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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.38, #4)

Prediction of subcellular location apoptosis proteins with ensemble classifier and feature selection by Quan Gu; Yong-Sheng Ding; Xiao-Ying Jiang; Tong-Liang Zhang (pp. 975-983).

Apoptosis proteins have a central role in the development and the homeostasis of an organism. These proteins are very important for understanding the mechanism of programmed cell death. The function of an apoptosis protein is closely related to its subcellular location. It is crucial to develop powerful tools to predict apoptosis protein locations for rapidly increasing gap between the number of known structural proteins and the number of known sequences in protein databank. In this study, amino acids pair compositions with different spaces are used to construct feature sets for representing sample of protein feature selection approach based on binary particle swarm optimization, which is applied to extract effective feature. Ensemble classifier is used as prediction engine, of which the basic classifier is the fuzzy K-nearest neighbor. Each basic classifier is trained with different feature sets. Two datasets often used in prior works are selected to validate the performance of proposed approach. The results obtained by jackknife test are quite encouraging, indicating that the proposed method might become a potentially useful tool for subcellular location of apoptosis protein, or at least can play a complimentary role to the existing methods in the relevant areas. The supplement information and software written in Matlab are available by contacting the corresponding author.

Keywords: Apoptosis protein subcellular location; Feature selection; Ensemble classifier; Fuzzy K-nearest neighbor classifier

Design, synthesis and biological evaluation of non-peptide PAR₁ thrombin receptor antagonists based on small bifunctional templates: arginine and phenylalanine side chain groups are keys for receptor activity by Maria-Eleni Androutsou; Mahmoud Saifeddine; Morley D. Hollenberg; John Matsoukas; George Agelis (pp. 985-990).

In the present study, we report the synthesis and biological evaluation of a series of new non-peptide PAR₁ mimetic receptor antagonists, based on conformational analysis of the S₄₂FLLR₄₆ tethered ligand (TL) sequence of PAR₁. These compounds incorporate the key pharmacophore groups in the TL sequence, guanidyl, amino and phenyl, which are essential for triggering receptor activity. Compounds 5 and 15 (50–100 μM) inhibited both TFLLR-amide (10 μM) and thrombin-mediated (0.5 and 1 U/ml; 5 and 10 μM) calcium signaling in a cultured human HEK cell assay.

Keywords: Thrombin; PAR₁ receptor; SFLLR; Pharmacophore; Mimetics

Molecular characterisation of the low-molecular weight glutenin subunit genes of tall wheatgrass and functional properties of one clone Ee34 by Fanguo Chen; Shuwei Liu; Feng Zhao; Chunhui Xu; Guangmin Xia (pp. 991-999).

Wild tall wheatgrass (Lophopyrum elongatum L., 2x = 14) is an important resource for improving bread wheat (Titicum aestivum L.), including HMW-GS and LMW-GS relevant to end-use quality of the wheat flour. A set of 14 distinct sequences were amplified from the genomic DNA of the tall wheatgrass, using degenerate primers targeted at Glu-3, the locus containing the genes encoding the low-molecular weight glutenin subunits (LMW-GS). Three sequences contained an internal stop codon and were classified as pseudogenes. The other 11 all consisted of a single intron-less intact open-reading frame. An alignment of deduced protein sequences showed that the primary structure of all 11 sequences was similar to that of wheat and other wheat-related grass Glu-3 genes. All 11 sequences carried the 14 amino acid residue N-terminal motif MESNIIISFLK/RPWL, and were classified as LMW-m genes, based on the identity of the first amino acid of the mature protein. All but one of the sequences contained seven cysteine residues (the exception had 6). Their repetitive domain differs significantly from that present in Glu-3 genes isolated from the close relative intermediate wheatgrass (Thinopyrum Intermedium, 6x). A phylogenetic analysis showed that the tall wheatgrass sequences were closely related to those of the intermediate wheatgrass, but only distantly so to those from decaploid tall wheatgrass. One of the 11 LMW-GS peptides with a free-cysteine residue was heterologously expressed in E. coli and purified in sufficient scale to perform a flour supplementation test. This showed that the dough strength of bread wheat flour was significantly increased by the presence of the tall wheatgrass LMW-GS.

Keywords: Lophopyrum elongatum (Host) A. Löve; LMW-GS gene; Flour quality; Dough micro-mixing test

Cystatin like thiol proteinase inhibitor from pancreas of Capra hircus: purification and detailed biochemical characterization by Medha Priyadarshini; Bilqees Bano (pp. 1001-1010).

A thiol proteinase inhibitor from Capra hircus (goat) pancreas (PTPI) isolated by ammonium sulphate precipitation (20–80%) and gel filtration chromatography on Sephacryl S-100HR, with 20.4% yield and 500-fold purification, gave molecular mass of 44 kDa determined by its electrophoretic and gel filtration behavior, respectively. The stokes radius, diffusion and sedimentation coefficients of PTPI were 27.3 Ǻ, 7.87 × 10⁻⁷ cm² s⁻¹ and 3.83 s, respectively. It was stable in pH range 3–10 and up to 70°C (critical temperature, E _a = 21 kJ mol⁻¹). Kinetic analysis revealed reversible and competitive mode of inhibition with PTPI showing the highest inhibitory efficiency against papain (K i = 5.88 nM). The partial amino acid sequence analysis showed that it shared good homology with bovine parotid and skin cystatin C. PTPI possessed 17.18% α helical content assessed by CD spectroscopy. The hydropathy plot of first 24 residues suggested that most amino acids of this stretch might be in the hydrophobic core of the protein.

Keywords: Goat pancreas cystatin; Kinetics of inhibition; Amino acid sequence; Mammalian cystatins; Characterization

Interactions of α₁–proteinase inhibitor with small ligands of therapeutic potential: binding with retinoic acid by Elena Karnaukhova (pp. 1011-1020).

