Amino Acids (v.35, #2)

DNA cleavage function of seryl-histidine dipeptide and its application by Y. Ma; X. Chen; M. Sun; R. Wan; C. Zhu; Y. Li; Y. Zhao (251-256).
The double-stranded DNA or circular plasmid DNA can be cleaved by the Ser-His dipeptide by hydrolysis of the DNA-phospho-diester bond. The proper sequential order of the amino acids serine and histidine is apparently crucial in its unique cleavage activity as compared to the other di- or tri-peptides containing one of these amino acids. An inverted sequence of this dipeptide to a His-Ser linkage renders the peptide ineffective in the cleavage of DNA. In addition to the DNA cleavage function, Ser-His is also capable of cleaving other molecules, e.g., proteins, esters and RNAs. The cooperative actions of the hydroxyl group and the basic groups in the serine and histidine or related amino acids can be found in contemporary enzymes, such as DNase, serine proteases, lipases, esterases, chymotrypsin, trypsin, and elastase, etc. The Ser-His and related oligopeptides might have played important roles in the evolution of enzyme functions.
Keywords: Keywords: Seryl-histidine dipeptide – DNA cleavage function – Evolution of enzymes

β-Lactoglobulin as source of bioactive peptides by B. Hernández-Ledesma; I. Recio; L. Amigo (257-265).
β-Lactoglobulin (β-Lg) is currently an important source of biologically active peptides. These peptides are inactive within the sequence of the precursor protein, but they can be released by in vivo or in vitro enzymatic proteolysis. Once released, these peptides play important roles in the human health, including antihypertensive, antioxidant and antimicrobial activities as well as opioid-like features and ability to decrease the body-cholesterol levels. Bioactive peptides derived from β-Lg are currently a point of intensive research. Their structure, biological significance and mechanism of action are briefly presented and discussed in this review.
Keywords: Keywords: β-Lactoglobulin – Bioactive peptides – Physiological activity

Reversible protein phosphorylation plays a crucial role in the regulation of signaling pathways that control various biological responses, such as cell growth, differentiation, invasion, metastasis and apoptosis. Proteomics is a powerful research approach for fully monitoring global molecular responses to the activation of signal transduction pathways. Identification of different phosphoproteins and their phosphorylation sites by functional proteomics provides informational insights into signaling pathways triggered by all kinds of factors. This review summarizes how functional proteomics can be used to answer specific questions related to signal transduction systems of interest. By examining our own example on identifying the novel phosphoproteins in signaling pathways activated by EB virus-encoded latent membrane protein 1 (LMP1), we demonstrated a functional proteomic strategy to elucidate the molecular activity of phosphorylated annexin A2 in LMP1 signaling pathway. Functional profiling of signaling pathways is promising for the identification of novel targets for drug discovery and for the understanding of disease pathogenesis.
Keywords: Keywords: Proteomics – Phosphoproteomics – Signal transduction – Phosphorylation

Experimental study on vasoactive intestinal peptide (VIP) and its diaminopropane bound (VIP-DAP) analog in solution by M. Caraglia; M. Carteni; A. Dicitore; D. Cassese; S. De Maria; P. Ferranti; G. Giuberti; A. Abbruzzese; P. Stiuso (275-281).
Bioactive peptides represent an exciting area of research in the fields of biochemistry and medicine and in particular the VIP/PACAP network appears to be of interest. Vasoactive intestinal peptide (VIP) is a pleiotropic factor that exerts a physiological regulatory influence and is involved in the pathogenesis of several human disorders. In this paper we have reported structural characterization of VIP by experimental and computational methods as well as a comparative analysis of the peptide with its transglutaminase catalyzed analog VIP-Diaminopropane (VIP-DAP).
Keywords: Keywords: Vasoactive intestinal peptide (VIP) – Transglutaminase (TGase) – Limited proteolysis – Computational study – VPAC receptors

Focus on cyclo(His-Pro): history and perspectives as antioxidant peptide by A. Minelli; I. Bellezza; S. Grottelli; F. Galli (283-289).
Cyclo(His-Pro) is an endogenous cyclic dipeptide structurally related to tyreotropin-releasing hormone that was originally discovered in brain. In the central nervous system it has been described to exert multiple biological activities, which seem to be related to a presynaptic dopaminergic mechanism and include among the others a leptin-like function. It can be found in several body fluids and in the gastrointestinal tract where it has been suggested to act as a gut peptide with influence on the entero-insular axis. The oral administration of cyclo(His-Pro) and zinc was described to improve with a synergistic mechanism the glycaemic control in diabetes.The most intriguing function of this cyclic dipeptide is related with its neuroprotective role that was first reported in traumatic injuries of the spinal cord, and then confirmed in other models of experimental injuries of the nervous system. The mechanism that lies behind the neuroprotective activity of cyclo(His-Pro) remain poorly understood. Recent in vitro studies on rat pheochromocytoma PC12 cells have shown that it is a protective factor against stress stimuli and there is early pre-clinical evidence strongly suggesting that it enhances the expression of small heat shock proteins and antioxidant protection at the cellular level.Future research is underway to better characterize the possible use of this cyclic dipeptide in the therapy of neurodegenerative and metabolic disorders.
Keywords: Keywords: CHP – Presynaptic dopaminergic mechanism – Diabetes – ERK 1/2 – p-38MAPK – Small hsp – Neuroprotection

