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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.43, #3)

Special issue in quantitative mass spectrometric proteomics by Karin Barnouin (pp. 1005-1007).

Advances in phosphopeptide enrichment techniques for phosphoproteomics by Luisa Beltran; Pedro R. Cutillas (pp. 1009-1024).

Phosphoproteomics is increasingly used to address a wide range of biological questions. However, despite some success, techniques for phosphoproteomics are not without challenges. Phosphoproteins are present in cells in low abundance relative to their unphosphorylated counterparts; therefore phosphorylated proteins (or phosphopeptides after protein digestion) are rarely detected in standard shotgun proteomics experiments. Thus, extraction of phosphorylated polypeptides from complex mixtures is a critical step in the success of phosphoproteomics experiments. Intense research over the last decade has resulted in the development of powerful techniques for phosphopeptide enrichment prior to analysis by mass spectrometry. Here, we review how the development of IMAC, MOAC, chemical derivatization and antibody affinity purification and chromatography is contributing to the evolution of phosphoproteomics techniques. Although further developments are needed for the technology to reach maturity, current state-of-the-art techniques can already be used as powerful tools for biological research.

Keywords: Mass spectrometry; Proteomics; Systems biology; Cell signaling

Enrichment techniques employed in phosphoproteomics by Jan Fíla; David Honys (pp. 1025-1047).

Rapid changes of protein phosphorylation play a crucial role in the regulation of many cellular processes. Being post-translationally modified, phosphoproteins are often present in quite low abundance and tend to co-exist with their unphosphorylated isoform within the cell. To make their identification more practicable, the use of enrichment protocols is often required. The enrichment strategies can be performed either at the level of phosphoproteins or at the level of phosphopeptides. Both approaches have their advantages and disadvantages. Most enriching strategies are based on chemical modifications, affinity chromatography to capture peptides and proteins containing negatively charged phosphate groups onto a positively charged matrix, or immunoprecipitation by phospho-specific antibodies.In this article, the most up-to-date enrichment techniques are discussed, taking into account their optimization, and highlighting their advantages and disadvantages. Moreover, these methods are compared to each other, revealing their complementary nature in providing comprehensive coverage of the phosphoproteome.

Keywords: Phosphoproteomics; Enrichment; IMAC; MOAC; Titanium dioxide (TiO₂); Antibodies

Quantitative proteomics to decipher ubiquitin signaling by Ping-Chung Chen; Chan Hyun Na; Junmin Peng (pp. 1049-1060).

Ubiquitin signaling plays an essential role in controlling cellular processes in eukaryotes, and the impairment of ubiquitin regulation contributes to the pathogenesis of a wide range of human diseases. During the last decade, mass spectrometry-based proteomics has emerged as an indispensable approach for identifying the ubiquitinated proteome (ubiquitinome), ubiquitin modification sites, the linkages of complex ubiquitin chains, as well as the interactome of ubiquitin enzymes. In particular, implementation of quantitative strategies allows the detection of dynamic changes in the ubiquitinome, enhancing the ability to differentiate between function-relevant protein targets and false positives arising from biological and experimental variations. The profiling of total cell lysate and the ubiquitinated proteome in the same sets of samples has become a powerful tool, revealing a subset of substrates that are modulated by specific physiological and pathological conditions, such as gene mutations in ubiquitin signaling. This strategy is equally useful for dissecting the pathways of ubiquitin-like proteins.

Keywords: Ubiquitin; Proteasome; E3; DUB; Mass spectrometry; Proteomics; SILAC

Analysing signalling networks by mass spectrometry by Claus Jørgensen; Marie Locard-Paulet (pp. 1061-1074).

Sequence analysis of the human genome and the association of genetic aberrations with diseases have provided a rough framework whereby the impact of individual genotypes can be assessed. To fully understand the effect of individual and co-occurring genetic aberrations, as well as their individual and collected contribution to the development of diseases, it is critical to analyse the matching proteome and to determine how the organisation, expression level and function of protein networks are affected. Sensitive mass spectrometric platforms in combination with innovative workflows allow qualitative and quantitative analyses of the cellular as well as the extracellular proteome. Importantly, in addition to specifically identifying the content of the proteome, several aspects of the proteomic organisation can be analysed including protein complexes, protein modifications, enzymatic activities and subcellular/organelle localisation. Together, these measurements will provide novel insight into the biological effect of disease-causing mutations ultimately coupling genotype and phenotype.

Keywords: Signal transduction; Mass spectrometry; Protein kinase signalling

Key issues in the acquisition and analysis of qualitative and quantitative mass spectrometry data for peptide-centric proteomic experiments by Andrew J. Thompson; Mika Abu; Diane P. Hanger (pp. 1075-1085).

Proteomic technologies have matured to a level enabling accurate and reproducible quantitation of peptides and proteins from complex biological matrices. Analysis of samples as diverse as assembled protein complexes, whole cell lysates or sub-cellular proteomes from cell cultures, and direct analysis of animal and human tissues and fluids demonstrate the incredible versatility of the fundamental nature of the technique that forms the basis of most proteomic applications today (mass spectrometry). Determining the mass of biomolecules and their fragments or related products with high accuracy can convey a highly specific assay for detection and identification. Importantly, ion currents representative of these specifically identified analytes can be accurately quantified with the correct application of smart isobaric tagging chemistries, heavy and light isotopically derivatised samples or standards, or by careful application of workflows to compare unlabelled samples in so-called ‘label-free’ and targeted selected reaction monitoring experiments. In terms of exploring biology, a myriad of protein changes and modifications are being increasingly probed and quantified, including diverse chemical changes from relatively decisive modifications such as protein splicing and truncation, to more transient dynamic modifications such as phosphorylation, acetylation and ubiquitination. Proteomic workflows can be complex beasts and several key considerations to ensure effective applications have been outlined in the recent literature. The past year has witnessed the publication of several excellent reviews that thoroughly describe the fundamental principles underlying the state of the art. This review further elaborates on specific critical issues introduced by these publications and raises other important unaddressed considerations and new developments that directly impact on the effectiveness of proteomic technologies, in particular for, but not necessarily exclusive to peptide-centric experiments. These factors are discussed both in terms of qualitative analyses, including dynamic range and sampling issues, and developments to improve the translation of peptide fragmentation data into peptide and protein identities, as well as quantitative analyses, including data normalisation and the utility of ontology or functional annotation, the effects of modified peptides, and considered experimental design to facilitate the use of robust statistical methods.

Keywords: Proteomics; Peptide-centric; Mass spectrometry; Quantitation; Data analysis

Current challenges in software solutions for mass spectrometry-based quantitative proteomics by Salvatore Cappadona; Peter R. Baker; Pedro R. Cutillas; Albert J. R. Heck; Bas van Breukelen (pp. 1087-1108).

