2 fmol and 10.8 to 191 fmol, respectively. The calibration curves for the NBD-amino acid had good linearity within the range of 40 fmol to 40 pmol when 6-aminocaproic acid was used as an internal standard. Using only conventional instruments, the 21 amino acids could be analyzed within 10 min. This method was found to be suitable for the quantification of the contents of amino acids in mouse plasma and adrenal gland samples.

Keywords: Plasma; Adrenal gland; 4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F); Temperature gradient

Cellular hypomethylation is associated with impaired nitric oxide production by cultured human endothelial cells by M. Barroso; M. S. Rocha; R. Esse; I. Gonçalves Jr.; A. Q. Gomes; T. Teerlink; C. Jakobs; H. J. Blom; J. Loscalzo; I. Rivera; I. Tavares de Almeida; R. Castro (pp. 1903-1911).

Hyperhomocysteinemia (HHcy) is a risk factor for vascular disease, but the underlying mechanisms remain incompletely defined. Reduced bioavailability of nitric oxide (NO) is a principal manifestation of underlying endothelial dysfunction, which is an initial event in vascular disease. Inhibition of cellular methylation reactions by S-adenosylhomocysteine (AdoHcy), which accumulates during HHcy, has been suggested to contribute to vascular dysfunction. However, thus far, the effect of intracellular AdoHcy accumulation on NO bioavailability has not yet been fully substantiated by experimental evidence. The present study was carried out to evaluate whether disturbances in cellular methylation status affect NO production by cultured human endothelial cells. Here, we show that a hypomethylating environment, induced by the accumulation of AdoHcy, impairs NO production. Consistent with this finding, we observed decreased eNOS expression and activity, but, by contrast, enhanced NOS3 transcription. Taken together, our data support the existence of regulatory post-transcriptional mechanisms modulated by cellular methylation potential leading to impaired NO production by cultured human endothelial cells. As such, our conclusions may have implications for the HHcy-mediated reductions in NO bioavailability and endothelial dysfunction.

Keywords: S-adenosylhomocysteine; Methylation; Endothelial dysfunction; Nitric oxide bioavailability; Endothelial nitric oxide synthase

On the Thermus thermophilus HB8 potential pathogenicity triggered from rhamnolipids secretion: morphological alterations and cytotoxicity induced on fibroblastic cell line by A. A. Pantazaki; T. Choli-Papadopoulou (pp. 1913-1926).

A limited number of bacterial strains usually grown under nutrient limitation secrete rhamnolipids (RLs), which are recorded as virulence factors that are implicated in the pathogenicity of a microorganism. The non-pathogenic T. thermophilus HB8 produces extracellular rhamnolipids (TthRLs) under defined cultivation conditions using sunflower seed oil and sodium gluconate as carbon sources. In particular, the secreted TthRLs have been isolated, purified and identified with ATR–FTIR. Their effects on the cells’ viability were examined when they were supplemented in a culture of human skin fibroblasts. Purified TthRLs triggered a sequence of rapid and pronounced morphological alterations characterized by transformation of fibroblast shape from polygonal to fusiform; retraction with cytoplasm condensation, rounding up, distortion of nuclei and loss of lamellar processes, and finally disruption of membrane. The addition of TthRLs in the cultured fibroblasts caused cytotoxicity, in contrast to that of rhamnose that stimulated viability, as it was assessed by MTT test. These results revealed that among the constituents of RLs that are implicated in the cytotoxicity, it has to be attributed to the lipidic chain variation and not to the carbohydrate part. TthRLs cytotoxicity on fibroblasts is comparable, and provoked similar effects, to that caused by saponin white, a known surfactant. TthRLs secretion might be a crucial point for the transformation of a non-pathogenic bacterium to a pathogenic one under certain environmental conditions favoring their secretion. RLs secretion in the microorganism’s world might be a general route for the passage in the pathogenicity to ensure their survival under nutrient limitation conditions.

Keywords: Rhamnolipids; Thermus thermophilus ; Fibroblasts; MTT cytotoxicity; Morphological alterations

Taurine in adipocytes prevents insulin-mediated H₂o₂ generation and activates Pka and lipolysis by Guadalupe Piña-Zentella; Gilda de la Rosa-Cuevas; Héctor Vázquez-Meza; Enrique Piña; Martha Zentella de Piña (pp. 1927-1935).