Human amniotic fluid stem cells: a new perspective by N. Siegel; M. Rosner; M. Hanneder; A. Freilinger; M. Hengstschläger (291-293).
The discovery of amniotic fluid stem cells initiated a new and very promising field in stem cell research. In the last four years amniotic fluid stem cells have been shown to express markers specific to pluripotent stem cells, such as Oct-4. Due to their high proliferation potential, amniotic fluid stem cell lineages can be established. Meanwhile, they have been shown to harbor the potential to differentiate into cells of all three embryonic germ layers. It will be a major aim for the future to define the potential of this new source of stem cells for therapies related to specific diseases.
Keywords: Keywords: Amniotic fluid stem cells – Oct-4 – Differentiation

Protein–RNA interactions play a key role in a number of biological processes such as protein synthesis, mRNA processing, assembly and function of ribosomes and eukaryotic spliceosomes. A reliable identification of RNA-binding sites in RNA-binding proteins is important for functional annotation and site-directed mutagenesis. We developed a novel method for the prediction of protein residues that interact with RNA using support vector machine (SVM) and position-specific scoring matrices (PSSMs). Two cases have been considered in the prediction of protein residues at RNA-binding surfaces. One is given the sequence information of a protein chain that is known to interact with RNA; the other is given the structural information. Thus, five different inputs have been tested. Coupled with PSI-BLAST profiles and predicted secondary structure, the present approach yields a Matthews correlation coefficient (MCC) of 0.432 by a 7-fold cross-validation, which is the best among all previous reported RNA-binding sites prediction methods. When given the structural information, we have obtained the MCC value of 0.457, with PSSMs, observed secondary structure and solvent accessibility information assigned by DSSP as input. A web server implementing the prediction method is available at the following URL: http://210.42.106.80/printr/ .
Keywords: Keywords: Protein–RNA interactions – RNA-binding sites – Support vector machine – Multiple sequence alignment

Serum phenylalanine in patients post trauma and with sepsis correlate to neopterin concentrations by M. Ploder; G. Neurauter; A. Spittler; K. Schroecksnadel; E. Roth; D. Fuchs (303-307).
Increased blood concentrations of phenylalanine in patients with trauma and sepsis are common but unexplained. We examined the potential relationship between serum concentrations of phenylalanine and the immune activation marker neopterin in 84 specimens of 18 patients (14 males and 4 females) post-trauma during 12–14 days of follow up. Compared to healthy controls, average phenylalanine and neopterin concentrations were elevated in patients, and there existed a positive correlation between concentrations of the two analytes (r s = 0.375, p < 0.001). No such association existed between neopterin and tyrosine concentrations (r s = −0.018), but neopterin concentrations correlated to the phenylalanine to tyrosine ratio (r s = 0.328, p = 0.001).Increased phenylalanine implies insufficient conversion by phenylalanine (4)-hydroxylase (PAH). Oxidative stress due to immune activation and inflammation may destroy cofactor 5,6,7,8-tetrahydrobiopterin and impair PAH activity. This assumption is further supported by the correlation found between higher neopterin concentrations and higher phenylalanine to tyrosine ratio, which estimates efficacy of PAH.
Keywords: Keywords: Phenylalanine – Neopterin – Immune activation – Oxidative stress – Trauma

X-ray sequence ambiguities of Sclerotium rolfsii lectin resolved by mass spectrometry by G. J. Sathisha; Y. K. Subrahmanya Prakash; V. B. Chachadi; N. N. Nagaraja; S. R. Inamdar; D. D. Leonidas; H. S. Savithri; B. M. Swamy (309-320).
X-ray crystallography, although a powerful technique for determining the three-dimensional structure of proteins, poses inherent problems in assigning the primary structure in residues Asp/Asn and Glu/Gln since these cannot be distinguished decisively in the electron density maps. In our recently published X-ray crystal structure of the Sclerotium rolfsii lectin (SRL) at 1.1 Å resolution, amino acid sequence was initially deduced from the electron density map and residues Asp/Asn and Glu/Gln were assigned by considering their hydrogen bonding potential within their structural neighborhood. Attempts to verify the sequence by Edman sequencing were not successful as the N terminus of the protein was blocked. Mass spectrometry was applied to verify and resolve the ambiguities in the SRL X-ray crystal structure deduced sequence. From the Matrix assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI TOF-MS) and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis of tryptic and chymotryptic peptides of SRL, we could confirm and correct the sequence at five locations with respect to Asp/Asn and Glu/Gln. Analysis data also confirmed the positions of Leu/Ile, Gln/Lys residues and the sequence covering 118 of the total 141 residues accounting to 83.68% of the earlier deduced sequence of SRL.
Keywords: Keywords: Sclerotium rolfsii lectin – Amino acid sequence – Mass spectrometry – Protein crystal structure

In the Post Genome Age, there is an urgent need to develop the reliable and effective computational methods to predict the subcellular localization for the explosion of newly found proteins. Here, a novel method of pseudo amino acid (PseAA) composition, the so-called “amino acid composition distribution” (AACD), is introduced. First, a protein sequence is divided equally into multiple segments. Then, amino acid composition of each segment is calculated in series. After that, each protein sequence can be represented by a feature vector. Finally, the feature vectors of all sequences thus obtained are further input into the multi-class support vector machines to predict the subcellular localization. The results show that AACD is quite effective in representing protein sequences for the purpose of predicting protein subcellular localization.
Keywords: Keywords: Protein subcellular localization – Amino acid composition distribution – Pseudo amino acid composition – Support vector machines

