Amino Acids (v.48, #7)

Methionine restriction on lipid metabolism and its possible mechanisms by Xihong Zhou; Liuqin He; Dan Wan; Huansheng Yang; Kang Yao; Guoyao Wu; Xin Wu; Yulong Yin (1533-1540).
Methionine restriction (MR) exerts many beneficial effects, such as increasing longevity, decreasing oxidative damage and alleviating inflammatory responses. Much attention has been recently focused on the effects of MR on metabolic health, especially lipid metabolism, since the increasing incidence of obesity, insulin resistance and type 2 diabetes causes a worldwide health problem. In general, MR is considered to increase de novo lipogenesis, lipolysis and fatty acid oxidation, with a result of reduced fat accumulation. However, different responses in lipid metabolism between adipose tissue and liver are declared. Therefore, in this review, we will focus on the changes of lipid metabolism responses to dietary MR. Moreover, the comparison of alterations of fat metabolism responses to dietary MR between adipose tissue and liver, and the comparison of changes between rodents and pigs is made to illustrate the tissue- and species-specific responses. In addition, the possible mechanisms that might be engaged in the regulation of MR diet on lipid metabolism are also discussed.
Keywords: Methionine restriction; Fat accumulation; Adipose tissue; Liver

Catabolism and safety of supplemental l-arginine in animals by Zhenlong Wu; Yongqing Hou; Shengdi Hu; Fuller W. Bazer; Cynthia J. Meininger; Catherine J. McNeal; Guoyao Wu (1541-1552).
l-arginine (Arg) is utilized via multiple pathways to synthesize protein and low-molecular-weight bioactive substances (e.g., nitric oxide, creatine, and polyamines) with enormous physiological importance. Furthermore, Arg regulates cell signaling pathways and gene expression to improve cardiovascular function, augment insulin sensitivity, enhance lean tissue mass, and reduce obesity in humans. Despite its versatile roles, the use of Arg as a dietary supplement is limited due to the lack of data to address concerns over its safety in humans. Data from animal studies are reviewed to assess arginine catabolism and the safety of long-term Arg supplementation. The arginase pathway was responsible for catabolism of 76–85 and 81–96 % Arg in extraintestinal tissues of pigs and rats, respectively. Dietary supplementation with Arg–HCl or the Arg base [315- and 630-mg Arg/(kg BW d) for 91 d] had no adverse effects on male or female pigs. Similarly, no safety issues were observed for male or female rats receiving supplementation with 1.8- and 3.6-g Arg/(kg BW d) for at least 91 d. Intravenous administration of Arg–HCl to gestating sheep at 81 and 180 mg Arg/(kg BW d) is safe for at least 82 and 40 d, respectively. Animals fed conventional diets can well tolerate large amounts of supplemental Arg [up to 630-mg Arg/(kg BW d) in pigs or 3.6-g Arg/(kg BW d) in rats] for 91 d, which are equivalent to 573-mg Arg/(kg BW d) for humans. Collectively, these results can help guide studies to determine the safety of long-term oral administration of Arg in humans.
Keywords: Amino acids; Nutrition; Catabolism; Health; Dietary supplementation