Human α₁–proteinase inhibitor (α₁–PI), also known as α₁-antitrypsin, is the most abundant plasma serine protease inhibitor (serpin). It is best recognized for inhibition of neutrophil elastase. The α₁–PI interactions with non-protease ligands were investigated mainly in regards to those molecules that may block the aggregation of α₁–PI Z mutant. The objective of this study was to evaluate the potential of α₁–PI to bind small non-peptide ligands of pharmaceutical interest that may attain additional properties to currently available α₁–PI therapeutic preparations. Among putative ligands of bio-medical interest examined in this study, all-trans retinoic acid (RA) was selected due to its recently proposed roles in the lungs, and as an efficient optical probe. The results of this study, including absorption spectroscopy data, fluorescence quenching and the protein-induced chirality of the visible circular dichroism strongly suggest that α₁–PI does bind RA in vitro to non-covalent complexes of up to two moles of RA per one mole of the protein. To our knowledge, this is the first report that provides experimental evidence of the α₁–PI potential towards bi-functional drugs via a combination with RA, or potentially other molecules of pharmaceutical interest, that ultimately, may enhance currently available α₁–PI therapies.

Keywords: Alpha-1 proteinase inhibitor; Antitrypsin; Retinoic acid

Effects of glutamine supplementation on kidney of diabetic rat by Tatiana Carolina Alba-Loureiro; Rodolfo Favaro Ribeiro; Telma Maria Tenório Zorn; Claudia J. Lagranha (pp. 1021-1030).

Glutamine is the most important donor of NH₃ in kidney playing an important role in acid–base buffering system. Besides this effect, glutamine presents many other relevant functions in the whole body, such as a precursor of arginine in adult and neonates. In addition to these effects, some studies have shown that glutamine can potentiate renal disease. In the present study, the effect of short-term treatment (15 days) with glutamine on control and diabetic rats was investigated. Using biochemical, histological and molecular biology analysis from control and diabetic rats we verified that glutamine supplementation increase in pro-inflammatory interleukins (IL)-1β and IL-6 content in renal cortex and induce alteration in glomerular characteristics. This study showed that short-term treatment with glutamine in association with increased glucose levels could cause important alterations in glomerular morphology that may result in fast progression of kidney failure.

Keywords: Kidney; Diabetes; Glutamine supplementation

Quantification of thyrotropin-releasing hormone by liquid chromatography–electrospray mass spectrometry by Angela Chambery; Valeria Severino; Antimo Di Maro; Antimo D’Aniello; Menotti Ruvo; Augusto Parente (pp. 1031-1041).

Thyrotropin-releasing hormone (TRH) is involved in a wide range of biological responses. It has a central role in the endocrine system and regulates several neurobiological activities. In the present study, a rapid, sensitive and selective liquid chromatography–mass spectrometry method for the identification and quantification of TRH has been developed. The methodology takes advantage of the specificity of the selected-ion monitoring acquisition mode with a limit of detection of 1 fmol. Furthermore, the MS/MS fragmentation pattern of TRH has been investigated to develop a selected reaction monitoring (SRM) method that allows the detection of a specific b2 product ion at m/z 249.1, corresponding to the N-terminus dipeptide pyroglutamic acid–histidine. The method has been tested on rat hypothalami to evaluate its suitability for the detection within very complex biological samples.

Keywords: Thyrotropin-releasing hormone; Liquid chromatography; Mass spectrometry; Selected-ion monitoring; Selected reaction monitoring

Complete sequencing of the recombinant granulocyte-colony stimulating factor (filgrastim) and detection of biotinylation by mass spectrometry by Kareem Eldin A. M. Ahmed; Wei-Qiang Chen; Julius Paul Pradeep John; Sung Ung Kang; Gert Lubec (pp. 1043-1049).

Granulocyte-colony stimulating factor stimulates production and antibacterial function of neutrophiles. Therapy using the recombinant protein drug represents a major step forward in oncology. The protein has not been, however, completely sequenced at the protein level and this formed the rationale of the current study. Recombinant G-CSF (filgrastim) was run on two-dimensional gel electrophoresis (2DE), the protein was in-gel digested with trypsin and chymotrypsin, and peptides were analysed on Nano-ESI-LC–MS/MS (high performance ion trap, HCT). Bioinformatic tools used were Mascot v2.2 and Modiro^TM v1.1 softwares. A single spot was detected on 2DE and peptides resulting from in-gel digestion were unambiguously identified by the MS/MS approach leading to complete sequencing when both searching engines were applied. N-terminal methionine loss, N-terminal methionine oxidation and amidination were observed. Both softwares identified modifications. Complete sequencing by a non-sophisticated and rapid gel-based mass spectrometry approach confirmed the primary structure predicted from nucleic acid sequences. A chemical modification of glutamine 26 with the interim name PentylamineBiotin (Unimod accession number #800) compatible with biotinylation with 5-(biotinamido) pentylamine by the producer was detected by both softwares. Although there is some evidence that biotinylated G-CSF analogues are active, it remains open whether this modification may be responsible for the side effects observed or lead to changes of antigenicity.

Keywords: Two-dimensional electrophoresis; Filgrastim; PentylamineBiotin; Complete sequencing; Mascot; Modiro

β-alanine elevates dopamine levels in the rat nucleus accumbens: antagonism by strychnine by Mia Ericson; Rhona B. C. Clarke; PeiPei Chau; Louise Adermark; Bo Söderpalm (pp. 1051-1055).