Chemotactic tripeptides incorporating at position 2 α-aminoacid residues with unsaturated side chains by G. Lucente; M. P. Paradisi; C. Giordano; A. Sansone; D. Torino; S. Spisani (329-338).
New N-For-Met-Leu-Phe-OMe (fMLF-OMe) analogues incorporating three different γ-δ-didehydro-α-aminoacid residues (namely: Alg = (S)-Allylglycine; Dag = Diallylglycine; Cpg = 1-Aminocyclopent-3-ene-1-carboxylic acid) replacing the native (S)-Leucine have been synthesized and their activity towards human neutrophils has been evaluated in comparison with that shown by the reference tripeptide fMLF-OMe. Chemotaxis, lysozyme release and superoxide anion production have been measured. 1H NMR titration experiments and NOESY spectrum of the Cpg containing model 10 have been discussed in order to ascertain the preferred solution conformations. A fully extended (C5) conformation at position 2 and a folded conformation with two consecutive γ-turns (C7 structure) have been proposed for the Dag and Cpg containing tripeptides, respectively.
Keywords: Keywords: Aminoacids with unsaturated side chains – Chemotactic peptides – Cyclopentenyl aminoacids – Conformation – Grubbs reaction – Human neutrophils

Protein expression of BACE1, BACE2 and APP in Down syndrome brains by M. S. Cheon; M. Dierssen; S. H. Kim; G. Lubec (339-343).
Down syndrome (DS) is the most common human chromosomal abnormality caused by an extra copy of chromosome 21. The phenotype of DS is thought to result from overexpression of a gene or genes located on the triplicated chromosome or chromosome region. Several reports have shown that the neuropathology of DS comprises developmental abnormalities and Alzheimer-like lesions such as senile plaques. A key component of senile plaques is amyloid β-peptide which is generated from the amyloid precursor protein (APP) by sequential action of β-secretases (BACE1 and BACE2) and γ-secretase. While BACE1 maps to chromosome 11, APP and BACE2 are located on chromosome 21. To challenge the gene dosage effect and gain insight into the expressional relation between β-secretases and APP in DS brain, we evaluated protein expression levels of BACE1, BACE2 and APP in fetal and adult DS brain compared to controls. In fetal brain, protein expression levels of BACE2 and APP were comparable between DS and controls. BACE1 was increased, but did not reach statistical significance. In adult brain, BACE1 and BACE2 were comparable between DS and controls, but APP was significantly increased. We conclude that APP overexpression seems to be absent during the development of DS brain up to 18–19 weeks of gestational age. However, its overexpression in adult DS brain could lead to disturbance of normal function of APP contributing to neurodegeneration. Comparable expression of BACE1 and BACE2 speaks against the hypothesis that increased β-secretase results in (or even underlies) increased production of amyloidogenic Aβ fragments.Furthermore, current data indicate that the DS phenotype cannot be fully explained by simple gene dosage effect.
Keywords: Keywords: Amyloid precursor protein – β-site APP-cleaving enzyme – Down syndrome – Protein expression

Identifying a protein’s subcellular localization is an important step to understand its function. However, the involved experimental work is usually laborious, time consuming and costly. Computational prediction hence becomes valuable to reduce the inefficiency. Here we provide a method to predict protein subcellular localization by using amino acid composition and physicochemical properties. The method concatenates the information extracted from a protein’s N-terminal, middle and full sequence. Each part is represented by amino acid composition, weighted amino acid composition, five-level grouping composition and five-level dipeptide composition. We divided our dataset into training and testing set. The training set is used to determine the best performing amino acid index by using five-fold cross validation, whereas the testing set acts as the independent dataset to evaluate the performance of our model. With the novel representation method, we achieve an accuracy of approximately 75% on independent dataset. We conclude that this new representation indeed performs well and is able to extract the protein sequence information. We have developed a web server for predicting protein subcellular localization. The web server is available at http://aaindexloc.bii.a-star.edu.sg .
Keywords: Keywords: Subcellular localization – Support vector machine – Amino acid indices

Handling and detoxification of metals by enzymes is a major issue that is not in the focus of current biomedical research concepts. The finding of the presence of arsenic (+3 Oxidation State) methyltransferase (AS3MT) in neuroblastoma cells NE-115 as a high abundance protein made us investigate primary structure of AS3MT reflecting an example of metal-handling in eucaryotes. Proteins extracted from NE-115 cells were run on 2-DE followed by two different mass spectrometrical methods. High sequence coverage was obtained by multiple protease digestion and a sequence conflict was solved at arginine 335.These findings are important when future studies on this enzyme are designed at the protein level and in particular, when antibodies against this protein will be generated.
Keywords: Keywords: Arsenite methyltransferase – MALDI-TOF-TOF – Nano-LC-ESI-MS/MS – Neuroblastoma cells