d-Aspartate drinking solution alleviates pain and cognitive impairment in neuropathic mice by Enza Palazzo; Livio Luongo; Francesca Guida; Ida Marabese; Rosaria Romano; Monica Iannotta; Francesca Rossi; Antimo D’Aniello; Luigi Stella; Federica Marmo; Alessandro Usiello; Andrea de Bartolomeis; Sabatino Maione; Vito de Novellis (1553-1567).
d-Aspartate (d-Asp) is a free d-amino acid detected in multiple brain regions and putative precursor of endogenous N-methyl-d-aspartate (NMDA) acting as agonist at NMDA receptors. In this study, we investigated whether d-Asp (20 mM) in drinking solution for 1 month affects pain responses and pain-related emotional, and cognitive behaviour in a model of neuropathic pain induced by the spared nerve injury (SNI) of the sciatic nerve in mice. SNI mice developed mechanical allodynia and motor coordination impairment 30 days after SNI surgery. SNI mice showed cognitive impairment, anxiety and depression-like behaviour, reduced sociability in the three chamber sociability paradigm, increased expression of NR2B subunit of NMDA receptor and Homer 1a in the medial prefrontal cortex (mPFC). The expression of (post synaptic density) PSD-95 and Shank 1was instead unaffected in the mPFC of the SNI mice. Treatment with d-Asp drinking solution, started right after the SNI (day 0), alleviated mechanical allodynia, improved cognition and motor coordination and increased social interaction. d-Asp also restored the levels of extracellular d-Asp, Homer 1a and NR2B subunit of the NMDA receptor to physiological levels and reduced Shank1 and PSD-95 protein levels in the mPFC. Amitriptyline, a tricyclic antidepressant used also to alleviate neuropathic pain in humans, reverted mechanical allodynia and cognitive impairment, and unlike d-Asp, was effective in reducing depression and anxiety-like behaviour in the SNI mice and increased PSD protein level. Altogether these findings demonstrate that d-Asp improves sensorial, motor and cognitive-like symptoms related to chronic pain possibly through glutamate neurotransmission normalization in neuropathic mice.
Keywords: d-Aspartate; Spared nerve injury; Mechanical allodynia; Pain-related affective and cognitive behaviour; NMDA receptor; Mice

Sequence characterization and glycosylation sites identification of donkey milk lactoferrin by multiple enzyme digestions and mass spectrometry by Serafina Gallina; Vincenzo Cunsolo; Rosaria Saletti; Vera Muccilli; Antonella Di Francesco; Salvatore Foti; Andrea Maria Lorenzten; Peter Roepstorff (1569-1580).
Lactoferrin, a protein showing an array of biochemical properties, including immuno-modulation, iron-binding ability, as well as antioxidant, antibacterial and antiviral activities, but which may also represent a potential milk allergen, was isolated from donkey milk by ion exchange chromatography. The characterization of its primary structure, by means of enzymatic digestions, SPITC derivatization of tryptic digest, reversed-phase high performance liquid chromatography, electrospray and matrix-assisted laser desorption/ionization mass spectrometry, is reported. Our results allowed the almost complete characterization of donkey lactoferrin sequence, that, at least for the covered sequence, differs from the horse genomic deduced sequence (UniProtKB Acc. Nr. O77811) by five point substitutions located at positions 91 (Arg → His), 328 (Thr → Ile/Leu), 466 (Ala → Gly), 642 (Asn → Ser) and 668 (Ser → Ala). Analysis of the glycosylated protein showed that glycans in donkey lactoferrin are linked to the protein backbone via an amide bond to asparagine residues located at the positions 137, 281 and 476.
Keywords: Lactoferrin; Sequence characterization; Glycosylation sites; Donkey milk; Mass spectrometry

S-adenosyl-l-methionine (SAM) is an amino acid involved in a number of physiological processes in the nervous system. Some evidence suggests a therapeutic potential of SAM in hypertension. In this study we investigated the effect of intracerebroventricular (ICV) infusions of SAM on arterial blood pressure in rats. Mean arterial blood pressure (MABP) and heart rate (HR) were measured at baseline and during ICV infusion of either SAM or vehicle (aCSF; controls) in conscious, male normotensive Wistar Kyoto rats (WKY) and Spontaneously Hypertensive Rats (SHR). MABP and HR were not affected by the vehicle. WKY rats infused with SAM (10 μM, 100 μM and 1 mM) showed a biphasic hemodynamic response i.e., mild hypotension and bradycardia followed by a significant increase in MABP and HR. On the contrary, SHR infused with SAM showed a dose-dependent hypotensive response. In separate series of experiments, pretreatment with hexamethonium, a ganglionic blocker as well as pretreatment with glibenclamide, a KATP channel blocker reduced the hemodynamic effects of SAM. SAM may affect the nervous control of arterial blood pressure via the autonomic nervous system and KATP channel-dependent mechanisms.
Keywords: Blood pressure; Brain; Hypertension; S-adenosyl-l-methionine