Among many actions assigned to taurine (Tau), the most abundant amino acid in numerous mammalian tissues, it prevents high-fat diet-induced obesity with increasing resting energy expenditure. To sustain this Tau action, the goal of the present study was to explore the acute effects of Tau on baseline and on adrenaline, insulin and their second messengers to modulate lipolysis in white adipose tissue (WAT) cells from rat epididymis. The Tau effects in this topic were compared with those recorded with Gly, Cys and Met. Tau on its own did not modify baseline lipolysis. Tau raised isoproterenol- and dibutyryl-cAMP (Bt₂cAMP)-activated glycerol release. Gly diminished Bt₂cAMP-activated glycerol release, and Cys and Met had no effect. Cyclic AMP-dependent activation of protein kinase A (PKA) in cell-free extracts decreased slightly by Gly and was unaltered by Cys, Met, and Tau. PKA catalytic activity in cell-free extracts was stimulated by Tau and unchanged by Cys, Gly and Met. Gly and Tau effects on PKA disappeared when these amino acids were withdrawn by gel filtration. Insulin-mediated NADPH-oxidase (NOX) raises H₂O₂ pool, which promotes PKA subunit oxidation, and precludes its cAMP activation; thus, lipolysis is diminished. Tau, but not Cys, Gly and Met, inhibited, by as much as 70%, insulin-mediated H₂O₂ pool increase. These data suggested that Tau raised lipolysis in adipocytes by two mechanisms: stimulating cAMP-dependent PKA catalytic activity and favoring PKA activation by cAMP as a consequence of lowering the H₂O₂ pool.

Keywords: Hydrogen peroxide; Insulin; Lipolysis; Obesity; PKA; Taurine

N-substituted aminomethanephosphonic and aminomethane-P-methylphosphinic acids as inhibitors of ureases by Łukasz Berlicki; Marta Bochno; Agnieszka Grabowiecka; Arkadiusz Białas; Paulina Kosikowska; Paweł Kafarski (pp. 1937-1945).

Small unextended molecules based on the diamidophosphate structure with a covalent carbon-to-phosphorus bond to improve hydrolytic stability were developed as a novel group of inhibitors to control microbial urea decomposition. Applying a structure-based inhibitor design approach using available crystal structures of bacterial urease, N-substituted derivatives of aminomethylphosphonic and P-methyl-aminomethylphosphinic acids were designed and synthesized. In inhibition studies using urease from Bacillus pasteurii and Canavalia ensiformis, the N,N-dimethyl derivatives of both lead structures were most effective with dissociation constants in the low micromolar range (K i = 13 ± 0.8 and 0.62 ± 0.09 μM, respectively). Whole-cell studies on a ureolytic strain of Proteus mirabilis showed the high efficiency of N,N-dimethyl and N-methyl derivatives of aminomethane-P-methylphosphinic acids for urease inhibition in pathogenic bacteria. The high hydrolytic stability of selected inhibitors was confirmed over a period of 30 days using NMR technique.

Keywords: Urease inhibition; Transition state analogues; Diamidophosphate; Aminophosphonic acid; Aminophosphinic acid

Detecting thermophilic proteins through selecting amino acid and dipeptide composition features by Songyot Nakariyakul; Zhi-Ping Liu; Luonan Chen (pp. 1947-1953).

Detecting thermophilic proteins is an important task for designing stable protein engineering in interested temperatures. In this work, we develop a simple but efficient method to classify thermophilic proteins from mesophilic ones using the amino acid and dipeptide compositions. Since most of the amino acid and dipeptide compositions are redundant, we propose a new forward floating selection technique to select only a useful subset of these compositions as features for support vector machine-based classification. We test the proposed method on a benchmark data set of 915 thermophilic and 793 mesophilic proteins. The results show that our method using 28 amino acid and dipeptide compositions achieves an accuracy rate of 93.3% evaluated by the jackknife cross-validation test, which is higher not only than the existing methods but also than using all amino acid and dipeptide compositions.

Keywords: Amino acid composition; Dipeptide composition; Feature selection; Floating search method; Protein thermostability

Synthesis of the amino acid conjugates of epi-jasmonic acid by N. Ogawa; Y. Kobayashi (pp. 1955-1966).