The role of heme oxygenase-1 in down regulation of PGE2 production by taurine chloramine and taurine bromamine in J774.2 macrophages by R. Olszanecki; M. Kurnyta; R. Biedroń; P. Chorobik; M. Bereta; J. Marcinkiewicz (359-364).
Taurine chloramine (TauCl) and taurine bromamine (TauBr), products of myeloperoxidase halide system, exert anti-inflammatory properties. TauCl was demonstrated to inhibit the production of a variety of pro-inflammatory mediators including cyclooxygenase-2 (COX-2) dependent production of prostaglandin E2 (PGE2). Recently we have demonstrated that both major leukocyte haloamines, TauCl and TauBr, induced expression of HO-1 in non-activated and LPS-activated J774.2 macrophages. In this study, we have shown that TauCl and TauBr, at non-cytotoxic concentrations, inhibited the production of (PGE2) without altering the expression of COX-2 protein, in LPS/IFN-γ stimulated J774.2 cells. The inhibitory effect of TauCl and TauBr was reversed by chromium III mesoporhyrin (CrMP), an inhibitor of HO-1 activity. Our data suggest that HO-1 might participate in anti-inflammatory effects of TauCl/TauBr possibly by inhibition of COX-2 activity and decrease of PGE2 production.
Keywords: Keywords: Inflammation – Macrophages – Heme oxygenase-1 – Taurine bromamine – Taurine chloramine – Prostaglandin E2 – Cyclooxygenase-2

This is the continuation of our studies on the prediction of mutation engineered by randomness in proteins from influenza A virus. In our previous studies, we have demonstrated that randomness plays a role in engineering mutations because the measures of randomness in protein are different before and after mutations. Thus we built a cause-mutation relationship to count the mutation engineered by randomness, and conducted several concept-initiated studies to predict the mutations in proteins from influenza A virus, which demonstrated the possibility of prediction of mutations along this line of thought. On the other hand, these concept-initiated studies indicate the directions forwards the enhancement of predictability, of which we need to use the neural network instead of logistic regression that was used in those concept-initiated studies to enhance the predictability. In this proof-of-concept study, we attempt to apply the neural network to modeling the cause-mutation relationship to predict the possible mutation positions, and then we use the amino acid mutating probability to predict the would-be-mutated amino acids at predicted positions. The results confirm the possibility of use of internal cause-mutation relationship with neural network model to predict the mutation positions and use of amino acid mutating probability to predict the would-be-mutated amino acids.
Keywords: Keywords: Amino acid – Hemagglutinin – Influenza – Mutation – Neural network

Cleavage mechanism of the H5N1 hemagglutinin by trypsin and furin by X.-L. Guo; L. Li; D.-Q. Wei; Y.-S. Zhu; K.-C. Chou (375-382).
The cleavage property of hemagglutinin (HA) by different proteases was the prime determinant for influenza A virus pathogenicity. In order to understand the cleavage mechanism, molecular modeling tools were utilized to study the coupled model systems of the proteases, i.e., trypsin and furin and peptides of the cleavage sites specific to H5N1 and H1 HAs, which constitute models of HA precursor in complex with cleavage proteases. The peptide segments ‘RERRRKKR ↓ G’ and ‘SIQSR ↓ G’ from the high pathogenic H5N1 H5 and the low pathogenic H1N1 H1 cleavage sites were docking to the trypsin and furin active pockets, respectively. It was observed through the docking studies that trypsin was able to recognize and cleave both the high pathogenic and low pathogenic hemagglutinin, while furin could only cleave the high pathogenic hemagglutinin. An analysis of binding energies indicated that furin got most of its selectivity due to the interactions with P1, P4, and P6, while having less interaction with P2 and little interactions with P3, P5, P7, and P8. Some mutations of H5N1 H5 cleavage sequence fitted less well into furin and would reduce high pathogenicity of the virus. These findings hint that we should focus at the subsites P1, P4, and P6 for developing drugs against H5N1 viruses.
Keywords: Keywords: Trypsin – Furin – H5N1 hemagglutinin – Cleavage mechanism – Pathogenicity

Apoptosis proteins play an important role in the development and homeostasis of an organism. The accurate prediction of subcellular location for apoptosis proteins is very helpful for understanding the mechanism of apoptosis and their biological functions. However, most of the existing predictive methods are designed by utilizing a single classifier, which would limit the further improvement of their performances. In this paper, a novel predictive method, which is essentially a multi-classifier system, has been proposed by combing a dual-layer support vector machine (SVM) with multiple compositions including amino acid composition (AAC), dipeptide composition (DPC) and amphiphilic pseudo amino acid composition (Am-Pse-AAC). As a demonstration, the predictive performance of our method was evaluated on two datasets of apoptosis proteins, involving the standard dataset ZD98 generated by Zhou and Doctor, and a larger dataset ZW225 generated by Zhang et al. With the jackknife test, the overall accuracies of our method on the two datasets reach 94.90% and 88.44%, respectively. The promising results indicate that our method can be a complementary tool for the prediction of subcellular location.
Keywords: Keywords: Subcellular location – Apoptosis protein – Dual-layer support vector machine – Amino acid composition – Dipeptide composition – Amphiphilic pseudo amino acid composition