Mass spectrometry-based proteomics has evolved as a high-throughput research field over the past decade. Significant advances in instrumentation, and the ability to produce huge volumes of data, have emphasized the need for adequate data analysis tools, which are nowadays often considered the main bottleneck for proteomics development. This review highlights important issues that directly impact the effectiveness of proteomic quantitation and educates software developers and end-users on available computational solutions to correct for the occurrence of these factors. Potential sources of errors specific for stable isotope-based methods or label-free approaches are explicitly outlined. The overall aim focuses on a generic proteomic workflow.

Keywords: LC–MS; Quantitative proteomics; Quantification software; Stable isotope labeling; Label-free

Analytical strategies for the global quantification of intact proteins by Timothy S. Collier; David Charles Muddiman (pp. 1109-1117).

The quantification of intact proteins is a relatively recent development in proteomics. In eukaryotic organisms, proteins are present as multiple isoforms as the result of variations in genetic code, alternative splicing, post-translational modification and other processing events. Understanding the identities and biological functions of these isoforms and how their concentrations vary across different states is the central goal of proteomics. To date, the bulk of proteomics research utilizes a “bottom-up” approach, digesting proteins into their more manageable constitutive peptides, but sacrificing information about the specific isoform and combinations of post-translational modifications present on the protein. Very specific strategies for protein quantification such as the enzyme-linked immunosorbent assay and Western blot are commonplace in laboratories and clinics, but impractical for the study of global biological changes. Herein, we describe strategies for the quantification of intact proteins, their distinct advantages, and challenges to their employment. Techniques contained in this review include the more traditional and widely employed methodology of differential gel electrophoresis and more recently developed mass spectrometry-based techniques including metabolic labeling, chemical labeling, and label-free methodologies.

Keywords: Proteomics; Intact proteins; Quantitation; Mass spectrometry

Analysis of the Plasmodium falciparum proteasome using Blue Native PAGE and label-free quantitative mass spectrometry by Nicole Sessler; Karsten Krug; Alfred Nordheim; Benjamin Mordmüller; Boris Macek (pp. 1119-1129).

Detailed knowledge of the composition of protein complexes is crucial for the understanding of their structure and function; however, appropriate techniques for compositional analyses of complexes largely rely on elaborate tagging, immunoprecipitation, cross-linking and purification strategies. The proteasome is a prototypical protein complex and therefore an excellent model to assess new methods for protein complex characterisation. Here we evaluated the applicability of Blue Native (BN) PAGE in combination with label-free protein quantification and protein correlation profiling (PCP) for the investigation of proteasome complexes directly from biological samples. Using the purified human 20S proteasome we showed that the approach can accurately detect members of a complex by clustering their gel migration profiles. We applied the approach to address proteasome composition in the schizont stage of the malaria parasite Plasmodium falciparum. The analysis, performed in the background of the whole protein extract, revealed that all subunits comigrated and formed a tight cluster with a single maximum, demonstrating presence of a single form of the 20S proteasome. This study shows that BN PAGE in combination with label-free quantification and PCP is applicable to the analysis of multiprotein complexes directly from complex protein mixtures.

Keywords: BN PAGE; PCP; Protein complex; LTQ Orbitrap XL; Proteasome; Plasmodium falciparum

Human relaxin-2: historical perspectives and role in cancer biology by Vinojini B. Nair; Chrishan S. Samuel; Frances Separovic; Mohammed Akhter Hossain; John D. Wade (pp. 1131-1140).

One of the most recognised and studied family of peptide hormones is the insulin superfamily. Within this family is the relaxin subfamily which comprises seven members: relaxin-1, -2 and -3 and insulin-like peptides 3, 4, 5 and 6. Besides exhibiting sequence similarities, each member exists as an active A–B heterodimer linked by three disulfide bonds. This mini-review is divided into three broad themes: an overview of all insulin superfamily members (including structural similarities); roles of each superfamily member and finally, a focus on the pleiotropic peptide hormone, human relaxin-2. In addition to promoting vasodilatory effects leading to evaluation in Phase III clinical trials for the treatment of acute heart failure, relaxin has recently been shown to be highly expressed by cancer cells, aiding in their proliferation, invasiveness and metastasis. These contrary effects of relaxin are discussed together with current efforts in the development of relaxin antagonists that may possess future therapeutic potential for the treatment of certain cancers.

Keywords: H2 relaxin; Relaxin; RXFP1; Cancer; Tumour development

Advances in the study of protein–DNA interaction by Yu-Hang Cai; He Huang (pp. 1141-1146).

Protein–DNA interaction plays an important role in many biological processes. The classical methods and the novel technologies advanced have been developed for the interaction of protein–DNA. Recent developments of these methods and research achievements have been reviewed in this paper.

Keywords: Protein–DNA interaction; Biotechnology; EMSA; SELEX

PD-sauvagine: a novel sauvagine/corticotropin releasing factor analogue from the skin secretion of the Mexican giant leaf frog, Pachymedusa dacnicolor by Yu Zhou; Yingchun Jiang; Ran Wang; Bing Bai; Mei Zhou; Tianbao Chen; Jiqun Cai; Lei Wang; Chris Shaw (pp. 1147-1156).

Sauvagine is a potent and broad-spectrum biologically active peptide of 40 amino acid residues originally isolated from the skin of the South American frog, Phyllomedusa sauvagei. Since its discovery, no additional sauvagine structures have been reported. Following the discovery of sauvagine, peptides with similar primary structures/activities were identified in mammalian brain [corticotropin-releasing factor (CRF) and urocortin]. Here, we report the identification of a second sauvagine from the Mexican giant leaf frog, Pachymedusa dacnicolor, which displays primary structural features of both sauvagine and CRF. A cDNA encoding the peptide precursor was “shotgun” cloned from a cDNA library constructed from lyophilised skin secretion by 3′- and 5′-RACE reactions. From this, the primary structure of a 38-mer peptide was deduced and this was located in reverse phase HPLC fractions of skin secretion and both its mass and structure were confirmed by mass spectrometry. The biological activities of synthetic replicates of PD-sauvagine and sauvagine were compared using two different mammalian smooth muscle preparations and the novel peptide was found to be more potent in both. Bioinformatic analyses of PD-sauvagine revealed that it shared different regional sequence identities with both sauvagine and CRF.

Keywords: Amphibian; Skin; Peptide; Molecular cloning; Smooth muscle

Modulation of innate immune-related pathways in nicotine-treated SH-SY5Y cells by Wen-Yan Cui; Ju Wang; Jinxue Wei; Junran Cao; Sulie L. Chang; Jun Gu; Ming D. Li (pp. 1157-1169).