Traf2- and Nck-interacting kinase (TNIK) is involved in the anti-cancer mechanism of dovitinib in human multiple myeloma IM-9 cells by Hae Jung Chon; Yura Lee; Kyoung Jun Bae; Byung Jin Byun; Soon Ae Kim; Jiyeon Kim (1591-1599).
Traf2- and Nck-interacting kinase (TNIK) is a member of the germinal center kinase family. TNIK was first identified as a kinase that is involved in regulating cytoskeletal organization in many types of cells, and it was recently proposed as a novel therapeutic target in several types of human cancers. Although previous studies suggest that TNIK plays a pivotal role in cancer cell survival and prognosis, its function in hematological cancer cell survival has not been investigated. Here we investigated the relationship between TNIK function and cell viability in multiple myeloma IM-9 cells using TNIK small interfering RNA (siRNA) transfection and dovitinib treatment. Treatment of IM-9 cells with TNIK siRNA and dovitinib treatment reduced cell proliferation. The ATP competing kinase assay and western blot analysis showed that dovitinib strongly inhibited both the interaction of TNIK with ATP (K i, 13 nM) and the activation of Wnt signaling effectors such as β-catenin and TCF4. Dovitinib also induced caspase-dependent apoptosis in IM-9 cells without significant cytotoxicity in PBMCs. Our results provide new evidence that TNIK may be involved in the proliferation of multiple myeloma IM-9 cells and in the anti-cancer activity of dovitinib via inhibition of the endogenous Wnt signaling pathway.
Keywords: TNIK; Dovitinib; Wnt signaling; Multiple myeloma; IM-9; Apoptosis

Effects of taurine on gut microbiota and metabolism in mice by Haining Yu; Zhengzhao Guo; Shengrong Shen; Weiguang Shan (1601-1617).
As being a necessary amino acid, taurine plays an important role in the regulation of neuroendocrine functions and nutrition. In this study, effects of taurine on mice gut microbes and metabolism were investigated. BALB/C mice were randomly divided into three experimental groups: The first group was administered saline (CK), the second was administered 165 mg/kg natural taurine (NE) and the third one administered 165 mg/kg synthetic taurine (CS). Gut microbiota composition in mice feces was analyzed by metagenomics technology, and the content of short-chain fatty acids (SCFA) in mice feces was detected by gas chromatography (GC), while the concentrations of lipopolysaccharide (LPS) and superoxide dismutase (SOD) were detected by a LPS ELISA kit and a SOD assay kit, respectively. The results showed that the effect of taurine on gut microbiota could reduce the abundance of Proteobacteria, especially Helicobacter. Moreover, we found that the SCFA content was increased in feces of the NE group while LPS content was decreased in serum of the NE group; the SOD activity in serum and livers of the NE and CS groups were not changed significantly compare to that of the CK group. In conclusion, taurine could regulate the gut micro-ecology, which might be of benefit to health by inhibiting the growth of harmful bacteria, accelerating the production of SCFA and reducing LPS concentration.
Keywords: Taurine; Gut microbiota; Metagenomics technology; SCFA; LPS; SOD activity

Decreased cysteine uptake by EAAC1 gene deletion exacerbates neuronal oxidative stress and neuronal death after traumatic brain injury by Bo Young Choi; In Yeol Kim; Jin Hee Kim; Bo Eun Lee; Song Hee Lee; A. Ra Kho; Hee Jae Jung; Min Sohn; Hong Ki Song; Sang Won Suh (1619-1629).
Excitatory amino acid carrier type 1 (EAAC1), a high-affinity glutamate transporter, can expend energy to move glutamate into neurons. However, under normal physiological conditions, EAAC1 does not have a great effect on glutamate clearance but rather participates in the neuronal uptake of cysteine. This process is critical to maintaining neuronal antioxidant function by providing cysteine for glutathione synthesis. Previous study showed that mice lacking EAAC1 show increased neuronal oxidative stress following transient cerebral ischemia. In the present study, we sought to characterize the role of EAAC1 in neuronal resistance after traumatic brain injury (TBI). Young adult C57BL/6 wild-type or EAAC1/ mice were subjected to a controlled cortical impact model for TBI. Neuronal death after TBI showed more than double the number of degenerating neurons in the hippocampus in EAAC1/ mice compared with wild-type mice. Superoxide production, zinc translocation and microglia activation similarly showed a marked increase in the EAAC1/ mice. Pretreatment with N-acetyl cysteine (NAC) reduced TBI-induced neuronal death, superoxide production and zinc translocation. These findings indicate that cysteine uptake by EAAC1 is important for neuronal antioxidant function and survival following TBI. This study also suggests that administration of NAC has therapeutic potential in preventing TBI-induced neuronal death.
Keywords: EAAC1; Cysteine; Zinc; TBI; Reactive oxygen species; Microglia