A molecular docking study was performed on several structurally diverse A2A AR antagonists, including xanthines, and non-xanthine type antagonists to investigate their binding modes with A2A adenosine receptor (AR), one of the four subtypes of AR, which is currently of great interest as a target for therapeutic intervention, in particular for Parkinson’s disease. The high-affinity binding site was found to be a hydrophobic pocket with the involvement of hydrogen bonding interactions as well as π–π stacking interactions with the ligands. The detailed binding modes for both xanthine and non-xanthine type A2A antagonists were compared and the essential features were extracted and converted to database searchable queries for virtual screening study of novel A2A AR antagonists. Findings from this study are helpful for elucidating the binding pattern of A2A AR antagonists and for the design of novel active ligands.
Keywords: Keywords: Adenosine receptors – A2A AR antagonists – Binding mode – Docking – Pharmacophore – Virtual screening

Heme oxygenase-1 participates in the anti-inflammatory activity of taurine chloramine by B. Muż; E. Kontny; J. Marcinkiewicz; W. Maśliński (397-402).
Interleukin-6 (IL-6) and interleukin-8 (IL-8) are implicated in the pathogenesis of rheumatic diseases. In affected joints fibroblast-like synoviocytes (FLS) are the major source of these pro-inflammatory cytokines. We have previously found that production of both cytokines is inhibited in vitro by taurine chloramine (Tau-Cl). Heme oxygenase (HO-1) activity was also reported to restrict synthesis of various inflammatory mediators, including IL-6 and IL-8. The aim of present study was to investigate whether this enzyme activity is implicated in the mechanism of Tau-Cl suppressive effect. We have shown that in rheumatoid FLS both hemin (known HO-1 inducer) and Tau-Cl significantly up-regulate HO-1 expression at the mRNA and protein levels and simultaneously inhibit IL-1β-triggered production of pro-inflammatory cytokines. However, the inhibitory potency of these compounds differs, because hemin is more potent inhibitor of IL-8 than IL-6 production, while Tau-Cl exerts opposite effect. Importantly, pretreatment of the cells with HO-1 inhibitor completely reverses the inhibitory effect of hemin on both cytokines production. However, in Tau-Cl treated cells this inhibitor fully restores only IL-8 secretion but has weaker effect on IL-6 response. Thus, the present results: (i) support HO-1 activity to be relevant to negatively control production of pro-inflammatory cytokines, and (ii) underline implication of HO-1 in mediating Tau-Cl inhibitory action.
Keywords: Keywords: Interleukins (IL) – Fibroblast-like synoviocytes (FLS) – Rheumatoid arthritis (RA) – Heme oxygenase-1 (HO-1) – Taurine chloramine (Tau-Cl)

The effect of taurine on mesenteric blood flow and organ injury in sepsis by A. Erdem; A. M. Sevgili; F. Akbiyik; P. Atilla; N. Cakar; Z. D. Balkanci; A. B. Iskit; M. O. Guc (403-410).
Endotoxin decreases mesenteric blood flow and inflicts organ injury via free radicals. We investigated whether taurine, an endogenous antioxidant and vasodilator, could attenuate the deleterious effects of endotoxin in a mouse model of sepsis. Swiss albino mice were allocated into four groups and treated either with taurine (150 mg/kg, i.p. at 0th, 8th, 16th h) or its solvent sterile saline (NaCl 0.9%, w/v) while E. coli endotoxin (20 mg/kg, i.p.) or its solvent saline were also given at 8th h. At 24th h the animals were anaesthetized and the mesenteric blood flow was measured by using perivascular ultrasonic Doppler-flowmeter. The animals were then exsanguinated, the spleen, liver, and kidneys were isolated for histopathological examination. Thiobarbituric acid-reacting substances (TBARS), glutathione, and myeloperoxidase activity were determined in the liver samples. Endotoxin significantly decreased the mesenteric blood flow and glutathione levels in liver while TBARS and myeloperoxidase activity were increased. However, taurine did not block the deleterious effects of endotoxin nor it did attenuate the histopathological injury. Therefore, we concluded that endotoxin-induced organ injury via free radicals is resistant to blockade by taurine.
Keywords: Keywords: Endothelin – Taurine – Sepsis – Mesenteric ischemia – Doppler-flowmeter

The aim of this work was to modify the method of Ladd and Buttler (1972), by substituting Tris–HCl buffer (pH 8.52) with demineralized water (DEMI H2O), in order to assess its suitability for measurement of casein-protease activity at pH levels close to those of real soil in H2O. Measurements were undertaken over a range of incubation temperatures from 3 to 49 °C. Testing was performed on one organic soil and two different mineral soils. The substitution of Tris–HCl buffer by DEMI H2O at 49 °C decreased casein-protease activity to 67.25% in mineral soil and to 53.76% in organic soil. With decreasing temperature casein-protease activity decreased the most in organic soil, i.e., 0.07% of original its value at 3 °C. The incubation period was extended to maximally 336 h at 3 °C to totally obtain >10.0% of L-tyrosine equivalents released at optimum or close to optimum temperature and pH conditions. The Q10 values of casein-protease activity measured after substituting Tris–HCl buffer with DEMI H2O were unexpectedly high. Between the temperatures of 3 and 49 °C Q10 ranged from 3.46 to 4.25, whereas between 3 and 25 °C Q10 ranged from 6.78 to 11.08. Therefore, the modified method of Ladd and Buttler (1972) presented can be used for measurement of soil casein-protease activity under pH conditions close to that of real soil pH and at an averaged soil temperatures measured in the field. This modification makes possible an expression of soil casein-protease activity potential – when being combined with measurements of casein-protease activity under optimum or close to optimum temperature and pH conditions, if high concentration of casein is present.
Keywords: Keywords: Soil proteases – Enzyme temperature dependence – Q10