Glycine receptors (GlyRs) in the nucleus accumbens (nAc) have recently been suggested to be involved in the reinforcing and dopamine-elevating properties of ethanol via a neuronal circuitry involving the VTA. Apart from ethanol, both glycine and taurine have the ability to modulate dopamine output via GlyRs in the same brain region. In the present study, we wanted to explore whether yet another endogenous ligand for the GlyR, β-alanine, had similar effects. To this end, we monitored dopamine in the nAc by means of in vivo microdialysis and found that local perfusion of β-alanine increased dopamine output. In line with previous observations investigating ethanol, glycine and taurine, the competitive GlyR antagonist strychnine completely blocked the dopamine elevation. The present results suggest that β-alanine has the ability to modulate dopamine levels in the nAc via strychnine-sensitive GlyRs, and are consistent with previous studies suggesting the importance of this receptor for modulating dopamine output.

Keywords: Nucleus accumbens; Dopamine; Glycine receptor; Rat

Analogues of both Leu- and Met-enkephalin containing a constrained dipeptide isostere prepared from a Baylis-Hillman adduct by Roberta Galeazzi; Gianluca Martelli; Eleonora Marcucci; Mario Orena; Samuele Rinaldi; Roberta Lattanzi; Lucia Negri (pp. 1057-1065).

An efficient route was developed for the synthesis of the Fmoc-protected dipeptide 4, isostere of Gly-Gly containing an α-methylene β-amino acid; the conformationally restricted analogues of Leu-enkephalin, 3a, and Met-enkephalin, 3b, respectively, were prepared by changing 4 for Gly²-Gly³ in the native compounds 3a and 3b whose biological activities were significantly lower than the parent compounds.

Keywords: Isostere; Dipeptide; Conformational restriction; Baylis–Hillman; Activity

NMDA receptor activation induces differential epigenetic modification of Bdnf promoters in hippocampal neurons by Feng Tian; Ann M. Marini; Robert H. Lipsky (pp. 1067-1074).

Transcriptional regulation of the gene encoding brain-derived neurotrophic factor (BDNF) has been widely studied. However, an understanding of mechanisms modifying chromatin, events that are essential for controlling transcription, is rudimentary. We focused on two activation-dependent regions of the Bdnf gene physically linked to known transcription sites for exons 1 and 4. Using chromatin immunoprecipitation assays, we determined that N-methyl-d-aspartate (NMDA) receptor activation derepressed promoters 1 and 4-mediated transcription. This derepression correlated with reduced occupancy by histone deacetylase 1 and methyl cytosine-binding protein 2 of each promoter region near known transcription start sites in cultured hippocampal neurons. These changes did not occur at all sites upstream of transcription initiation. Taken together, these findings suggest that histone and other DNA-binding proteins are involved in remodeling of chromatin at some, but not all sites, within Bdnf promoters 1 and 4 and are associated with NMDA receptor-dependent increases in transcription.

Keywords: Brain-derived neurotrophic factor; BDNF; Chromatin remodeling; Epigenetic modification; Histone; Methylation; HDAC; MeCP2

Label-free quantitation, an extension to 2DB by Jens Allmer (pp. 1075-1087).

Determining the differential expression of proteins under different conditions is of major importance in proteomics. Since mass spectrometry-based proteomics is often used to quantify proteins, several labelling strategies have been developed. While these are generally more precise than label-free quantitation approaches, they imply specifically designed experiments which also require knowledge about peptides that are expected to be measured and need to be modified. We recently designed the 2DB database which aids storage, analysis, and publication of data from mass spectrometric experiments to identify proteins. This database can aid identifying peptides which can be used for quantitation. Here an extension to the database application, named MSMAG, is presented which allows for more detailed analysis of the distribution of peptides and their associated proteins over the fractions of an experiment. Furthermore, given several biological samples in the database, label-free quantitation can be performed. Thus, interesting proteins, which may warrant further investigation, can be identified en passant while performing high-throughput proteomics studies.

Keywords: Quantitation; Quantification; Label-free; Software; MS/MS; Spectral count

Cysteine, thiourea and thiocyanate interactions with clays: FT-IR, Mössbauer and EPR spectroscopy and X-ray diffractometry studies by Henrique de Santana; Andrea Paesano Jr.; Antonio C. S. da Costa; Eduardo di Mauro; Ivan G. de Souza; Flávio F. Ivashita; Cláudio M. D. de Souza; Cássia T. B. V. Zaia; Dimas A. M. Zaia (pp. 1089-1099).

The present study examined the adsorption of cysteine, thiourea and thiocyanate on bentonite and montmorillonite at two different pHs (3.00, 8.00). The conditions used here are closer to those of prebiotic earth. As shown by FT-IR, Mössbauer and EPR spectroscopy and X-ray diffractometry, the most important finding of this work is that cysteine and thiourea penetrate into the interlayer of the clays and reduce Fe³⁺ to Fe²⁺, and as consequence, cystine and c,c′-dithiodiformamidinium ion are formed. This mechanism resembles that which occurs with aconitase. This is a very important result for prebiotic chemistry; we should think about clays not just sink of molecules, but as primitive vessels of production of biomolecules. At pH 8.00, an increasing expansion was observed in the following order for both minerals: thiourea > thiocyanate > cysteine. At pH 3.00, the same order was not observed and thiourea had an opposite behavior, being the compound producing the lowest expansion. Mössbauer spectroscopy showed that at pH 8.00, the proportion of Fe²⁺ ions in bentonite increased, doubling for thiourea, or more than doubling for cysteine, in both clays. However, at pH 3.00, cysteine and thiourea did not change significantly the relative amount of Fe²⁺ and Fe³⁺ ions, when compared to clays without adsorption. For thiocyanate, the amount of Fe²⁺ produced was independent of the pH or clay used, probably because the interlayers of clays are very acidic and HSCN formed does not reduce Fe³⁺ to Fe²⁺. For the interaction of thiocyanate with the clays, it was not possible to identify any potential compound formed. For the samples of bentonite and montmorillonite at pH 8.00 with cysteine, EPR spectroscopy showed that intensity of the lines due to Fe³⁺ decreased because the reaction of Fe³⁺/cysteine. Intensity of EPR lines did not change when the samples of bentonite at pH 3.00 with and without cysteine were compared. These results are in accordance with those obtained using Mössbauer and FT-IR spectroscopy.