Although nicotine has a broad impact on both the central and peripheral nervous systems, the molecular mechanisms remain largely unknown, especially at the signaling pathway level. To investigate that aspect, we employed both conventional molecular techniques, such as quantitative real-time PCR and Western blotting analysis, and high-throughput microarray approach to identify the genes and signaling pathways that are modulated by nicotine. We found 14 pathways significantly altered in SH-SY5Y neuroblastoma cells. Of these, the Toll-like receptor pathway (TLR; p = 2.57 × 10⁻⁴) is one of the most important innate immune pathways. The death receptor pathway (DR; p = 8.71 × 10⁻⁴), whose transducers coordinate TLR signals and help conduct the host immune response to infection, was also significantly changed by nicotine. Furthermore, we found that several downstream pathways of TLR and DR signaling, such as PI3K/AKT signaling (p = 9.55 × 10⁻⁶), p38 signaling (p = 2.40 × 10⁻⁶), and ERK signaling (p = 1.70 × 10⁻⁴), were also significantly modulated by nicotine. Interestingly, most of the differentially expressed genes in these pathways leading to nuclear factor κB (NF-κB) activation and those important inhibitors of pathways leading to apoptosis, including FLIP and Bcl-2, were up-regulated by nicotine. Taken together, our findings demonstrate that nicotine can regulate multiple innate immune-related pathways, and our data thus provide new clues to the molecular mechanisms underlying nicotine’s regulatory effects on neurons.

Keywords: Nicotine; Signaling; Immune system; SH-SY5Y

Effects of leucine and citrulline versus non-essential amino acids on muscle protein synthesis in fasted rat: a common activation pathway? by Servane Le Plénier; Stéphane Walrand; Richard Noirt; Luc Cynober; Christophe Moinard (pp. 1171-1178).

Leucine (LEU) is recognized as a major regulator of muscle protein synthesis (MPS). Citrulline (CIT) is emerging as a potent new regulator. The aim of our study was to compare MPS modulation by CIT and LEU in food-deprived rats and to determine whether their action was driven by similar mechanisms. Rats were either freely fed (F, n = 10) or food deprived for 18 h. Food-deprived rats were randomly assigned to one of four groups and received per os, i.e., gavage, saline (S, n = 10), l-leucine (1.35 g/kg, LEU, n = 10), l-citrulline (1.80 g/kg CIT, n = 10) or isonitrogenous non-essential amino acids (NEAA, n = 10). After gavage, the rats were injected with a flooding dose of [¹³C] valine to determine MPS. The rats were killed 50 min after the injection of the flooding dose. Blood was collected for amino acid, glucose and insulin determinations. Tibialis anterior muscles were excised for determination of MPS and for Western blot analyses of the PI3K/Akt, mTORC1, ERK1/2/MAPK pathways and AMP kinase component. MPS was depressed by 61% in starved rats (Saline vs. Fed, P < 0.05). Administration of amino acids (NEAA, LEU or CIT) completely abolished this decrease (NEAA, CIT, LEU vs. Fed, NS). Food deprivation affected the phosphorylation status of the mTORC1 pathway and AMP kinase (Saline vs. Fed, P < 0.05). LEU and CIT administration differently stimulated the mTORC1 pathway (LEU > CIT). LEU but not CIT increased the phosphorylation of rpS6 at serine 235/236. Our findings clearly demonstrated that both CIT and LEU were able to stimulate MPS, but this effect was likely related to the nitrogen load. LEU, CIT and NEAA may have different actions on MPS in this model as they share different mTORC1 regulation capacities.

Keywords: mTORC1 pathway; Fasting; Insulin

Continuous exposure to l-arginine induces oxidative stress and physiological tolerance in cultured human endothelial cells by Srinidi Mohan; Chia-Ching Wu; Soyoung Shin; Ho-Leung Fung (pp. 1179-1188).

The therapeutic benefits of l-arginine (ARG) supplementation in humans, often clearly observed in short-term studies, are not evident after long-term use. The mechanisms for the development of ARG tolerance are not known and cannot be readily examined in humans. We have developed a sensitive in vitro model using a low glucose/low arginine culture medium to study the mechanisms of ARG action and tolerance using two different human endothelial cells, i.e., Ea.hy926 and human umbilical venous endothelial cells. Cultured cells were incubated with different concentrations of ARG and other agents to monitor their effects on endothelial nitric oxide synthase (eNOS) expression and function, as well as glucose and superoxide (O ₂ ^·− ) accumulation. Short-term (2 h) exposure to at least 50 μM ARG moderately increased eNOS activity and intracellular glucose (p < 0.05), with no change in eNOS mRNA or protein expression. In contrast, 7-day continuous ARG exposure suppressed eNOS expression and activity. This was accompanied by increase in glucose and O ₂ ^·− accumulation. Co-incubation with 100 μM ascorbic acid, 300 U/ml polyethylene glycol-superoxide dismutase (PEG-SOD), 100 μM l-lysine or 30 μM 5-chloro-2-(N-2,5-dichlorobenenesulfonamido)-benzoxazole (a fructose-1,6-bisphosphatase inhibitor) prevented the occurrence of cellular ARG tolerance. Short-term co-incubation of ARG with PEG-SOD improved cellular nitrite accumulation without altering cellular ARG uptake. These studies suggest that ARG-induced oxidative stress may be a primary causative factor for the development of cellular ARG tolerance.

Keywords: Endothelial nitric oxide synthase; Oxidative stress; Nitric oxide; Glucose accumulation

Plant aminoaldehyde dehydrogenases oxidize a wide range of nitrogenous heterocyclic aldehydes by Jan Frömmel; Miroslav Soural; Martina Tylichová; David Kopečný; Gabriel Demo; Michaela Wimmerová; Marek Šebela (pp. 1189-1202).

The metabolic degradation of aldehydes is catalyzed by oxidoreductases from which aldehyde dehydrogenases (EC 1.2.1) comprise nonspecific or substrate-specific enzymes. The latter subset is represented, e.g., by NAD⁺-dependent aminoaldehyde dehydrogenases (AMADHs; EC 1.2.1.19) oxidizing a group of naturally occurring ω-aminoaldehydes including polyamine oxidation products. Recombinant isoenzymes from pea (PsAMADH1 and 2) and tomato (LeAMADH1 and 2) were subjected to kinetic measurements with synthetic aldehydes containing a nitrogenous heterocycle such as pyridinecarbaldehydes and their halogenated derivatives, (pyridinylmethylamino)-aldehydes, pyridinyl propanals and aldehydes derived from purine, 7-deazapurine and pyrimidine to characterize their substrate specificity and significance of the resulting data for in vivo reactions. The enzymatic production of the corresponding carboxylic acids was analyzed by liquid chromatography coupled to electrospray ionization mass spectrometry. Although the studied AMADHs are largely homologous and supposed to have a very similar active site architecture, significant differences were observed. LeAMADH1 displayed the broadest specificity oxidizing almost all compounds followed by PsAMADH2 and 1. In contrast, LeAMADH2 accepted only a few compounds as substrates. Pyridinyl propanals were converted by all isoenzymes, usually better than pyridinecarbaldehydes and aldehydes with fused rings. The K _m values for the best substrates were in the range of 10⁻⁵–10⁻⁴ M. Nevertheless, the catalytic efficiency values (V _max/K _m) reached only a very small fraction of that with 3-aminopropanal (except for LeAMADH1 activity with two pyridine-derived compounds). Docking experiments using the crystal structure of PsAMADH2 were involved to discuss differences in results with position isomers or alkyl chain homologs.