The TES ether of the C6-hydroxy derivative of naturally occurring epi-jasmonic acid (epi-JA) was designed as epimerization-free equivalent of epi-JA. The TES ether was synthesized from (1R,4S)-4-hydroxycyclopent-2-enyl acetate in 13 steps. The acid part of the ether was activated with ClCO₂Bu i and subjected to condensation with l-amino acid at room temperature for 48 h. The TES group in the condensation product was removed in HCO₂H (0°C, 30 min) and the resulting hydroxyl group was oxidized with Jones reagent (acetone, 0°C, 30 min) to furnish the amino acid conjugate of epi-JA. The amino acids examined are l-isoleucine, l-leucine, l-alanine, l-valine, and d-allo-isoleucine, which afforded the conjugates in 48–68% yields with 89–96% diastereomeric purity over the trans isomers. Similarly, the possible three stereoisomers of epi-JA were condensed with l-isoleucine successfully, producing the corresponding stereoisomers in good yields.

Keywords: epi-Jasmonic acid; Amino acid conjugate; Isoleucine; Asymmetric synthesis; Stereoselective

An investigation into possible xenobiotic–endobiotic inter-relationships involving the amino acid analogue drug, S-carboxymethyl-l-cysteine and plasma amino acids in humans by Glyn B. Steventon; Stephen C. Mitchell; Santigo Angulo; Coral Barbas (pp. 1967-1973).

The amino acid derivative, S-carboxymethyl-l-cysteine, is an anti-oxidant agent extensively employed as adjunctive therapy in the treatment of human pulmonary conditions. A major biotransformation route of this drug, which displays considerable variation in capacity in man, involves the oxidation of the sulfide moiety to the inactive S-oxide metabolite. Previous observations have indicated that fasted plasma l-cysteine concentrations and fasted plasma l-cysteine/free inorganic sulfate ratios were correlated with the degree of sulfoxidation of this drug and that these particular parameters may be used as endobiotic biomarkers for this xenobiotic metabolism. It has been proposed also that the enzyme, cysteine dioxygenase, was responsible for the drug sulfoxidation. Further in this theme, the degree of S-oxidation of S-carboxymethyl-l-cysteine in 100 human volunteers was investigated with respect to it potential correlation with fasted plasma amino acid concentrations. Extensive statistical analyses showed no significant associations or relationships between the degree of drug S-oxidation and fasted plasma amino acid concentrations, especially with respect to the sulfur-containing compounds, methionine, l-cysteine, l-cysteine sulfinic acid, taurine and free inorganic sulfate, also the derived ratios of l-cysteine/l-cysteine sulfinic acid and l-cysteine/free inorganic sulfate. It was concluded that plasma amino acid levels or derived ratios cannot be employed to predict the degree of S-oxidation of S-carboxymethyl-l-cysteine (or vice versa) and that it is doubtful if the enzyme, cysteine dioxygenase, has any involvement in the metabolism of this drug.

Keywords: Metabolomics; S-oxidation; S-carboxymethyl-l-cysteine; l-Cysteine/sulfate ratio; Cysteine dioxygenase

Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of ^99mTc-labeled recombinant Affibody molecules by Mohamed Altai; Helena Wållberg; Anna Orlova; Maria Rosestedt; Seyed Jalal Hosseinimehr; Vladimir Tolmachev; Stefan Ståhl (pp. 1975-1985).

Affibody molecules constitute a novel class of molecular display selected affinity proteins based on non-immunoglobulin scaffold. Preclinical investigations and pilot clinical data have demonstrated that Affibody molecules provide high contrast imaging of tumor-associated molecular targets shortly after injection. The use of cysteine-containing peptide-based chelators at the C-terminus of recombinant Affibody molecules enabled site-specific labeling with the radionuclide ^99mTc. Earlier studies have demonstrated that position, composition and the order of amino acids in peptide-based chelators influence labeling stability, cellular processing and biodistribution of Affibody molecules. To investigate the influence of the amino acid order, a series of anti-HER2 Affibody molecules, containing GSGC, GEGC and GKGC chelators have been prepared and characterized. The affinity to HER2, cellular processing of ^99mTc-labeled Affibody molecules and their biodistribution were investigated. These properties were compared with that of the previously studied ^99mTc-labeled Affibody molecules containing GGSC, GGEC and GGKC chelators. All variants displayed picomolar affinities to HER2. The substitution of a single amino acid in the chelator had an appreciable influence on the cellular processing of ^99mTc. The biodistribution of all ^99mTc-labeled Affibody molecules was in general comparable, with the main difference in uptake and retention of radioactivity in excretory organs. The hepatic accumulation of radioactivity was higher for the lysine-containing chelators and the renal retention of ^99mTc was significantly affected by the amino acid composition of chelators. The order of amino acids influenced renal uptake of some conjugates at 1 h after injection, but the difference decreased at later time points. Such information can be helpful for the development of other scaffold protein-based imaging and therapeutic radiolabeled conjugates.