The purpose of the study is to identify the sites of modification when fibronectin reacts with glycolaldehyde or methylglyoxal as a model system for aging of Bruch’s membrane. A synthetic peptide consisting of the α5β1 integrin binding region of fibronectin was incubated with glycolaldehyde for 12 h or with methylglyoxal for 1 h at 37 °C. After tryptic digestion, the samples were analyzed with liquid chromatography–mass spectrometry (LC/MS). Tandem MS was used to determine the sites of modification. The adducts, aldoamine and N ε-carboxymethyl-lysine, attached preferably at lysine residues when the fibronectin peptide reacted with glycolaldehyde. When the fibronectin peptide reacted with methylglyoxal, modifications occurred at lysine and arginine residues. At lysine residues, N ε-carboxyethyl-lysine adducts were present. At arginine residues, hydroimidazolone and tetrapyrimidine adducts were present. Several advanced glycation endproducts were generated when fibronectin was glycated via glycolaldehyde and methylglyoxal. These results can help explain the structural changes Bruch’s membrane undergoes during aging.
Keywords: Bruch’s membrane; Fibronectin; Retinal pigment epithelium; Glycation; Aging

In vivo monitoring of angiogenesis in a mouse hindlimb ischemia model using fluorescent peptide-based probes by Subin Park; Jangwook Lee; Mi-hee Jo; Jin Hee Na; Sung-Gurl Park; Hyeon-Ki Jang; Sun-Woong Kang; Jong-Ho Kim; Byung-Soo Kim; Jae Hyung Park; Ick Chan Kwon; Ju Hee Ryu; Kwangmeyung Kim (1641-1654).
Vascular endothelial growth factor receptor (VEGFR) and matrix metalloproteinase (MMP) are up-regulated in ischemic tissue and play pivotal roles in promoting angiogenesis. The purpose of the present study was to evaluate two fluorophore-conjugated peptide probes specific to VEGFR and MMP for dual-targeted in vivo monitoring of angiogenesis in a murine model of hindlimb ischemia. To this end, VEGFR-Probe and MMP-Probe were developed by conjugating distinct near-infrared fluorophores to VEGFR-binding and MMP substrate peptides, respectively. VEGFR-Probe exhibited specific binding to VEGFR on HUVECs, and self-quenched MMP-Probe produced strong fluorescence intensity in the presence of MMPs in vitro. Subsequently, VEGFR-Probe and MMP-Probe were successfully utilized for time course in vivo visualization of VEGFR or MMP, respectively. Simultaneous visualization provided information regarding the spatial distribution of these proteins, including areas of co-localization. This dual-targeted in vivo imaging approach will be useful for understanding the detailed mechanism of angiogenesis and for evaluating therapeutic angiogenesis.
Keywords: Vascular endothelial growth factor receptor; Matrix metalloproteinase; Molecular imaging; Angiogenesis; Fluorescence imaging