Keywords: Adsorption; FT-IR spectroscopy; Mössbauer spectroscopy; X-ray diffractometry; EPR spectroscopy; Clays

Generation of reactive oxygen species by beta amyloid fibrils and oligomers involves different intra/extracellular pathways by Cenini Giovanna; Cristina Cecchi; Anna Pensalfini; Sara Anna Bonini; Giulia Ferrari-Toninelli; Gianfranco Liguri; Maurizio Memo; Daniela Uberti (pp. 1101-1106).

A neuropathological characteristic of Alzheimer’s disease is the extracellular accumulation of amyloid beta peptide (Aβ) in neuritic plaques. Recent evidences suggested that soluble Aβ oligomers are the predominant neurotoxic species for neurons. Thus, considerable attention has been paid to discriminate the cytotoxic pathways of Aβ pre-fibrillar aggregates and mature fibrils. We showed that the mechanisms by which Aβ oligomers and fibrils generated reactive oxygen species differ in terms of site of production and kinetics, suggesting the involvement of different intra/extracellular pathways.

Keywords: Bet amyloid; Neurotoxicity; Free radicals; Oligomers

Generation of reactive oxygen species by beta amyloid fibrils and oligomers involves different intra/extracellular pathways by Giovanna Cenini; Cristina Cecchi; Anna Pensalfini; Sara Anna Bonini; Giulia Ferrari-Toninelli; Gianfranco Liguri; Maurizio Memo; Daniela Uberti (pp. 1107-1107).

Post-exercise carbohydrate plus whey protein hydrolysates supplementation increases skeletal muscle glycogen level in rats by Masashi Morifuji; Atsushi Kanda; Jinichiro Koga; Kentaro Kawanaka; Mitsuru Higuchi (pp. 1109-1115).

Recent studies showed that a combination of carbohydrate and protein was more effective than carbohydrate alone for replenishing muscle glycogen after exercise. However, it remains to be unclear whether the source or degree of hydrolysis of dietary protein influences post-exercise glycogen accumulation. The aim of this study was to compare the effect of dietary protein type on glycogen levels in the post-exercise phase, and to investigate the effects of post-exercise carbohydrate and protein supplementation on phosphorylated enzymes of Akt/PKB and atypical PKCs. Male Sprague-Dawley rats, trained for 3 days, swam with a 2% load of body weight for 4 h to deplete skeletal muscle glycogen. Immediately after the glycogen-depleting exercise, one group was killed, whereas the other groups were given either glucose or glucose plus protein (whey protein, whey protein hydrolysates (WPH), casein hydrolysates or branched-chain amino acid (BCAA) solutions. After 2 h, the rats were killed, and the triceps muscles quickly excised. WPH caused significant increases in skeletal muscle glycogen level (5.01 ± 0.24 mg/g), compared with whey protein (4.23 ± 0.24 mg/g), BCAA (3.92 ± 0.18 mg/g) or casein hydrolysates (2.73 ± 0.22 mg/g). Post-exercise ingestion of glucose plus WPH significantly increased both phosphorylated Akt/PKB (131%) and phosphorylated PKCζ (154%) levels compared with glucose only. There was a significant positive correlation between skeletal muscle glycogen content and phosphorylated Akt/PKB (r = 0.674, P < 0.001) and PKCζ (r = 0.481, P = 0.017). Post-exercise supplementation with carbohydrate and WPH increases skeletal muscle glycogen recovery by activating key enzymes such as Akt/PKB and atypical PKCs.

Keywords: Whey protein hydrolysates; Skeletal muscle glycogen; Exercise

Transgenic manipulation of a single polyamine in poplar cells affects the accumulation of all amino acids by Sridev Mohapatra; Rakesh Minocha; Stephanie Long; Subhash C. Minocha (pp. 1117-1129).

The polyamine metabolic pathway is intricately connected to metabolism of several amino acids. While ornithine and arginine are direct precursors of putrescine, they themselves are synthesized from glutamate in multiple steps involving several enzymes. Additionally, glutamate is an amino group donor for several other amino acids and acts as a substrate for biosynthesis of proline and γ-aminobutyric acid, metabolites that play important roles in plant development and stress response. Suspension cultures of poplar (Populus nigra × maximowiczii), transformed with a constitutively expressing mouse ornithine decarboxylase gene, were used to study the effect of up-regulation of putrescine biosynthesis (and concomitantly its enhanced catabolism) on cellular contents of various protein and non-protein amino acids. It was observed that up-regulation of putrescine metabolism affected the steady state concentrations of most amino acids in the cells. While there was a decrease in the cellular contents of glutamine, glutamate, ornithine, arginine, histidine, serine, glycine, cysteine, phenylalanine, tryptophan, aspartate, lysine, leucine and methionine, an increase was seen in the contents of alanine, threonine, valine, isoleucine and γ-aminobutyric acid. An overall increase in percent cellular nitrogen and carbon content was also observed in high putrescine metabolizing cells compared to control cells. It is concluded that genetic manipulation of putrescine biosynthesis affecting ornithine consumption caused a major change in the entire ornithine biosynthetic pathway and had pleiotropic effects on other amino acids and total cellular carbon and nitrogen, as well. We suggest that ornithine plays a key role in regulating this pathway.

Keywords: Genetic manipulation; Polyamines; Ornithine decarboxylase; Poplar

Mass spectrometrical characterisation of mouse and rat synapsin isoforms 2a and 2b by Sung Ung Kang; Ming Zhang; Miguel Burgos; Gert Lubec (pp. 1131-1143).