Keywords: Aldehyde; Aminoaldehyde dehydrogenase; 7-Deazapurine; Pyridine; Pyrimidine; Purine

Membrane-destabilizing activity of pH-responsive cationic lysine-based surfactants: role of charge position and alkyl chain length by Daniele Rubert Nogueira; Montserrat Mitjans; M. Carmen Morán; Lourdes Pérez; M. Pilar Vinardell (pp. 1203-1215).

Many strategies for treating diseases require the delivery of drugs into the cell cytoplasm following internalization within endosomal vesicles. Thus, compounds triggered by low pH to disrupt membranes and release endosomal contents into the cytosol are of particular interest. Here, we report novel cationic lysine-based surfactants (hydrochloride salts of N^ε- and N^α-acyl lysine methyl ester) that differ in the position of the positive charge and the length of the alkyl chain. Amino acid-based surfactants could be promising novel biomaterials in drug delivery systems, given their biocompatible properties and low cytotoxic potential. We examined their ability to disrupt the cell membrane in a range of pH values, concentrations and incubation times, using a standard hemolysis assay as a model of endosomal membranes. Furthermore, we addressed the mechanism of surfactant-mediated membrane destabilization, including the effects of each surfactant on erythrocyte morphology as a function of pH. We found that only surfactants with the positive charge on the α-amino group of lysine showed pH-sensitive hemolytic activity and improved kinetics within the endosomal pH range, indicating that the positive charge position is critical for pH-responsive behavior. Moreover, our results showed that an increase in the alkyl chain length from 14 to 16 carbon atoms was associated with a lower ability to disrupt cell membranes. Knowledge on modulating surfactant-lipid bilayer interactions may help us to develop more efficient biocompatible amino acid-based drug delivery devices.

Keywords: Lysine-based surfactants; Hemolysis; pH-sensitivity; Membrane disruption; Drug delivery

Synthesis and biological activity of new series of N-modified analogues of the N/OFQ(1–13)NH₂ with aminophosphonate moiety by Petar T. Todorov; Polina I. Mateeva; Rositza N. Zamfirova; Nikola D. Pavlov; Emilia D. Naydenova (pp. 1217-1223).

New series of N-modified analogues of the N/OFQ(1–13)NH₂ with aminophosphonate moiety have been synthesized and investigated for biological activity. These peptides were prepared by solid-phase peptide synthesis—Fmoc-strategy. The N/OFQ(1–13)NH₂ analogues were tested for agonistic activity in vitro on electrically stimulated rat vas deferens smooth-muscle preparations isolated from Wistar albino rats. Our study has shown that the selectivity of the peptides containing 1-[(methoxyphosphono)methylamino]cycloalkanecarboxylic acids to the N-side of Phe is not changed—they remain selective agonists of NOP receptors. The derivative with the largest ring (NOC-6) demonstrated efficacy similar to that of N/OFQ(1–13)NH₂, but in a 10-fold higher concentration. The agonistic activity of newly synthesized N-modified analogues of N/OFQ(1–13)NH₂ with aminophosphonate moiety was investigated for the first time.

Keywords: Nociceptin analogues; Nociceptin/orphanin FQ; NOP receptor; SPPS; Aminophosphonates; Rat vas deferens

Synthesis of hydroxydiamines and triamines via reductive cleavage of N–N bond in substituted pyrazolidines by Ludmila A. Sviridova; Galina A. Golubeva; Alexander N. Tavtorkin; Konstantin A. Kochetkov (pp. 1225-1231).

Aliphatic polyamines, being a versatile class of organic compounds, are widely used in many fields of medicine and organic chemistry. However, the general approach to the synthesis of chiral aliphatic polyamines has been still undeveloped. Here, we describe a new method for the synthesis of chiral trifunctional amino compounds, namely hydroxydiamines and triamines. The initial compounds, namely substituted hydroxy- or aminopyrazolidines and pyrazolines, are readily available using convenient stereoselective methods developed earlier by us. The proposed method allows synthesizing of chiral diaminoalcohols and triamines, which are the analogs of a well-known anti-TB drug, namely ethambutol, and cannot be obtained alternatively. The key step of the synthesis is N–N bond cleavage in substituted hydroxy- or aminopyrazolidines and pyrazolines with borane-tetrahydrofuran complex; other known methods for N–N bond cleavage turned out to be ineffective. The main advantage of the proposed method is the retention of a certain configuration of stereocenters in the course of the reaction. Six new chiral diasteomerically pure substituted hydroxydiamines and triamines and the enantiomerically pure triamine with four chiral centers were synthesized and characterized using NMR, IR and mass spectroscopy, as well as elemental analysis.

Keywords: Hydroxydiamines; Triamines; Ethambutol; Pyrazolines; Pyrazolidines; Diasteomerically pure polyamines

Protective effects of N-acetylcysteine on intestinal functions of piglets challenged with lipopolysaccharide by Yongqing Hou; Lei Wang; Wei Zhang; Zhenguo Yang; Binying Ding; Huiling Zhu; Yulan Liu; Yinsheng Qiu; Yulong Yin; Guoyao Wu (pp. 1233-1242).

The neonatal small intestine is susceptible to damage by endotoxin, but effective methods for prevention and treatment are lacking. N-acetylcysteine (NAC) is a widely used precursor of l-cysteine for animal cells and plays an important role in protecting cells against oxidative stress. This study was conducted with the lipopolysaccharide (LPS)-challenged piglet model to determine the effects of NAC on intestinal function. Eighteen piglets were randomly allocated into control, LPS and LPS + NAC groups. The control and LPS groups were fed a corn- and soybean meal-based diet, and the LPS + NAC group was fed the basal diet +500 mg/kg NAC. On days 10, 13 and 20 of the trial, the LPS and LPS + NAC groups received intraperitoneal administration of LPS (100 μg/kg BW), whereas the control piglets received saline. On day 20 of the trial, d-xylose (0.1 g/kg BW) was orally administrated to all piglets 2 h after LPS or saline injection, and blood samples were collected 1 h thereafter. One hour blood xylose test was used to measure intestinal absorption capacity and mucosal integrity, and diamine oxidase (DAO) was used as a marker of intestinal injury. On day 21 of the trial, pigs were killed to obtain the intestinal mucosa. Compared to the control, LPS challenge reduced (P < 0.05) the concentrations of d-xylose (a marker of intestinal absorption) in plasma, activities of DAO in the jejunal mucosa, the ratio of villus height to crypt depth in the jejunal mucosa, RNA/DNA and protein/DNA in the jejunal and ileal mucosae, while increasing (P < 0.05) DAO activity in plasma and caspase-3 expression in the intestinal mucosa. The adverse effects of LPS were partially ameliorated (P < 0.05) by NAC supplementation. Moreover, NAC prevented the LPS-induced decrease in claudin-1 and occludin expression in the jejunal and ileal mucosae. Collectively, these results indicate that dietary NAC supplementation alleviates the mucosal damage and improves the absorptive function of the small intestine in LPS-challenged piglets.