Keywords: Affibody molecule; Technetium-99m; Molecular imaging; HER2; C-terminal cysteine; Peptide-based chelator

Effect of leucine supplementation on indices of muscle damage following drop jumps and resistance exercise by Tyler J. Kirby; N. Travis Triplett; Tracie L. Haines; Jared W. Skinner; Kimberly R. Fairbrother; Jeffrey M. McBride (pp. 1987-1996).

The purpose of this study was to determine the effect of leucine supplementation on indices of muscle damage following eccentric-based resistance exercise. In vitro, the amino acid leucine has been shown to reduce proteolysis and stimulate protein synthesis. Twenty-seven untrained males (height 178.6 ± 5.5 cm; body mass 77.7 ± 13.5 kg; age 21.3 ± 1.6 years) were randomly divided into three groups; leucine (L) (n = 10), placebo (P) (n = 9) and control (C) (n = 8). The two experimental groups (L and P) performed 100 depth jumps from 60 cm and six sets of ten repetitions of eccentric-only leg presses. Either leucine (250 mg/kg bm) or placebo was ingested 30 min before, during and immediately post-exercise and the morning of each recovery day following exercise. Muscle function was determined by peak force during an isometric squat and by jump height during a static jump at pre-exercise (PRE) and 24, 48, 72, and 96 h post-exercise (24, 48, 72, 96 h). Additionally, at these time points each group’s serum levels of creatine kinase (CK) and myoglobin (Mb) along with perceived feelings of muscle soreness were determined. None of the C group dependent variables was altered by the recurring testing procedures. Peak force was significantly decreased across all time points for both experimental groups. The L group experienced an attenuated drop in mean peak force across all post-exercise time points compared to the P group. Jump height significantly decreased from PRE for both the L and P group at 24 h and 48 h. CK and Mb was significantly elevated from PRE for both experimental groups at 24 h. Muscle soreness increased across all time points for the both the L and P group, and the L group experienced a significantly higher increase in mean muscle soreness post-exercise. Following exercise-induced muscle damage, high-dose leucine supplementation may help maintain force output during isometric contractions, however, not force output required for complex physical tasks thereby possibly limiting its ergogenic effectiveness.

Keywords: Creatine kinase; Myoglobin; Muscle soreness; Force output

Fabrication of biodegradable poly(ester-amide)s based on tyrosine natural amino acid by Amir Abdolmaleki; Shadpour Mallakpour; Sedigheh Borandeh; Mohammad R. Sabzalian (pp. 1997-2007).

N,N′-Bis[2-(methyl-3-(4-hydroxyphenyl)propanoate)]isophthaldiamide (5), a novel diol monomer containing chiral group, was prepared by the reaction of S-tyrosine methyl ester (3) with isophthaloyl dichloride (4a). A new family of optically active and potentially biodegradable poly(ester-amide)s (PEAs) based on tyrosine amino acid were prepared by the polycondensation reaction of diol monomer 5 with several aromatic diacid chlorides. The resulting new polymers were obtained in good yields with inherent viscosities ranging between 0.25 and 0.42 dL/g and are soluble in polar aprotic solvents. They showed good thermal stability and high optical purity. The synthetic compounds were characterized and studied by FT-IR, ¹H-NMR, specific rotation, elemental and thermogravimetric analysis (TGA) techniques and typical ones by ¹³C-NMR, differential scanning calorimetry (DSC), X-ray diffraction (XRD), and field emission scanning electron microscopy (FE-SEM) analysis. Soil burial test of the diphenolic monomer 5, and obtained PEA6a, and soil enzymatic assay showed that the synthesized diol and its polymer are biologically active and probably biodegradable in soil environment.

