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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.43, #6)

Protein and non-protein biomarkers in melanoma: a critical update by Nadine Tandler; Birgit Mosch; Jens Pietzsch (pp. 2203-2230).

Melanoma is the most malignant type of all skin neoplasms. Its worldwide incidence has steadily increased during the past decades, suggesting a probable melanoma ‘epidemic’. Although current clinical, morphologic, and histopathologic methods provide insights into disease behavior and outcome, melanoma is still an unpredictable disease. Once in an advanced stage, it remains a disastrous affliction with scarce therapeutic options. Therefore, significant efforts need to be made in finding informative biomarkers or surrogate markers that could aid or improve early diagnosis of melanoma, its correct staging, the discrimination of other pathological conditions as well as indicate patients’ prognosis or the most appropriate therapeutic regimes. Ideally these markers are secreted into body fluids and easily amenable to the design of non-invasive clinical tests. A critical view on the current debate on serologic protein markers, e.g., lactate dehydrogenase, tyrosinase, and melanoma inhibiting activity, and some selected non-protein markers, e.g., 5-S-cysteinyl-dopa and circulating nucleic acids, will be offered and novel innovative approaches currently being explored will be discussed. Special emphasis is put on the S100 family of calcium binding proteins that is more and more emerging as a potentially important group of both molecular key players and biomarkers in the etiology, progression, manifestation, and therapy of neoplastic disorders, including malignant melanoma. Notably, S100B and, possibly, other S100 proteins like S100A4 are assumed to fulfill requirements which make them strong biomarker candidates in melanoma. Moreover, S100 proteins receive attention as possible targets of therapeutic intervention moving closer to clinical impact.

Keywords: Diagnostic markers; Melanomata; Molecular targets; Pigment cells; Prognostic markers; Serological markers; Skin cancer; Stem cell-like markers; S100 proteins; Therapy monitoring

On the segregation of protein ionic residues by charge type by Michael S. Parker; Ambikaipakan Balasubramaniam; Steven L. Parker (pp. 2231-2247).

Based on ubiquitous presence of large ionic motifs and clusters in proteins involved in gene transcription and protein synthesis, we analyzed the distribution of ionizable sidechains in a broad selection of proteins with regulatory, metabolic, structural and adhesive functions, in agonist, antagonist, toxin and antimicrobial peptides, and in self-excising inteins and intron-derived proteins and sequence constructs. All tested groups, regardless of taxa or sequence size, show considerable segregation of ionizable sidechains into same type charge (homoionic) tracts. These segments in most cases exceed half of the sequence length and comprise more than two-thirds of all ionizable sidechains. This distribution of ionic residues apparently reflects a fundamental advantage of sorted electrostatic contacts in association of sequence elements within and between polypeptides, as well as in interaction with polynucleotides. While large ionic densities are encountered in highly interactive proteins, the average ionic density in most sets does not change appreciably with size of the homoionic segments, which supports the segregation as a modular feature favoring association.

Keywords: Ionic segregation; Homobasic tract; Homoacidic tract; Homoionic zipper

The role of taurine in renal disorders by Xiaobin Han; Russell W. Chesney (pp. 2249-2263).

This article examines the actions of taurine on models of renal dysfunction, the potential mechanisms of taurine action and the possible clinical significance of these findings. Our laboratory has written previously on the role of taurine in renal function and we have focused upon the normal physiology of the kidney and on the mechanisms and regulation of the renal transport of taurine. This review is a distinct change of emphasis in that we describe a number of studies which have evaluated various aspects of renal dysfunction, including hypertension and proteinuria, specific glomerular and tubular disorders, acute and chronic renal conditions, urinary tract conditions including infection and nephrolithiasis, and diabetic nephropathy. The subject of chronic kidney disease and renal transplantation will also be examined relative to β amino acid. The studies evaluated will be mainly recent ones, recognizing that older reviews of the role of this taurine in the kidney are available.

Keywords: Taurine; Renal function; Glomerular nephritis; Acute kidney injury; Diabetic nephropathy; Chronic kidney disease

Enhanced leishmanicidal activity of cryptopeptide chimeras from the active N1 domain of bovine lactoferrin by Tânia Silva; María Ángeles Abengózar; María Fernández-Reyes; David Andreu; Kamran Nazmi; Jan G. M. Bolscher; Margarida Bastos; Luis Rivas (pp. 2265-2277).