l-Carnitine protects against apoptosis of murine MC3T3-E1 osteoblastic cells by H. Xie; S.-Y. Tang; H. Li; X.-H. Luo; L.-Q. Yuan; D. Wang; E.-Y. Liao (419-423).
l-Carnitine (LC), an amino acid with a major role in cellular energy metabolism, has positive effects on bone metabolism. However, the effect of LC on apoptosis of osteoblast in vitro has not been reported. The aim of this study was to investigate the action of LC on apoptosis of mouse osteoblastic cell line MC3T3-E1. Cell apoptosis was measured by sandwich-enzyme-immunoassay. Release of cytochrome c from mitochondria into cytosol and Bcl-2, Bax protein levels were determined by Western blot analysis. The enzyme substrate was used to assess the activation of caspase-3 and caspase-9. LC inhibited MC3T3-E1 cell apoptosis induced by serum deprivation. Our study also shows that LC decreased cytochrome c release and caspase-3 and caspase-9 activation in serum-deprived MC3T3-E1 cells. Furthermore, LC protected against MC3T3-E1 cell apoptosis induced by the glucocorticoid (GC) dexamethasone (Dex).
Keywords: Keywords: l-Carnitine – Osteoblast – Apoptosis – Glucocorticoid

Objective. The effect of creatine supplementation upon plasma levels of pro-inflammatory cytokines: Interleukin (IL) 1β and IL-6, Tumor Necrosis Factor α (TNFα), and Interferon α (INFα) and Prostaglandin E2 (PGE2) after a half-ironman competition were investigated. Methods. Eleven triathletes, each with at least three years experience of participation in this sport were randomly divided between the control and experimental groups. During 5 days prior to competition, the control group (n = 6) was supplemented with carbohydrate (20 g · d−1) whereas the experimental group (n = 5) received creatine (20 g · d−1) in a double-blind trial. Blood samples were collected 48 h before and 24 and 48 h after competition and were used for the measurement of cytokines and PGE2. Results. Forty-eight hours prior to competition there was no difference between groups in the plasma concentrations (pg · ml−1, mean ± SEM) of IL-6 (7.08 ± 0.63), TNFα (76.50 ± 5.60), INFα (18.32 ± 1.20), IL-1β (23.42 ± 5.52), and PGE2 (39.71 ± 3.8). Twenty-four and 48 h after competition plasma levels of TNFα, INFα, IL-1β and PGE2 were significantly increased (P < 0.05) in both groups. However, the increases in these were markedly reduced following creatine supplementation. An increase in plasma IL-6 was observed only after 24 h and, in this case, there was no difference between the two groups. Conclusion. Creatine supplementation before a long distance triathlon competition may reduce the inflammatory response induced by this form of strenuous of exercise.
Keywords: Keywords: Long distance triathlon – Muscle inflammation – Muscle damage – Eccentric contraction – Pro-inflammatory cytokines

Amino acids supply in culture media is not a limiting factor in the matrix synthesis of engineered cartilage tissue by K. W. Ng; J. G. DeFrancis; L. E. Kugler; T.-A. N. Kelly; M. M. Ho; C. J. O’Conor; G. A. Ateshian; C. T. Hung (433-438).
Increased amino acid supplementation (0.5×, 1.0×, and 5.0× recommended concentrations or additional proline) was hypothesized to increase the collagen content in engineered cartilage. No significant differences were found between groups in matrix content or dynamic modulus. Control constructs possessed the highest compressive Young’s modulus on day 42. On day 42, compared to controls, decreased type II collagen was found with 0.5×, 1.0×, and 5.0× supplementation and significantly increased DNA content found in 1.0× and 5.0×. No effects were observed on these measures with added proline. These results lead us to reject our hypothesis and indicate that the low collagen synthesis in engineered cartilage is not due to a limited supply of amino acids in media but may require a further stimulatory signal. The results of this study also highlight the impact that culture environment can play on the development of engineered cartilage.
Keywords: Keywords: Agarose – Cartilage tissue engineering – Chondrocyte – Amino acids – Cell proliferation – Dedifferentiation – Cell culture media

The purpose of this study was to determine the effect of adding fenugreek extract (FG) to post-exercise carbohydrate feeding on glycogen resynthesis and subsequent exercise performance in normoglycemic male endurance athletes. A muscle biopsy sample was obtained from the vastus lateralis from subjects prior to exercise for 5 h at 50% of peak cycling power (52.1 ± 3.3% of VO2 peak). A second muscle biopsy sample was obtained immediately after exercise. Immediately after and 2 h after the second biopsy subjects ingested either an oral dose of dextrose (GLU) (1.8 g · kg BW−1) or GLU with FG containing 1.99 ± 0.20 mg · kg−1 4-hydroxyisoleucine (GLU + FG) in a randomized, cross-over, double blind design. At 4 h post-exercise a third biopsy was taken and subjects received a standardised meal along with FG or a placebo capsule. At 15 h post-exercise subjects underwent their final muscle biopsy before completing a simulated 40 km cycling time trial. There was no difference in muscle glycogen at any time between GLU and GLU + FG. Additionally, 40 km time trial performance was similar for average power output (221 ± 28 vs. 213 ± 16 watts) and for time to completion (69.7 ± 3.7 vs. 70.5 ± 2.2 min) for the GLU and GLU + FG, respectively. Despite earlier data to the contrary, the present results do not support an effect of fenugreek supplementation on glycogen resynthesis, even though this may have been the result of differences in experimental protocol.
Keywords: Keywords: Glycogen – Fenugreek – Trigonella foenum-graecum – 4-Hydroxyisoleucine – Exercise