Keywords: N-acetylcysteine; Intestinal functions; Piglets; Lipopolysaccharide

Homocysteine enhances clot-promoting activity of endothelial cells via phosphatidylserine externalization and microparticles formation by Jiuxin Zhu; Rui Xie; Xianmei Piao; Yunlong Hou; Chongbao Zhao; Guofen Qiao; Baofeng Yang; Jialan Shi; Yanjie Lu (pp. 1243-1250).

Total elevated plasma homocysteine (Hcy) is a risk factor for thromboembolism. Vascular endothelium is important to regulate coagulation, but the impact of Hcy on the clot-promoting activity (CPA) of endothelial cells has not been fully understood. In our study, human umbilical vein endothelial cells (HUVECs) were treated with Hcy (8, 20, 80, 200, 800 μmol/L) for 24 h. Annexin V was utilized to detect phosphatidylserine (PS) externalization and endothelial microparticles (MPs) formation. CPA was assessed by recalcification time and purified clotting complex tests. We found that Hcy enhanced the externalized PS and consequent CPA of HUVECs in a dose-dependent fashion, effect of Hcy had statistical significance at 800 μmol/L. In addition, Hcy also increased the shedding of procoagulant endothelial MPs. Blocking of PS with 128 nmol/L annexin V reduced approximately 70% CPA of HUVECs and endothelial MPs, but human anti-tissue factor antibody had little inhibitive effect. Our results showed that Hcy increased CPA of HUVECs via PS externalization and MPs release. Our present study has implications for hyperhomocysteinemia-related hypercoagulability.

Keywords: Homocysteine; Endothelial cells; Phosphatidylserine; Microparticles; Clot-promoting activity; Annexin V

Detection of transglutaminase activity using click chemistry by Remon van Geel; Marjoke F. Debets; Dennis W. P. M. Löwik; Ger J. M. Pruijn; Wilbert C. Boelens (pp. 1251-1263).

Transglutaminase 2 (TG2) is a Ca²⁺-dependent enzyme able to catalyze the formation of ε(γ-glutamyl)-lysine crosslinks between polypeptides, resulting in high molecular mass multimers. We have developed a bioorthogonal chemical method for the labeling of TG2 glutamine-donor proteins. As amine-donor substrates we used a set of azide- and alkyne-containing primary alkylamines that allow, after being crosslinked to glutamine-donor proteins, specific labeling of these proteins via the azide-alkyne cycloaddition. We demonstrate that these azide- and alkyne-functionalized TG2 substrates are cell permeable and suitable for specific labeling of TG2 glutamine-donor substrates in HeLa and Movas cells. Both the Cu(I)-catalyzed and strain promoted azide-alkyne cycloaddition proved applicable for subsequent derivatization of the TG2 substrate proteins with the desired probe. This new method for labeling TG2 substrate proteins introduces flexibility in the detection and/or purification of crosslinked proteins, allowing differential labeling of cellular proteins.

Keywords: Transglutaminase; Click chemistry; Azide; Alkyne; Cycloaddition; Protein modification

l-Arginine improves multiple physiological parameters in mice exposed to diet-induced metabolic disturbances by Christoffer Clemmensen; Andreas N. Madsen; Sanela Smajilovic; Birgitte Holst; Hans Bräuner-Osborne (pp. 1265-1275).

l-Arginine (l-Arg) is a conditionally essential amino acid and a natural constituent of dietary proteins. Studies in obese rats and type 2 diabetic humans have indicated that dietary supplementation with l-Arg can diminish gain in white adipose tissue (WAT) and improve insulin sensitivity. However, the effects of l-Arg on glucose homeostasis, body composition and energy metabolism remain unclear. In addition, no studies have, to our knowledge, examined whether l-Arg has beneficial effects as a dietary supplement in the mouse model. In the present study, we investigated the effects of l-Arg supplementation to male C57BL/6 mice on an array of physiological parameters. l-Arg supplemented mice were maintained on a low-protein diet and body composition, appetite regulation, glucose tolerance, insulin sensitivity and energy expenditure were evaluated. A significant reduction in epididymal WAT was observed in l-Arg supplemented mice compared with mice fed an isocaloric control diet. Surprisingly, the l-Arg supplemented animals were hyperphagic corresponding to a highly significant decrease in feed efficiency, as body weight developed in a similar pattern in both experimental groups. Glucose homeostasis experiments revealed a major effect of l-Arg supplementation on glucose tolerance and insulin sensitivity, interestingly, independent of a parallel regulation in whole-body adiposity. Increased l-Arg ingestion also raised energy expenditure; however, no concurrent effect on locomotor activity, substrate metabolism or expression of uncoupling proteins (UCP1 and UCP2) in adipose tissues was displayed. In conclusion, dietary l-Arg supplementation substantially affects an array of metabolic-associated parameters including a reduction in WAT, hyperphagia, improved insulin sensitivity and increased energy expenditure in mice fed a low-protein diet.

Keywords: l-Arginine; Diet; Amino acids; Low protein; Insulin sensitivity; Obesity; Diabetes

Diffuse reflectance spectroscopy of fibrous proteins by Keith R. Millington (pp. 1277-1285).

UV–visible diffuse reflectance (DR) spectra of the fibrous proteins wool and feather keratin, silk fibroin and bovine skin collagen are presented. Natural wool contains much higher levels of visible chromophores across the whole visible range (700–400 nm) than the other proteins and only those above 450 nm are effectively removed by bleaching. Both oxidative and reductive bleaching are inefficient for removing yellow chromophores (450–400 nm absorbers) from wool. The DR spectra of the four UV-absorbing amino acids tryptophan, tyrosine, cystine and phenylalanine were recorded as finely ground powders. In contrast to their UV–visible spectra in aqueous solution where tryptophan and tyrosine are the major UV absorbing species, surprisingly the disulphide chromophore of solid cystine has the strongest UV absorbance measured using the DR remission function F(R)_∞. The DR spectra of unpigmented feather and wool keratin appear to be dominated by cystine absorption near 290 nm, whereas silk fibroin appears similar to tyrosine. Because cystine has a flat reflectance spectrum in the visible region from 700 to 400 nm and the powder therefore appears white, cystine absorption does not contribute to the cream colour of wool despite the high concentration of cystine residues near the cuticle surface. The disulphide absorption of solid l-cystine in the DR spectrum at 290 nm is significantly red shifted by ~40 nm relative to its wavelength in solution, whereas homocystine and lipoic acid showed smaller red shifts of 20 nm. The large red shift observed for cystine and the large difference in intensity of absorption in its UV–visible and DR spectra may be due to differences in the dihedral angle between the crystalline solid and the solvated molecules in solution.