Keywords: Optically active polymer; Tyrosine amino acid; Biodegradable; Poly(ester-amide); Polycondensation

Characterization of MRNP34, a novel methionine-rich nacre protein from the pearl oysters by Benjamin Marie; Caroline Joubert; Corinne Belliard; Alexandre Tayale; Isabelle Zanella-Cléon; Frédéric Marin; Yannick Gueguen; Caroline Montagnani (pp. 2009-2017).

Nacre of the Pinctada pearl oyster shells is composed of 98% CaCO₃ and 2% organic matrix. The relationship between the organic matrix and the mechanism of nacre formation currently constitutes the main focus regarding the biomineralization process. In this study, we isolated a new nacre matrix protein in P. margaritifera and P. maxima, we called Pmarg- and Pmax-MRNP34 (methionine-rich nacre protein). MRNP34 is a secreted hydrophobic protein, which is remarkably rich in methionine, and which is specifically localised in mineralizing the epithelium cells of the mantle and in the nacre matrix. The structure of this protein is drastically different from those of the other nacre proteins already described. This unusual methionine-rich protein is a new member in the growing list of low complexity domain containing proteins that are associated with biocalcifications. These observations offer new insights to the molecular mechanisms of biomineralization.

Keywords: Matrix protein; Methionine-rich; Biomineralization; Mollusc; Nacre; Calcifying mantle

Electrostatics of folded and unfolded bovine β-lactoglobulin by Ivano Eberini; Cristina Sensi; Alberto Barbiroli; Franco Bonomi; Stefania Iametti; Monica Galliano; Elisabetta Gianazza (pp. 2019-2030).

We report on electrophoretic, spectroscopic, and computational studies aimed at clarifying, at atomic resolution, the electrostatics of folded and unfolded bovine β-lactoglobulin (BLG) with a detailed characterization of the specific aminoacids involved. The procedures we used involved denaturant gradient gel electrophoresis, isoelectric focusing, electrophoretic titration curves, circular dichroism and fluorescence spectra in the presence of increasing concentrations of urea (up to 8 M), electrostatics computations and low-mode molecular dynamics. Discrepancy between electrophoretic and spectroscopic evidence suggests that changes in mobility induced by urea are not just the result of changes in gyration radius upon unfolding. Electrophoretic titration curves run across a pH range of 3.5–9 in the presence of urea suggest that more than one aminoacid residue may have anomalous pK _a value in native BLG. Detailed computational studies indicate a shift in pKa of Glu44, Glu89, and Glu114, mainly due to changes in global and local desolvation. For His161, the formation of hydrogen bond(s) could add up to desolvation contributions. However, since His161 is at the C terminus, the end-effect associated to the solvated form strongly influences its pK _a value with extreme variation between crystal structures on one side and NMR or low-mode molecular dynamics structures on the other. The urea concentration effective in BLG unfolding depends on pH, with higher stability of the protein at lower pH.

Keywords: Bovine β-lactoglobulin; Electrostatics; Unfolding; Urea; Electrophoresis; Molecular modeling

Comparisons of treatment means when factors do not interact in two-factorial studies by Jiawei Wei; Raymond J. Carroll; Kathryn K. Harden; Guoyao Wu (pp. 2031-2035).

Scientists in the fields of nutrition and other biological sciences often design factorial studies to test the hypotheses of interest and importance. In the case of two-factorial studies, it is widely recognized that the analysis of factor effects is generally based on treatment means when the interaction of the factors is statistically significant, and involves multiple comparisons of treatment means. However, when the two factors do not interact, a common understanding among biologists is that comparisons among treatment means cannot or should not be made. Here, we bring this misconception into the attention of researchers. Additionally, we indicate what kind of comparisons among the treatment means can be performed when there is a nonsignificant interaction among two factors. Such information should be useful in analyzing the experimental data and drawing meaningful conclusions.

Keywords: Factor level means; Interaction; Main effects; Multiple comparison; Treatment means; Two-factor studies

May GABA transaminase inhibitors improve stereotyped behaviors in Rett syndrome? by Ahmad Ghanizadeh (pp. 2037-2038).

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.42, #5)