A novel sample preparation protocol for gas chromatographic (GC) analysis of amino acid enantiomers in peptides was developed. It comprises traditional acid hydrolysis, a novel treatment of the analytes with a fluoroalkyl chloroformate and GC/FID separation of enantiomers on a chiral capillary column. The major improvements consist in that the derivatization step proceeds in organic-aqueous media within seconds and the amino acid derivatives are volatile enough to suit the temperature range of the chiral Chirasil-Val® capillary column. The approach was found beneficial for chiral analysis of pharmaceutically important Carbetocin peptide.
Keywords: Keywords: D,L-amino acids – Peptides – Chloroformates – GC – Chiral separation

We examined the effects of orally administered glycine on myofibrillar proteolysis in food-deprived chicks. Food-deprived (24 h) chicks were orally administered 57, 113, and 225 mg glycine/100 g body weight and killed after 2 h. The plasma Nτ-methylhistidine concentration, used as myofibrillar proteolysis, was decreased by glycine. We also examined the expression of proteolytic-related genes by real-time PCR of cDNA from chick skeletal muscles. The mRNA expression of atrogin-1/MAFbx, proteasome C2 subunit, m-calpain large subunit, and cathepsin B was decreased by glycine in a dose-dependent manner. The plasma corticosterone concentration was also decreased by glycine, but the plasma insulin concentration was unaffected. These results indicate that orally administered glycine suppresses myofibrillar proteolysis and expression of proteolytic-related genes of skeletal muscle by decreasing the plasma corticosterone concentration in chicks.
Keywords: Keywords: Glycine – Myofibrillar proteolysis – Atrogin-1/MAFbx – Corticosterone – Skeletal muscle – Chick

Taurine, a known antioxidant and neuroprotector has been investigated for its free radical scavenging action in vitro in isolated mitochondria, and tested whether it protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurodegeneration in mice. Taurine (0.1–10 mM) did not affect 1-methyl-4-phenyl pyridinium-induced hydroxyl radical production in isolated mitochondria. Systemic administration of taurine (250 mg/kg, i.p.) caused a small, but significant loss of dopamine levels in the striatum of mice. Taurine failed to reverse MPTP-induced striatal dopamine depletion, but caused significant increase in dopamine turnover in these animals. In the light of the present study it may be suggested that consumption of taurine may neither help in scavenging of neurotoxic hydroxyl radicals in the brain mitochondria, nor would it help in blocking the process of neurodegeneration.
Keywords: Keywords: Taurine – Striatum – Hydroxyl radical – Parkinson’s disease – Mitochondria – MPTP

A series of novel naphthoquinone fused cyclic α-aminophosphonates, 2-alkoxy-3,4-dihydro-2H-naphtho[2,3-e][1,4,2]oxazaphosphinane-5,10-dione 2-oxide 317 and naphthoquinone fused cyclic α-aminophosphonic monoester 18 were synthesized for the first time. These cyclic α-aminophosphonates were evaluated for antitumor activity on four human tumor cell lines, and three of them showed significant cytotoxicity (IC50: 0.019–5.15 µM) comparable to that of the reference drug doxorubicin. Furthermore, inhibition assays for topoisomerase II-mediated relaxation of supercoiled DNA indicated that the naphthoquinone fused cyclic aminophosphonates were catalytic inhibitors of topoisomerase II.
Keywords: Keywords: Amino acid – Aminoalkylphosphonic acid – Topoisomerase II inhibitors – Mannich-type reaction – Phosphorus heterocycles

Dietary amino acid taurine ameliorates liver injury in chronic hepatitis patients by Y. H. Hu; C. L. Lin; Y. W. Huang; P. E. Liu; D.-F. Hwang (469-473).
The effect of dietary amino acid taurine on the liver function of chronic hepatitis patients was investigated. The 24 chronic hepatitis patients with 2–5 times over normal activities of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) were selected and equally divided into taurine treatment and control groups. In taurine treatment group, each patient took 2 g taurine 3 times a day for three months, and then stopped treatment for 1 month. Patients taking placebo without taurine for 4 months served as a control group. ALT and AST activities and levels of cholesterol, triglyceride and thiobarbituric acid relative substances of serum plasma in the taurine group were all decreased at the end of three month treatment. The study suggested that dietary amino acid taurine may ameliorate liver injury for chronic hepatitis patients.
Keywords: Keywords: Alanine aminotransferase – Antioxidation – Hapatitis patient – Liver function – Taurine