Synapsin 2 proteins are key elements of the synaptic machinery and still hold the centre stage in neuroscience research. Although fully sequenced at the nucleic acid level in mouse and rat, structural information on amino acid sequences and post-translational modifications (PTMs) is limited. Knowledge on protein sequences and PTMs, however, is mandatory for several purposes including conformational studies and the generation of antibodies. Hippocampal proteins from rat and mouse were extracted, run on two-dimensional gel electrophoresis and multi-enzyme digestion was carried out to generate peptides for mass spectrometrical analysis [nano-LC-ESI-(CID/ETD)-MS/MS]. As much as 12 synapsin 2 proteins (6 alpha and 6 beta isoforms) in the mouse and 13 synapsin 2 proteins (6 alpha and 7 beta isoforms) were observed in the rat. Protein sequences were highly identical to nucleic acid sequences, and only few amino acid exchanges probably representing polymorphisms or sequence conflicts were detected. Mouse and rat synapsins 2a differed in three amino acids, while rat and mouse synapsins 2b differed in two amino acids. As much as 13 phosphorylation sites were determined by MS/MS data in rat and mouse hippocampus and 5 were verified by phosphatase treatment. These findings are important for interpretation of previous results and design of future studies on synapsins.

Keywords: Amino acid substitutions; Multi-enzyme digestion; Synapsin 2 isoforms; Synapsin phosphorylation; Two-dimensional gel electrophoresis

Suppressive effect of dexamethasone on TIMP-1 production involves murine osteoblastic MC3T3-E1 cell apoptosis by Hui Xie; Ling-Li Tang; Xiang-Hang Luo; Xi-Yu Wu; Xian-Ping Wu; Hou-De Zhou; Ling-Qing Yuan; Er-Yuan Liao (pp. 1145-1153).

High dose glucocorticoid (GC) treatment induces osteoporosis partly via increasing osteoblast apoptosis. However, the mechanism of GC-induced apoptosis has not been fully elucidated. Osteoblast-derived tissue inhibitor of metalloproteinase-1 (TIMP-1) was recently reported to be involved in bone metabolism. Our previous study demonstrated that TIMP-1 suppressed apoptosis of the mouse bone marrow stromal cell line MBA-1 (pre-osteoblast) induced by serum deprivation. Therefore, we tested the effect of the GC dexamethasone (Dex) on TIMP-1 production in murine osteoblastic MC3T3-E1 cells and further determined whether this action is associated with Dex-induced osteoblast apoptosis. Dex decreased TIMP-1 production in MC3T3-E1 cells, and this effect was blocked by the glucocorticoid receptor (GR) antagonists, RU486 and RU40555. Recombinant TIMP-1 protein reduced caspase-3 activation and apoptosis induced by Dex in MC3T3-E1 cells. In addition, the pro-apoptotic effect of the Dex was augmented by suppression of TIMP-1 with siRNA. Furthermore, mutant TIMP-1, which has no inhibitory effects on MMPs, yet protects MC3T3-E1 cells against Dex-induced apoptosis. Our study demonstrates that Dex suppresses TIMP-1 production in osteoblasts through GR, and this effect is associated with its induction of osteoblast apoptosis. The anti-apoptotic action of TIMP-1 is independent of its inhibitory effects on MMPs activities. The decrease in TIMP-1 production caused by Dex may contribute to the mechanisms of Dex-induced bone loss.

Keywords: Dexamethasone; Tissue inhibitor of metalloproteinase-1; Apoptosis; Osteoblast

Inhibitors of N ^α-acetyl-l-ornithine deacetylase: synthesis, characterization and analysis of their inhibitory potency by J. Hlaváček; J. Pícha; V. Vaněk; J. Jiráček; J. Slaninová; V. Fučík; M. Buděšínský; D. Gilner; R. C. Holz (pp. 1155-1164).

A series of N ^α-acyl (alkyl)- and N ^α-alkoxycarbonyl-derivatives of l- and d-ornithine were prepared, characterized, and analyzed for their potency toward the bacterial enzyme N ^α-acetyl-l-ornithine deacetylase (ArgE). ArgE catalyzes the conversion of N ^α-acetyl-l-ornithine to l-ornithine in the fifth step of the biosynthetic pathway for arginine, a necessary step for bacterial growth. Most of the compounds tested provided IC₅₀ values in the μM range toward ArgE, indicating that they are moderately strong inhibitors. N ^α-chloroacetyl-l-ornithine (1g) was the best inhibitor tested toward ArgE providing an IC₅₀ value of 85 μM while N ^α-trifluoroacetyl-l-ornithine (1f), N ^α-ethoxycarbonyl-l-ornithine (2b), and N ^α-acetyl-d-ornithine (1a) weakly inhibited ArgE activity providing IC₅₀ values between 200 and 410 μM. Weak inhibitory potency toward Bacillus subtilis-168 for N ^α-acetyl-d-ornithine (1a) and N ^α-fluoro- (1f), N ^α-chloro- (1g), N ^α-dichloro- (1h), and N ^α-trichloroacetyl-ornithine (1i) was also observed. These data correlate well with the IC₅₀ values determined for ArgE, suggesting that these compounds might be capable of getting across the cell membrane and that ArgE is likely the bacterial enzymatic target.

Keywords: ArgE inhibitors; Acetylornithine derivatives; Synthesis; Inhibitory and antibacterial activity

Daily pattern of pituitary glutamine, glutamate, and aspartate content disrupted by cadmium exposure by Ana Caride; Belén Fernández Pérez; Teresa Cabaleiro; Anunciación Lafuente (pp. 1165-1172).