Keywords: Fibrous protein; Diffuse reflectance; Keratin; Silk; Collagen; Cystine

Changes in plasma phenylalanine, isoleucine, leucine, and valine are associated with significant changes in intracranial pressure and jugular venous oxygen saturation in patients with severe traumatic brain injury by Raphael N. Vuille-Dit-Bille; Riem Ha-Huy; John F. Stover (pp. 1287-1296).

Changes in plasma aromatic amino acids (AAA = phenylalanine, tryptophan, tyrosine) and branched chain amino acids (BCAA = isoleucine, leucine, valine) levels possibly influencing intracranial pressure (ICP) and cerebral oxygen consumption (SjvO₂) were investigated in 19 sedated patients up to 14 days following severe traumatic brain injury (TBI). Compared to 44 healthy volunteers, jugular venous plasma BCAA were significantly decreased by 35% (p < 0.001) while AAA were markedly increased in TBI patients by 19% (p < 0.001). The BCAA to AAA ratio was significantly decreased by 55% (p < 0.001) which persisted during the entire study period. Elevated plasma phenylalanine was associated with decreased ICP and increased SjvO₂, while higher plasma isoleucine and leucine levels were associated with increased ICP and higher plasma leucine and valine were linked to decreased SjvO₂. The amount of enterally administered amino acids was associated with significantly increased plasma levels with the exception of phenylalanine. Contrary to the initial assumption that elevated AAA and decreased BCAA levels are detrimental, increased plasma phenylalanine levels were associated with beneficial signs in terms of decreased ICP and reduced cerebral oxygen consumption reflected by increased SjvO₂; concomitantly, elevated plasma isoleucine and leucine levels were associated with increased ICP while leucine and valine were associated with decreased SjvO₂ following severe TBI, respectively. The impact of enteral nutrition on this observed pattern must be examined prospectively to determine if higher amounts of phenylalanine should be administered to promote beneficial effects on brain metabolism and if normalization of plasma BCAA levels is without cerebral side effects.

Keywords: Critical care; Monitoring; Neuromonitoring; Nutrition

Comparative syntheses of peptides and peptide thioesters derived from mouse and human prion proteins by Jaroslav Šebestík; Zbigniew Zawada; Martin Šafařík; Jan Hlaváček (pp. 1297-1309).

Prions are suspected as causative agents of several neuropathogenic diseases, even though the mode of their action is still not clear. A combination of chemical and recombinant syntheses can provide suitable probes for explanation of prions role in pathogenesis of neurodegenerative diseases. However, the prions contain several difficult sequences for synthesis by Fmoc/tBu approach. For that reason, the peptide thioesters as the key building blocks for chemical syntheses of proteins by native chemical ligation were employed. A scan of the mouse prion domain 93–231 was carried out in order to discover availability of derived thioesters as the suitable building blocks for a total chemical synthesis of the prion protein based probes. The synthesis on 2-chlorotritylchloride resin was utilized and after a deprotection of the samples for analysis, the peptide segments were purified and characterized. If the problems were detected during the synthesis, the segment was re-synthesized either using the special pseudoproline dipeptides or by splitting its molecule to two or three smaller segments, which were prepared easier. The protected segments, prepared correctly without any deletion and in sufficient amounts, were coupled either with EtSH after DIC/DMAP activation or with p-Ac-NH-Ph-SH using PyBOP activation to yield corresponding thioesters. In some special cases, the other techniques of thioester formation, like sulfonamide-safety catch and/or trimethylaluminium approach were utilized.

Keywords: Prion protein segments; Classical synthesis; Chemical ligation synthesis; Peptide thioesters

Glycation promotes the formation of genotoxic aggregates in glucose oxidase by Taqi Ahmed Khan; Samreen Amani; Aabgeena Naeem (pp. 1311-1322).

This study investigates the effect of pentose sugars (ribose and arabinose) on the structural and chemical modifications in glucose oxidase (GOD) as well as genotoxic potential of this modified form. An intermediate state of GOD was observed on day 12 of incubation having CD minima peaks at 222 and 208 nm, characteristic of α-helix and a few tertiary contacts with altered tryptophan environment and high ANS binding. All these features indicate the existence of molten globule state of the GOD with ribose and arabinose on day 12. GOD on day 15 of incubation forms β structures as revealed by CD and FTIR which may be due to its aggregation. Furthermore, GOD on day 15 showed a remarkable increase in Thioflavin T fluorescence at 485 nm. Comet assay of lymphocytes and plasmid nicking assay in presence of glycated GOD show DNA damage which confirmed the genotoxicity of advance glycated end products. Hence, our study suggests that glycated GOD results in the formation of aggregates and the advanced glycated end products, which are genotoxic in nature.

Keywords: AGEs; Aggregation; CD; Comet assay; Fluorescence; Molten globule; Plasmid nicking assay

Synthesis of biologically stable gold nanoparticles using imidazolium-based amino acid ionic liquids by Afsaneh Safavi; Sedigheh Zeinali; Mahdieh Yazdani (pp. 1323-1330).

A novel double-step reduction procedure for the synthesis of gold nanoparticles (AuNPs) using amino acid ionic liquids has been employed. 1-Dodecyl-3-methyl imidazolium tryptophan ([C₁₂mim]Trp) and 1-ethyl-3-methyl imidazolium tryptophan ([C₂mim]Trp) were used for this synthesis. The synthesized AuNPs were characterized by UV–vis spectroscopy, transmission electron microscopy and dynamic light scattering. The behavior of these AuNPs were also probed in a biological media. It was proven that AuNPs synthesized at [C₁₂mim]Trp have more stability than AuNPs synthesized at [C₂mim]Trp due to the longer alkyl chain of the imidazolium moiety. The solubility test shows that the resultant AuNPs have a hydrophilic nature. Finally, it was seen that due to the presence of a biomolecule, namely Trp, in the structure of AuNPs protecting shell, higher stability and biocompatibility was achieved in the biological media.

Keywords: Gold nanoparticle; Amino acid ionic liquid; Tryptophan; Double-step reduction; Biological stability

Oral intake of γ-aminobutyric acid affects mood and activities of central nervous system during stressed condition induced by mental tasks by A. Yoto; S. Murao; M. Motoki; Y. Yokoyama; N. Horie; K. Takeshima; K. Masuda; M. Kim; H. Yokogoshi (pp. 1331-1337).