SPAR: a random forest-based predictor for self-interacting proteins with fine-grained domain information by Xuhan Liu; Shiping Yang; Chen Li; Ziding Zhang; Jiangning Song (1655-1665).
Protein self-interaction, i.e. the interaction between two or more identical proteins expressed by one gene, plays an important role in the regulation of cellular functions. Considering the limitations of experimental self-interaction identification, it is necessary to design specific bioinformatics tools for self-interacting protein (SIP) prediction from protein sequence information. In this study, we proposed an improved computational approach for SIP prediction, termed SPAR (Self-interacting Protein Analysis serveR). Firstly, we developed an improved encoding scheme named critical residues substitution (CRS), in which the fine-grained domain–domain interaction information was taken into account. Then, by employing the Random Forest algorithm, the performance of CRS was evaluated and compared with several other encoding schemes commonly used for sequence-based protein–protein interaction prediction. Through the tenfold cross-validation tests on a balanced training dataset, CRS performed the best, with the average accuracy up to 72.01 %. We further integrated CRS with other encoding schemes and identified the most important features using the mRMR (the minimum redundancy maximum relevance) feature selection method. Our SPAR model with selected features achieved an average accuracy of 92.09 % on the human-independent test set (the ratio of positives to negatives was about 1:11). Besides, we also evaluated the performance of SPAR on an independent yeast test set (the ratio of positives to negatives was about 1:8) and obtained an average accuracy of 76.96 %. The results demonstrate that SPAR is capable of achieving a reasonable performance in cross-species application. The SPAR server is freely available for academic use at http://systbio.cau.edu.cn/zzdlab/spar/ .
Keywords: Self-interacting protein; Prediction; Machine learning; Feature selection; Domain–domain interaction

Catabolism of 64Cu and Cy5.5-labeled human serum albumin in a tumor xenograft model by Choong Mo Kang; Hyunjung Kim; Hyun-Jung Koo; Jin Won Park; Gwang Il An; Joon Young Choi; Kyung-Han Lee; Byung-Tae Kim; Yearn Seong Choe (1667-1675).
Human serum albumin (HSA), the most abundant protein in blood plasma, has been used as a drug carrier for the last few decades. Residualizingly radiolabeled serum albumin has been reported to be avidly taken up by tumors of sarcoma-bearing mice and to most likely undergo lysosomal degradation. In this study, we prepared 64Cu-1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) and Cy5.5-conjugated HSA (dual probe), and evaluated its tumor uptake and catabolism. Two dual probes were prepared using different DOTA conjugation sites of HSA (one via Lys residues and the other via the Cys residue). 64Cu-DOTA-Lys-HSA-Cy5.5 (dual probe-Lys) exhibited higher uptake by RR1022 sarcoma cells in vitro than 64Cu-DOTA-Cys-HSA-Cy5.5 (dual probe-Cys). In RR1022 tumor-bearing mice, the two dual probes showed a similar level of tumor uptake, but uptake of dual probe-Lys was reduced in the liver and spleen compared to dual probe-Cys, probably because of the presence of a higher number of DOTA molecules in the former. At 24 and 48 h after injection, dual probe-Lys was intact or partially degraded in blood, liver, kidney, and tumor samples, but 64Cu-DOTA-Lys was observed in the urine using radioactivity detection. Similarly, Cy5.5-Lys was observed in the urine using fluorescence detection. These results indicate that dual probe-Lys may be useful for predicting the catabolic fate of drug–HSA conjugates.
Keywords: Human serum albumin; PET/optical imaging; 64Cu-DOTA-Lys; Cy5.5-Lys; Tumor; Catabolism

A species of the poorly studied order Embioptera, the webspinner Oligotoma saundersii, is investigated for its complement of neuropeptides of the adipokinetic hormone (AKH) family. A methanolic extract of its corpora cardiaca (CC) is able to effect carbohydrate mobilization in the cockroach, Periplaneta americana, and liquid chromatography coupled to electrospray ionization mass spectrometry clearly identified one decapeptide as a member of the AKH family in the CC of O. saundersii. The primary structure of this peptide, code-named Olisa-AKH, is elucidated as pEVNFSPNWGG amide. It is a novel member of the AKH family and in its synthetic form it has strong hypertrehalosemic activity in the American cockroach. This effect may be explained by its near-identical structure compared with one of the endogenous cockroach AKH peptides. An analog with the reversed order of the proline and asparagine residues, viz. N6P7-Olisa-AKH, had negligible activity thus, shedding light on the requirements of the cockroach AKH receptor. From reversed-phase high-performance liquid chromatography experiments, we can conclude that the CC from an individual webspinner contains less than one pmol of Olisa-AKH. Comparison of the AKH sequences from the major orders of the Polyneoptera does not point to a close phylogenetic relationship between webspinners and stick insects.
Keywords: Insect; Webspinner; Embioptera; Adipokinetic peptide; Mass spectrometry; Metabolic bioassay