Beneficial impact of L-carnitine in liver: a study in a rat model of syndrome X by P. Rajasekar; P. Viswanathan; C. V. Anuradha (475-483).
The present study was designed to explore whether L-carnitine (CA) regulates insulin signaling and modulates the changes in liver in a well-characterized insulin resistant rat model. Adult male Wistar rats were divided into 4 groups. Groups I and IV animals received starch-based control diet, while groups II and III rats were fed a high fructose-diet (60 g/100 g). Groups III and IV animals additionally received CA (300 mg/kg/day i.p). After a period of 60 days hepatic tyrosine phosphorylation status was determined by assaying protein tyrosine phosphatase (PTP) and protein tyrosine kinase (PTK) activities. Oxidative damage was monitored by immunohistochemical localization of 4-hydroxynonenal (4-HNE), 3-nitrotyrosine (3-NT) and dinitrophenol (DNP)-protein adducts. In addition protein kinase C βII (PKC βII) expression, propidium iodide staining of isolated hepatocytes and histology of liver tissue were determined to examine liver integrity. Fructose-fed rats displayed reduced insulin action, increased expression of PKC βII, altered histology, fragmentation of hepatocyte nuclear DNA, and accumulation of oxidatively modified proteins. Simultaneous treatment with CA alleviated the abnormalities associated with fructose feeding. In summary the data suggest that elevated oxidative damage and PKC expression could in part induce insulin resistance and CA has beneficial impact on liver during insulin resistance with modulatory effects at the post-receptor level.
Keywords: Keywords: Insulin resistance – Carnitine – Protein tyrosine kinase – Protein tyrosine phosphatase – Protein kinase C – Oxidative damage – Apoptosis

Anterior gradient protein 2 homolog is a metastasis-inducing protein in a rat model of rat breast cancer and prognostic for outcome in hormonally treated breast cancer patients. Carrying out protein profiling in several mammalian cells and tissues, we detected this protein (synonym: secreted cement gland protein XAG-2 homolog) that was originally described in toad skin, in human bronchial epithelia.Tissues obtained from biopsies were homogenised and extracted proteins were run on two-dimensional gel electrophoresis. Following in-gel digestion with proteases trypsin, AspN, LysC and chymotrypsin, mass spectrometrical analysis was carried out by MALDI-TOF/TOF.The use of MS following multi-enzyme digestion of the protein resulted into 100% sequence coverage. MS/MS analysis enabled sequencing of 87% of the protein structure. This percentage does not include the signal peptide that was not observed in our protein due to processing. No posttranslational modifications were detectable and no sequence conflicts were observed.Complete analysis, unambiguous identification and characterisation of this biologically important protein could be shown, which is relevant for the definition of a marker protein that has been described so far by immunochemical methods only. Complete analysis is of importance as it forms the basis for all future work on this protein and, moreover, may serve as an analytical tool for further studies.
Keywords: Keywords: Anterior gradient protein 2 homolog – Human bronchial epithelia – Sequencing – MALDI-TOF/TOF

Evolutionary conservation derived from a multiple sequence alignment is a powerful indicator of the functional significance of a residue, and it can help to predict active sites, ligand-binding sites, and protein interaction interfaces. The results of the existing algorithms in identifying the residue’s conservation strongly depend on the sequence alignment, making the results highly variable. Here, by introducing the amino acid similarity matrix, we propose a novel gap-treating approach by combining the evolutionary information and von Neumann entropies to compute the residue conservation scores. It is indicated through a series of tested results that the new approach is quite encouraging and promising and may become a useful tool in complementing the existing methods.
Keywords: Keywords: Evolutionary conservation – Amino acid similarity matrix – von Neumann entropy – Functional residue – Sensitivity – Specificity

Micromolar concentration of kynurenic acid in rat small intestine by D. Kuc; W. Zgrajka; J. Parada-Turska; T. Urbanik-Sypniewska; W. A. Turski (503-505).
Kynurenic acid is an antagonist of glutamate and alpha-7 nicotinic acetylcholine receptors and an agonist of the g-protein-coupled receptor GPR35, which is predominantly expressed in immune and gastrointestinal tissues. In this study, we report that kynurenic acid is present in the lumen of rat small intestine in micromolar concentration sufficient to affect the GPR35 receptor. Moreover, we show that kynurenic acid can be produced by Escherichia coli. We suggest that kynurenic acid may modulate gastrointestinal function and integrity.
Keywords: Keywords: Kynurenic acid – Small intestine – Rat

3-Hydroxyaspartic acid and its derivatives are found both in the free form and as peptide constituents in various microorganisms and fungi. Considering the biological importance of this amino acid and its potential utility as a multifunctional building block in organic syntheses, we have developed a short-step, asymmetric synthetic route to a strategically protected 3-hydroxyaspartic acid derivative in enantiopure form. The key steps in the synthesis involve, Sharpless asymmetric aminohydroxylation of commercially available trans-ethyl cinnamate, and, utilization of the phenyl group as a masked carboxylic acid synthon towards construction of the complete structural framework of the title compound.
Keywords: Keywords: Non-proteinogenic amino acid – Sharpless asymmetric aminohydroxylation – Exhaustive oxidation – Orthogonally protected functional groups

Recently, Guo et al. have reported structural as well as the binding energy data of the particular interactions between the cleavage sites of hemagglutinin and serine proteases, trypsin and furin, using molecular docking approach. Due to a wrong assignment of protonation state on the histidine, one of the catalytic triad in the active site of both enzymes, their docking results are contradictory with the fundamental principle and previous theoretical studies of the known cleavage mechanism in serine proteases.
Keywords: Keywords: Catalytic triad – Cleavage mechanism – Serine proteases – Trypsin – Furin