γ-Aminobutyric acid (GABA) is a kind of amino acid contained in green tea leaves and other foods. Several reports have shown that GABA might affect brain protein synthesis, improve many brain functions such as memory and study capability, lower the blood pressure of spontaneously hypertensive rats, and may also have a relaxation effect in humans. However, the evidence for its mood-improving function is still not sufficient. In this study, we investigated how the oral intake of GABA influences human adults psychologically and physiologically under a condition of mental stress. Sixty-three adults (28 males, 35 females) participated in a randomized, single blind, placebo-controlled, crossover-designed study over two experiment days. Capsules containing 100 mg of GABA or dextrin as a placebo were used as test samples. The results showed that EEG activities including alpha band and beta band brain waves decreased depending on the mental stress task loads, and the condition of 30 min after GABA intake diminished this decrease compared with the placebo condition. That is to say, GABA might have alleviated the stress induced by the mental tasks. This effect also corresponded with the results of the POMS scores.

Keywords: γ-Aminobutyric acid; Electroencephalogram; Acute stress; Profile of Mood States

Metabolism and neurotoxicity of homocysteine thiolactone in mice: protective role of bleomycin hydrolase by Kamila Borowczyk; Joanna Tisończyk; Hieronim Jakubowski (pp. 1339-1348).

Genetic or nutritional disorders in homocysteine (Hcy) metabolism elevate Hcy-thiolactone and cause heart and brain diseases. Hcy-thiolactone has been implicated in these diseases because it has the ability to modify protein lysine residues and generate toxic N-Hcy-proteins with auto-immunogenic, pro-thrombotic, and amyloidogenic properties. Bleomycin hydrolase (Blmh) has the ability to hydrolyze l-Hcy-thiolactone (but not d-Hcy-thiolactone) to Hcy in vitro, but whether this reflects a physiological function has been unknown. Here, we show that Blmh ^−/− mice excreted in urine 1.8-fold more Hcy-thiolactone than wild-type Blmh ^+/+ animals (P = 0.02). Hcy-thiolactone was elevated 2.3-fold in brains (P = 0.004) and 2.0-fold in kidneys (P = 0.047) of Blmh ^−/− mice relative to Blmh ^+/+ animals. Plasma N-Hcy-protein was elevated in Blmh ^−/− mice fed a normal (2.3-fold, P < 0.001) or hyperhomocysteinemic diet (1.5-fold, P < 0.001), compared with Blmh ^+/+ animals. More intraperitoneally injected l-Hcy-thiolactone was recovered in plasma in Blmh ^−/− mice than in wild-type Blmh ^+/+ animals (83.1 vs. 39.3 μM, P < 0.0001). In Blmh ^+/+ mice injected intraperitoneally with d-Hcy-thiolactone, d,l-Hcy-thiolactone, or l-Hcy-thiolactone, 88, 47, or 6.3%, respectively, of the injected dose was recovered in plasma. The incidence of seizures induced by l-Hcy-thiolactone injections (3,700 nmol/g body weight) was higher in Blmh ^−/− than in Blmh ^+/+ mice (93.8 vs. 29.5%, P < 0.001). Using the Blmh null mice, we provide the first direct evidence that a specific Hcy metabolite, Hcy-thiolactone, rather than Hcy itself, is neurotoxic in vivo. Taken together, our findings indicate that Blmh protects mice against l-Hcy-thiolactone toxicity by metabolizing it to Hcy and suggest a mechanism by which Blmh might protect against neurodegeneration associated with hyperhomocysteinemia and Alzheimer’s disease.

Keywords: Bleomycin hydrolase; Homocysteine thiolactone; Brain; Seizures; Alzheimer’s disease

N-Succinimidyl 4-[¹⁸F]-fluoromethylbenzoate-labeled dimeric RGD peptide for imaging tumor integrin expression by Weihua Li; Lixin Lang; Gang Niu; Ning Guo; Ying Ma; Dale O. Kiesewetter; Baozhong Shen; Xiaoyuan Chen (pp. 1349-1357).

RGD peptides, radiolabeled with ¹⁸F, have been used in the clinic for PET imaging of tumor angiogenesis in cancer patients. RGD peptides are typically labeled using a prosthetic group such as N-succinimidyl 4-[¹⁸F]-fluorobenzoate ([¹⁸F]SFB) or 4-nitrophenyl 2-[¹⁸F]-fluoropropionate ([¹⁸F]NPFP). However, the complex radiosynthetic procedures have impeded their broad application in clinical studies. We previously radiolabeled proteins and peptides with the prosthetic group, N-succinimidyl 4-[¹⁸F]-fluoromethylbenzoate ([¹⁸F]SFMB), which was prepared in a simple one-step procedure. In this study, we labeled a PEGylated cyclic RGD peptide dimer, PEG₃-E[c(RGDyK)]₂ (PRGD2), using [¹⁸F]SFMB and evaluated for imaging tumor αvβ3 integrin expression with positron emission tomography (PET). [¹⁸F]SFMB was prepared in one step using [¹⁸F]fluoride displacement of a nitrobenzenesulfonate leaving group under mild reaction conditions followed by HPLC purification. The ¹⁸F-labeled peptide, [¹⁸F]FMBPRGD2 was prepared by coupling PRGD2 with [¹⁸F]SFMB in pH 8.6 borate buffer and purified with HPLC. The direct labeling on BMBPRGD2 was also attempted. A Siemens Inveon PET was used to image the uptake of the [¹⁸F]FMBPRGD2 into a U87MG xenograft mouse model. [¹⁸F]FMBPRGD2, was prepared with a 15% overall radiochemical yield (uncorrected) in a total synthesis time of 90 min, which was considerably shorter than the preparation of [¹⁸F]SFB- and [¹⁸F]NPFP-labeled RGD peptides. The direct labeling, however, was not successful. High quality microPET images using [¹⁸F]FMBPRGD2 clearly visualized tumors by 15 min with good target to background ratio. Early tracer accumulation in the bladder suggests fast renal clearance. No obvious bone uptake can be detected even at 4-h time point indicating that fluorine attachment is stable in mice. In conclusion, N-succinimidyl 4-[¹⁸F]-fluoromethylbenzoate ([¹⁸F]SFMB) prosthetic group can be a good alternative for labeling RGD peptides to image αvβ3 integrin expression and for labeling other peptides.

Keywords: Integrin αvβ3; RGD peptide dimer; Positron emission tomography; N-Succinimidyl 4-[¹⁸F]-fluoromethylbenzoate ([¹⁸F]SFMB)

Molecular mechanism underlying the cerebral effect of Gly-Pro-Glu tripeptide bound to l-dopa in a Parkinson’s animal model by Alba Minelli; C. Conte; I. Cacciatore; C. Cornacchia; F. Pinnen (pp. 1359-1367).