The natural amino acid l-α-proline (Pro) is a compatible osmolyte which accumulates in the cell cytoplasm to protect structure and function of various proteins and enzymes under abiotic stress, like for instance, freezing. It is assumed that the interactions of Pro with intracellular water play an important role in the protection mechanism. However, until now the details of these interactions are far from being fully understood. We present results of a theoretical study of the hydration of Pro zwitterion (Pro-ZW) in water in the temperature range of 298–248 K. The data were obtained by the integral equation method in the framework of the 1D- and 3D-RISM approaches. The structural data were analyzed in terms of radial and spatial distribution functions. The observed features of Pro-ZW hydration are discussed from the position of the biological role of Pro as a cryoprotectant. In particular, it was found that under cooling conditions this protectant is able to bind a significant amount of water molecules and, thus, is helping to keep water inside the cell.
Keywords: Amino acid; Proline; Hydration; Low temperatures; 1D- and 3D-RISM integral equation method

Carbonic anhydrases are producers of S-nitrosothiols from inorganic nitrite and modulators of soluble guanylyl cyclase in human platelets by Erik Hanff; Anke Böhmer; Maximilian Zinke; Stepan Gambaryan; Alexandra Schwarz; Claudiu T. Supuran; Dimitrios Tsikas (1695-1706).
Nitric oxide (NO), S-nitrosoglutathione (GSNO) and S-nitrosocysteine are highly potent signaling molecules, acting both by cGMP-dependent and cGMP-independent mechanisms. The NO metabolite nitrite (NO2 ) is a major NO reservoir. Hemoglobin, xanthine oxidoreductase and carbonic anhydrase (CA) have been reported to reduce/convert nitrite to NO. We evaluated the role and the physiological importance of CA for an extra-platelet CA/nitrite/NO/cGMP pathway in human platelets. Authentic NO was analyzed by an NO-sensitive electrode. GSNO and GS15NO were measured by liquid chromatography–tandem mass spectrometry (LC–MS/MS). cGMP was determined by LC–MS/MS or RIA. In reduced glutathione (GSH) containing aqueous buffer (pH 7.4), human and bovine erythrocytic CAII-mediated formation of GSNO from nitrite and GS15NO from 15N-nitrite. In the presence of l-cysteine and GSH, this reaction was accompanied by NO release. Incubation of nitrite with bovine erythrocytic CAII and recombinant soluble guanylyl cyclase resulted in cGMP formation. Upon incubation of nitrite with bovine erythrocytic CAII and washed human platelets, cGMP and P-VASPS239 were formed in the platelets. This study provides the first evidence that extra-platelet nitrite and erythrocytic CAII may modulate platelet function in a cGMP-dependent manner. The new nitrite-dependent CA activity may be a general principle and explain the cardioprotective effects of inorganic nitrite in the vasculature. We propose that nitrous acid (ONOH) is the primary CA-catalyzed reaction product of nitrite.
Keywords: Carbonic anhydrase; cGMP; Nitric oxide; Nitrite; S-Nitrosothiols; Platelets