Cadmium is a neurotoxic heavy metal and is considered endocrine disruptor. In this work, we investigate the effects of cadmium on the 24 h changes of aspartate, glutamate, and glutamine content in the pituitary. Adult male Sprague–Dawley rats were treated with 25 or 50 mg/l of cadmium chloride (CdCl₂) in the drinking water for 30 days. Metal exposure with the lowest dose induced the disappearance of the nocturnal peak of anterior pituitary amino acid content, and the appearance of a peak of glutamine concentration during the resting phase of the photoperiod. After exposure to 50 mg/l of CdCl₂, the peaks of anterior pituitary amino acid content at 12:00 and 00:00 h disappeared, and two minimal values at these same hours and a peak at 08:00 h appeared. In the posterior pituitary, cadmium treatment with the lowest dose induced the appearance of a peak of aspartate and glutamate concentration at 12:00 h, and the disappearance of the peak of glutamine content at 16:00 h. After exposure to 50 mg/l of CdCl₂ aspartate and glutamate daily pattern presented two maximal values between 00:00 and 04:00 h, and the metal abolished glutamine daily pattern. These results suggest that cadmium disrupted aspartate, glutamate, and glutamine daily pattern in the pituitary.

Keywords: Cadmium; Pituitary; Daily pattern; Aspartate; Glutamate; Glutamine

Enhancement of menadione stress tolerance in yeast by accumulation of hypotaurine and taurine: co-expression of cDNA clones, from Cyprinus carpio, for cysteine dioxygenase and cysteine sulfinate decarboxylase in Saccharomyces cerevisiae by Ken-ichi Honjoh; Kanae Matsuura; Takeshi Machida; Koutarou Nishi; Miki Nakao; Tomoki Yano; Takahisa Miyamoto; Masayoshi Iio (pp. 1173-1183).

Taurine is known to function as a protectant against various stresses in animal cells. In order to utilize taurine as a compatible solute for stress tolerance of yeast, isolation of cDNA clones for genes encoding enzymes involved in biosynthesis of taurine was attempted. Two types of cDNA clones corresponding to genes encoding cysteine dioxygenase (CDO1 and CDO2) and a cDNA clone for cysteine sulfinate decarboxylase (CSD) were isolated from Cyprinus carpio. Deduced amino acid sequences of the two CDOs and that of CSD showed high similarity to those of CDOs and those of CSDs from other organisms, respectively. The coding regions of CDO1, CDO2, and CSD were subcloned into an expression vector, pESC-TRP, for Saccharomyces cerevisiae. Furthermore, to enhance the efficiency of synthesis of taurine in S. cerevisiae, a CDO–CSD fusion was designed and expressed. Expression of CDO and CSD proteins, or the CDO–CSD fusion protein was confirmed by Western blot analysis. HPLC analysis showed that the expression of the proteins led to enhancement of the accumulation level of hypotaurine, a precursor of taurine, rather than taurine. The yeast cells expressing corresponding genes showed tolerance to oxidative stress induced by menadione, but not to freezing–thawing stress.

Keywords: Cyprinus carpio ; Hypotaurine; Saccharomyces cerevisiae ; Stress tolerance; Taurine

Computational structure–activity study directs synthesis of novel antitumor enkephalin analogs by M. Gredičak; F. Supek; M. Kralj; Z. Majer; M. Hollósi; T. Šmuc; K. Mlinarić-Majerski; Š. Horvat (pp. 1185-1191).

The capability of a Support Vector Machines QSAR model to predict the antiproliferative ability of small peptides was evaluated by screening a virtual library of enkephalin-like analogs modified by incorporation of the (R,S)-(1-adamantyl)glycine (Aaa) residue. From an initial set of 390 compounds, the peptides, Tyr-Aaa-Gly-Phe-Met (2), Tyr-Aaa-Gly-Phe-Phe (3), Phe-Aaa-Gly-Phe-Phe (4) and Phe-Aaa-Gly-Phe-Met (5) were selected, synthesized and their antitumor activity was tested and compared to that of Met-enkephalin (1). The antiproliferative activity correlated with the computational prediction and with the foldamer-forming ability of the studied peptides. The most active compounds were the hydrophobic peptides, Phe-Aaa-Gly-Phe-Phe (4) and Phe-Aaa-Gly-Phe-Met (5), having a greater propensity to adopt folded structures than the other peptides.

Keywords: Enkephalin analogs; (1-Adamantyl)glycine; QSAR study; CD spectroscopy; In vitro antiproliferative activity; Peptide synthesis

Effect of Red Bull energy drink on cardiovascular and renal function by Frances R. Ragsdale; Tyler D. Gronli; Narjes Batool; Nicole Haight; April Mehaffey; Erin C. McMahon; Thomas W. Nalli; Carla M. Mannello; Crystal J. Sell; Patrick J. McCann; Gary M. Kastello; Tisha Hooks; Ted Wilson (pp. 1193-1200).

Energy drink consumption has been anecdotally linked to the development of adverse cardiovascular effects in consumers, although clinical trials to support this link are lacking. The effects of Red Bull^® energy drink on cardiovascular and neurologic functions were examined in college-aged students enrolled at Winona State University. In a double-blind experiment where normal calorie and low calorie Red Bull^® were compared to normal and low calorie placebos, no changes in overall cardiovascular function nor blood glucose (mg/dL) were recorded in any participant (n = 68) throughout a 2-h test period. However, in the second experiment, nine male and twelve female participants subjected to a cold pressor test (CPT) before and after Red Bull^® consumption showed a significant increase in blood sugar levels pre- and post Red Bull^® consumption. There was a significant increase in diastolic blood pressure of the male volunteers immediately after submersion of the hand in the 5°C water for the CPT. Under the influence of Red Bull^®, the increase in diastolic pressure for the male participants during the CPT was negated. There were no significant changes in the blood pressure of the female participants for the CPT with or without Red Bull^®. Finally, the CPT was used to evaluate pain threshold and pain tolerance before and after Red Bull^® consumption. Red Bull^® consumption was associated with a significant increase in pain tolerance in all participants. These findings suggest that Red Bull^® consumption ameliorates changes in blood pressure during stressful experiences and increases the participants’ pain tolerance.