Oxidative stress is a critical contributing factor to neurodegenerative disorders. Therefore, the inhibition of ROS formation, responsible for chronic detrimental neuroinflammation, is an important strategy for preventing the neurodegenerative disease and for neuroprotective therapy. Gly-Pro-Glu (GPE) is the N-terminal tripeptide of insulin-like growth factor-I, which is naturally cleaved in the plasma and brain tissues. GPE has neuroprotective effects since it crosses the blood–CSF and the functional CSF–brain barriers and binds to glial cells. It has been shown that GPE improves motor behaviour in rats after 6-OHDA lesion, although it does not rescue dopaminergic neurons. Thus, we hypothesized that the GPE therapeutic efficacy in a Parkinson model might be improved by combining GPE to l-dopa. Here, we used an animal model that represents a progressive chronic Parkinson’s disease (PD) model, characterized by high levels of oxidative stress and inflammation. We showed that the co-drug, in which l-dopa is covalently linked to the GPE tripeptide, by down-regulating the expression of inflammatory genes, decreases the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced inflammatory response and, by up-regulating tyrosine hydroxylase, reduces MPTP-induced neurotoxicity. Furthermore, by determining the nuclear translocation/activation of Nrf2 and NF-κB, we showed that systemic administration of the co-drug activates Nrf2-induced antioxidant response while suppressing NF-κB inflammatory pathway. Data suggest that the binding of l-dopa to GPE tripeptide might represent a promising strategy to supply l-dopa to parkinsonian patients.

Keywords: MPTP; PD animal model; LD-GPE; Nrf2; NF-κB

Cis–trans peptide variations in structurally similar proteins by Agnel Praveen Joseph; Narayanaswamy Srinivasan; Alexandre G. de Brevern (pp. 1369-1381).

The presence of energetically less favourable cis peptides in protein structures has been observed to be strongly associated with its structural integrity and function. Inter-conversion between the cis and trans conformations also has an important role in the folding process. In this study, we analyse the extent of conservation of cis peptides among similar folds. We look at both the amino acid preferences and local structural changes associated with such variations. Nearly 34% of the Xaa-Proline cis bonds are not conserved in structural relatives; Proline also has a high tendency to get replaced by another amino acid in the trans conformer. At both positions bounding the peptide bond, Glycine has a higher tendency to lose the cis conformation. The cis conformation of more than 30% of β turns of type VIb and IV are not found to be conserved in similar structures. A different view using Protein Block-based description of backbone conformation, suggests that many of the local conformational changes are highly different from the general local structural variations observed among structurally similar proteins. Changes between cis and trans conformations are found to be associated with the evolution of new functions facilitated by local structural changes. This is most frequent in enzymes where new catalytic activity emerges with local changes in the active site. Cis–trans changes are also seen to facilitate inter-domain and inter-protein interactions. As in the case of folding, cis–trans conversions have been used as an important driving factor in evolution.

Keywords: Protein folds; cis peptides; ω dihedral; cis–trans isomerization; Structural alignment; Structural alphabet; Protein blocks; Protein Data Bank

Lyophilization is suitable for storage and shipment of fresh tissue samples without altering RNA and protein levels stored at room temperature by Yonghong Wu; Min Wu; Yanchun Zhang; Weiguang Li; Yan Gao; Zhihui Li; Zhaoyan Wang; Gert Lubec; Chenggang Zhang (pp. 1383-1388).

Lyophilization has been widely used for preservation, such as in food industry, pharmacy, biotechnology and tissues engineering, etc. However, there is no report on whether it could affect stability of RNA and protein levels in biological tissue samples. Herein we show that lyophilization can be used for storage of biological tissue samples without loss of bioactivities even stored at room temperature for 7–14 days. To address this issue, C57BL mouse tissues were prepared and dried by lyophilization and a baking method, respectively, followed by examination of morphological structure and total proteins by SDS-PAGE as well as gelatin zymography. Subsequently, the stability of RNAs and proteins, which were lyophilized and stored at room temperature (23°C) for 14 days was further examined by RT-PCR, SDS-PAGE and western blot. Results demonstrated that lyophilization did not alter total protein activities of various tissues, including enzyme activities, immunoreactivities and phosphorylation, and did not affect several RNAs in lyophilized tissues. Taken together, lyophilization may represent a valuable approach for preservation and long-distance shipment of biological samples, particularly for the international exchange of biological samples without altering their bioactivities.

Keywords: Lyophilization; Tissue storage; Tissue transferring; RNA; Protein; Bioactivity

Monosodium glutamate intake increases hemoglobin level over 5 years among Chinese adults by Zumin Shi; Baojun Yuan; Anne W. Taylor; Eleonora Dal Grande; Gary A. Wittert (pp. 1389-1397).

The aim of this analysis was to determine the relationship between monosodium glutamate (MSG) intake and change in hemoglobin (Hb) levels and the risk of anemia over 5 years in 1197 Chinese men and women who participated in the Jiangsu Nutrition Study (JIN). MSG intake and Hb were quantitatively assessed in 2002 and followed up in 2007. Diet and lifestyle factors were assessed at both time points. There was a positive association between MSG intake and increase in Hb among men but not women. In the multivariate model adjusting for demographic and lifestyle factors as well as baseline dietary pattern, the beta values and 95% confidence interval for Hb changes across quartiles of MSG intake were 0, 0.67(0.04–1.29), 0.99(0.38–1.60), 0.73(0.13–1.34) among men (p for trend 0.091); 0, −0.01(−0.45–0.43), 0.23(−0.25–0.71), and −0.45(−0.96–0.05) among women (p for trend 0.087). Among anemic participants at baseline, there was a significant inverse association between MSG intake and the risk of anemia at follow-up. Comparing extreme quartiles of MSG intake among those anemic at baseline, the relative risk for persistent anemia at follow-up was 0.49 (95% CI: 0.28–0.86, p < 0.01). The association was independent of dietary patterns and lifestyle factors. A dose–response relationship between MSG intake and increase in Hb levels among anemic participants was seen. MSG intake may have independent Hb-increasing effects, especially among men and those anemic at baseline.

Keywords: Anemia; Hemoglobin; Cohort study; Chinese; Monosodium glutamate

Synaptic localisation of agmatinase in rat cerebral cortex revealed by virtual pre-embedding by V. I. Madai; W. C. Poller; D. Peters; J. Berger; K. Paliege; R. Bernard; R. W. Veh; G. Laube (pp. 1399-1403).

Light microscopic evidence suggested a synaptic role for agmatinase, an enzyme capable of inactivating the putative neurotransmitter and endogenous anti-depressant agmatine. Using electron microscopy and an alternative pre-embedding approach referred to as virtual pre-embedding, agmatinase was localised pre- and postsynaptically, to dendritic spines, spine and non-spine terminals, and dendritic profiles. In dendritic spines, labelling displayed a tendency towards the postsynaptic density. These results further strengthen a synaptic role for agmatine and strongly suggest a regulatory role for synaptically expressed agmatinase.

Keywords: Agmatinase; Agmatine; Neurotransmission; Pre-embedding; Mood disorders

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.43, #3)