Determination of the safety of leucine supplementation in healthy elderly men by Betina Rasmussen; Erin Gilbert; Abrar Turki; Kenneth Madden; Rajavel Elango (1707-1716).
Leucine, a branched-chain amino acid (BCAA), has been shown to stimulate muscle protein synthesis, and thus has been proposed to prevent age-related muscle atrophy (sarcopenia). Therefore, leucine supplementation may have potential benefits in elderly populations to preserve muscle mass. The tolerable upper intake level (UL) for leucine intake in young men has recently been determined to be 500 mg kg−1 day−1, and increases in blood ammonia concentrations were seen at intake levels above 500 mg kg−1 day−1; the UL for leucine in elderly is unknown. The objective of the current study was to determine the safety of leucine supplementation in healthy elderly men. Six healthy elderly men (72.2 ± 3.5 years) received graded stepwise increases in leucine intakes ranging from 50 to 750 mg kg−1 day−1, on eight separate study days. Plasma and urinary biochemical variables, including blood ammonia, and an oral primed-continuous protocol of L-1-13C-Leucine was performed. Blood ammonia concentrations above normal values (35 µmol/L) were observed at leucine intakes >550 mg kg−1 day−1. Leucine oxidation measured as a F13CO2 (rate of label tracer oxidation) increased with increasing leucine intakes and started to plateau after 450 mg kg−1 day−1. Two-phased linear regression analysis of the F13CO2 data revealed a breakpoint of 431 mg kg−1 day−1 (R 2 = 0.73), suggesting that the upper limit to oxidize leucine was reached at that point. Taking the data together the upper limit for leucine intake in healthy elderly could be set similar to young men at 500 mg kg−1 day−1 or ~35 g/day for an individual weighing 70 kg.
Keywords: Leucine; Upper limit; Oxidation; Elderly; Ammonia

Epitope location for two monoclonal antibodies against human cystatin C, representing opposite aggregation inhibitory properties by Izabela Behrendt; Martyna Prądzińska; Marta Spodzieja; Aleksandra S. Kołodziejczyk; Sylwia Rodziewicz-Motowidło; Aneta Szymańska; Paulina Czaplewska (1717-1729).
Human cystatin C (hCC), like many other amyloidogenic proteins, dimerizes and possibly makes aggregates by subdomain swapping. Inhibition of the process should suppress the fibrillogenesis leading to a specific amyloidosis (hereditary cystatin C amyloid angiopathy, HCCAA). It has been reported that exogenous agents like monoclonal antibodies against cystatin C are able to suppress formation of cystatin C dimers and presumably control the neurodegenerative disease. We have studied in detail two monoclonal antibodies (mAbs) representing very different aggregation inhibitory potency, Cyst10 and Cyst28, to find binding sites in hCC sequence responsible for the immunocomplex formation and pave the way for possible immunotherapy of HCCAA. We used the epitope extraction/excision mass spectrometry approach with the use of different enzymes complemented by affinity studies with synthetic hCC fragments as a basic technique for epitope identification. The results were analyzed in the context of hCC structure allowing us to discuss the binding sites for both antibodies. Epitopic sequences for clone Cyst28 which is a highly potent dimerization inhibitor were found in N-terminus, loop 1 and 2 (L1, L2) and fragments of β2 and β3 strands. The crucial difference between conformational epitope sequences found for both mAbs seems to be the lack of interactions with hCC via N-terminus and the loop 1 in the case of mAb Cyst10. Presumably the interactions of mAbs with hCC via L1 and β sheet fragments make the hCC structure rigid and unable to undergo the swapping process.
Keywords: Mass spectrometry; Human cystatin C; Epitope; Monoclonal antibodies

Rapid quantitative method for the detection of phenylalanine and tyrosine in human plasma using pillar array columns and gradient elution by Yanting Song; Katsuya Takatsuki; Tetsushi Sekiguchi; Takashi Funatsu; Shuichi Shoji; Makoto Tsunoda (1731-1735).
This study reports a fast and quantitative determination method for phenylalanine (Phe) and tyrosine (Tyr) in human plasma using on-chip pressure-driven liquid chromatography. A pillar array column with low-dispersion turns and a gradient elution system was used. The separation of fluorescent derivatives of Phe, Tyr, and other hydrophobic amino acids was successfully performed within 140 s. Under the optimized conditions, Phe and Tyr in human plasma were quantified. The developed method is promising for rapid diagnosis in the clinical field.
Keywords: Microfluidics; 4-Fluoro-7-nitro-2,1,3-benzoxadiazole; Fluorescence; Quantitative determination; Amino acids

Erratum to: Hydration of amino acids: FTIR spectra and molecular dynamics studies by Aneta Panuszko; Beata Adamczak; Jacek Czub; Emilia Gojło; Janusz Stangret (1737-1737).