Keywords: Energy drink; Red Bull; Caffeine; EKG; Pain

Predicting subcellular location of apoptosis proteins based on wavelet transform and support vector machine by Jian-Ding Qiu; San-Hua Luo; Jian-Hua Huang; Xing-Yu Sun; Ru-Ping Liang (pp. 1201-1208).

Apoptosis proteins have a central role in the development and homeostasis of an organism. These proteins are very important for understanding the mechanism of programmed cell death. As a result of genome and other sequencing projects, the gap between the number of known apoptosis protein sequences and the number of known apoptosis protein structures is widening rapidly. Because of this extremely unbalanced state, it would be worthwhile to develop a fast and reliable method to identify their subcellular locations so as to gain better insight into their biological functions. In view of this, a new method, in which the support vector machine combines with discrete wavelet transform, has been developed to predict the subcellular location of apoptosis proteins. The results obtained by the jackknife test were quite promising, and indicated that the proposed method can remarkably improve the prediction accuracy of subcellular locations, and might also become a useful high-throughput tool in characterizing other attributes of proteins, such as enzyme class, membrane protein type, and nuclear receptor subfamily according to their sequences.

Keywords: Apoptosis protein; Subcellular location; Discrete wavelet transform; Support vector machines; Hydrophobicity

Toward quantitative characterization of the binding profile between the human amphiphysin-1 SH3 domain and its peptide ligands by Ping He; Wei Wu; Hai-Dong Wang; Kang Yang; Ke-Long Liao; Wei Zhang (pp. 1209-1218).

A new structure-based approach was proposed to quantitatively characterize the binding profile of human amphiphysin-1 (hAmph1) SH3 domain–peptide complexes. In this protocol, the protein/peptide atoms were classified into 16 types in terms of their physicochemical meaning and biological function, and then a 16 × 16 atom-pair interaction matrix was constructed to describe 256 atom-pair types between the SH3 domain and the peptide ligand, with atoms from peptide and SH3 domain served as the matrix columns and rows, respectively. Three non-covalent effects dominating SH3 domain–peptide binding as electrostatic, van der Waals (steric) and hydrophobic interactions were separately calculated for the 256 atom-pair types. As a result, 768 descriptors coding detailed information about SH3 domain–peptide interactions were yielded for further statistical modeling and analysis. Based on a culled data set consisting of 592 samples with known affinities, we employed this approach, coupled with partial least square (PLS) regression and genetic algorithm (GA), to predict and to interpret the peptide-binding behavior to SH3 domain. In comparison with the previous works, our method is more capable of capturing important factors in the SH3 domain-peptide binding, thus, yielding models with better statistical performance. Furthermore, the optimal GA/PLS model indicates that the electrostatic effect plays a crucial role in SH3 domain–peptide complexes, and steric contact and hydrophobic force also contribute significantly to the binding.

Keywords: hAmph1 SH3 domain; Peptide; Non-binding interaction; Atom pair; Quantitative structure–affinity relationship

Histamine modulates the cellular stress response in yeast by Basil Delitheos; Konstantinos Papamichael; Ekaterini Tiligada (pp. 1219-1226).

The cellular stress response is a universal protective reaction to adverse environmental or microenvironmental conditions, such as heat and drugs, associated in part with the highly conserved heat shock proteins (HSPs). Histamine is a key inflammatory mediator derived from l-histidine that governs vital cellular processes beyond inflammation, while recent evidence implies additional actions in both prokaryotes and eukaryotes. This study explored the possible role of histamine in the heat shock response in yeast, an established experimental model for the pharmacological investigation of the cellular stress response. The response was evaluated by determining growth and viability of post-logarithmic phase grown yeast cultures after heat shock at 53°C for 30 min. Thermal preconditioning at 37°C for 2 h served as a positive control. The effect of histamine was investigated following long-term administration through the post-logarithmic phase of growth or short-term administration for 2 h prior to heat shock. Short-term treatment with 1 mM histamine resulted in de novo protein synthesis-dependent acquisition of thermotolerance, while lower doses or long-term administration of histamine failed to induce the heat-resistant phenotype. Preliminary investigation of HSP104, HSP70 and HSP60 expression by western blotting showed an increase of these proteins after thermal preconditioning. However, a differential HSP and tubulin expression appeared to underlie the response of yeast cells to histamine. In conclusion, histamine was capable of inducing the adaptive phenotype, while the contribution of HSPs and tubulin and the potential implications remain largely elusive.

Keywords: Cellular stress response; Heat shock protein; Histamine; Tubulin; Yeast

l-Arginine stimulates proliferation and prevents endotoxin-induced death of intestinal cells by Bie Tan; Yulong Yin; Xiangfeng Kong; Peng Li; Xilong Li; Haijun Gao; Xinguo Li; Ruilin Huang; Guoyao Wu (pp. 1227-1235).

This study tested the hypothesis that l-arginine (Arg) may stimulate cell proliferation and prevent lipopolysaccharide (LPS)-induced death of intestinal cells. Intestinal porcine epithelial cells (IPEC-1) were cultured for 4 days in Arg-free Dulbecco’s modified Eagle’s-F12 Ham medium (DMEM-F12) containing 10, 100 or 350 μM Arg and 0 or 20 ng/ml LPS. Cell numbers, protein concentrations, protein synthesis and degradation, as well as mammalian target of rapamycin (mTOR) and Toll-like receptor 4 (TLR4) signaling pathways were determined. Without LPS, IPEC-1 cells exhibited time- and Arg-dependent growth curves. LPS treatment increased cell death and reduced protein concentrations in IPEC-1 cells. Addition of 100 and 350 μM Arg to culture medium dose-dependently attenuated LPS-induced cell death and reduction of protein concentrations, in comparison with the basal medium containing 10 μM Arg. Furthermore, supplementation of 100 and 350 μM Arg increased protein synthesis and reduced protein degradation in both control and LPS-treated IPEC-1 cells. Consistent with the data on cell growth and protein turnover, addition of 100 or 350 μM Arg to culture medium increased relative protein levels for phosphorylated mTOR and phosphorylated ribosomal protein S6 kinase-1, while reducing the relative levels of TLR4 and phosphorylated levels of nuclear factor-κB in LPS-treated IPEC-1 cells. These results demonstrate a protective effect of Arg against LPS-induced enterocyte damage through mechanisms involving mTOR and TLR4 signaling pathways, as well as intracellular protein turnover.

Keywords: Arginine; IPEC-1; Lipopolysaccharide; mTOR; TLR4; Protein turnover

A mouse protein interactome through combined literature mining with multiple sources of interaction evidence by Xiao Li; Haoyang Cai; Jiabao Xu; Sancheng Ying; Yizheng Zhang (pp. 1237-1252).

Protein–protein interactions (PPIs) play crucial roles in a number of biological processes. Recently, protein interaction networks (PINs) for several model organisms and humans have been generated, but few large-scale researches for mice have ever been made neither experimentally nor computationally. In the work, we undertook an effort to map a mouse PIN, in which protein interactions are hidden in enormous amount of biomedical literatures. Following a co-occurrence-based text-mining approach, a probabilistic model—naïve Bayesian was used to filter false-positive interactions by integrating heterogeneous kinds of evidence from genomic and proteomic datasets. A support vector machine algorithm was further used to choose protein pairs with physical interactions. By comparing with the currently available PPI datasets from several model organisms and humans, it showed that the derived mouse PINs have similar topological properties at the global level, but a high local divergence. The mouse protein interaction dataset is stored in the Mouse protein–protein interaction DataBase (MppDB) that is useful source of information for system-level understanding of gene function and biological processes in mammals. Access to the MppDB database is public available at http://bio.scu.edu.cn/mppi .

Keywords: Interactome; Mouse; Protein interaction network; Protein–protein interaction

Microwave-assisted synthesis and characterization of optically active poly (ester-imide)s incorporating l-alanine by Saeed Zahmatkesh; Abdol R. Hajipour (pp. 1253-1260).

Pyromellitic dianhydride (1) was reacted with L-alanine (2) to result [N,N′-(pyromellitoyl)-bis-l-alanine diacid] (3). This compound (3) was converted to N,N′-(pyromellitoyl)-bis-l-alanine diacyl chloride (4) by reaction with thionyl chloride. The microwave-assisted polycondensation of this diacyl chloride (4) with polyethyleneglycol-diol (PEG-200) and/or three synthetic aromatic diols furnish a series of new PEIs and Co-PEIs in a laboratory microwave oven (Milestone). The resulting polymers and copolymers have inherent viscosities in the range of 0.31–0.53 dl g⁻¹. These polymers are optically active, thermally stable and soluble in polar aprotic solvents such as DMF, DMSO, NMP, DMAc, and sulfuric acid. All of the above polymers were fully characterized by IR spectroscopy, ¹H NMR spectroscopy, elemental analyses, specific rotation and thermal analyses. Some structural characterizations and physical properties of these optically active PEIs and Co-PEIs have been reported.

Keywords: Optically active; Poly (ester-imide); Microwave-assisted; Pyromellitic dianhydride

EBP50 exerts tumor suppressor activity by promoting cell apoptosis and retarding extracellular signal-regulated kinase activity by Jun-Fang Zheng; Li-Cui Sun; Hua Liu; Yan Huang; Yang Li; Junqi He (pp. 1261-1268).

The expression of Ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) and the intragenic mutation of the ebp50 gene have been reported to correlate with human breast cancer development, but the exact impacts on breast cancer development and its molecular mechanism are not fully understood. In this study, we investigate the potential function of EBP50 through over-expression in the breast cancer cell line, MDA-MB-231, which has low EBP50 protein expression levels. The effects of EBP50 over-expression on cellular proliferation, anchorage-independent growth and apoptosis were examined. In addition, the activity of extracellular signal-regulated kinase (ERK) was also determined. Our results show that a decrease of cellular proliferation and attenuation of colony-forming ability were evident in MDA-MB-231 cells stably transfected with an EBP50 expressing plasmid (EBP-231) when compared with control cells. There was also a statistically significant increase in spontaneous apoptosis in EBP-231 cells accompanied by an attenuation in ERK activity. Altogether, our results suggest that restoring EBP50 expression could suppress breast cancer cell proliferation by promoting cell apoptosis and inhibiting ERK activity, and that EBP50 may be a target for development of diagnostics and therapeutics in breast cancer.

Keywords: Anchorage-independent growth; Colony formation; Cell cycle; Apoptosis; MAPK

Role of the guanidine group in the N-terminal fragment of PTH(1–11) by Andrea Caporale; Iwona Woznica; Elisabetta Schievano; Stefano Mammi; Evaristo Peggion (pp. 1269-1275).

A series of PTH hybrids containing a diamine [NH₂(CH₂) nNH₂; n = 4, 5, 6] in the C-terminal position was synthesized based on the H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH₂ (Har = homoarginine) template. The compounds were pharmacologically characterized at PTH1R receptors for agonist activity.

Keywords: SPPS; CD; Peptidomimetics; PTH

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.38, #4)