Two antimicrobial cryptopeptides from the N1 domain of bovine lactoferrin, lactoferricin (LFcin17–30) and lactoferrampin (LFampin265–284), together with a hybrid version (LFchimera), were tested against the protozoan parasite Leishmania. All peptides were leishmanicidal against Leishmania donovani promastigotes, and LFchimera showed a significantly higher activity over its two composing moieties. Besides, it was the only peptide active on Leishmania pifanoi axenic amastigotes, already showing activity below 10 μM. To investigate their leishmanicidal mechanism, promastigote membrane permeabilization was assessed by decrease of free ATP levels in living parasites, entrance of the vital dye SYTOX Green (MW = 600 Da) and confocal and transmission electron microscopy. The peptides induced plasma membrane permeabilization and bioenergetic collapse of the parasites. To further clarify the structural traits underlying the increased leishmanicidal activity of LFchimera, the activity of several analogues was assessed. Results revealed that the high activity of these hybrid peptides seems to be related to the order and sequence orientation of the two cryptopeptide moieties, rather than to their particular linkage through an additional lysine, as in the initial LFchimera. The incorporation of both antimicrobial cryptopeptide motifs into a single linear sequence facilitates chemical synthesis and should help in the potential clinical application of these optimized analogues.

Keywords: Leishmania ; Leishmanicidal activity; Antimicrobial peptides; Membrane permeabilization; Lactoferricin; Lactoferrampin; LFchimera

Chemical synthesis and biological evaluation of an antimicrobial peptide gonococcal growth inhibitor by John D. Wade; Feng Lin; Mohammed Akhter Hossain; Raymond M. Dawson (pp. 2279-2283).

Gonococcal growth inhibitor 1 (GGI-1) is a 44-residue peptide with potent anti-Legionella activity. It has been isolated from Staphylococcus haemolyticus but, to date, its chemical synthesis has not been reported. Acquisition of this peptide via this means would enable a more detailed examination of its antimicrobial properties. However, its synthesis represents a significant challenge because of two predicted “difficult sequences” within the peptide. Its successful solid-phase assembly is reported in this paper, and was accomplished by use of simple palliative measures including the introduction of a single pseudo-proline isostere in order to counteract on-resin aggregation. The peptide had moderate antimicrobial activity against Escherichia coli but was inactive against another Gram-negative bacterium and two Gram-positive bacteria (Bacillus species). It had significant haemolytic activity, with a H₅₀ (concentration of peptide that causes 50 % haemolysis) of 20 and 125 μM for two blood samples from different donors. An alternative therapeutic index to that proposed for GGI-1 in a recent publication is proposed.

Keywords: Antimicrobial peptide; “Difficult sequence”; Haemolytic activity; Gonococcal growth inhibitor 1; Solid phase peptide synthesis

Modafinil improves performance in the multiple T-Maze and modifies GluR1, GluR2, D2 and NR1 receptor complex levels in the C57BL/6J mouse by Sunetra Sase; Deeba Khan; Fernando Sialana; Harald Höger; Nina Russo-Schlaff; Gert Lubec (pp. 2285-2292).

Modafinil has been shown to modify behavioural and cognitive functions and to effect several brain receptors. Effects, however, were not observed at the receptor protein complex level and it was therefore the aim of the study to train mice in the multiple T-Maze (MTM) as a paradigm for spatial memory and to determine paralleling brain receptor complex levels. Sixty C57BL/6J mice were used in the study and divided into four groups (trained drug injected; trained vehicle injected; yoked drug injected; yoked vehicle injected). Animals obtained training for 4 days and were killed 6 h following the last training session on day 4. Hippocampi were dissected from the brain, membrane fractions were prepared by ultracentrifugation and were run on blue-native gels and immunoblotted with antibodies against major brain receptors. Modafinil treatment led to decreased latency and increased average speed, but not to changes in pathlength and number of correct decisions in the MTM. Drug effects were modifying receptor complexes of GluR1, GluR2, D2 and NR1. Training effects on receptor complex levels were observed for GluR3, D1 and nicotinic acetylcholine receptor alpha 7 (Nic7). GluR1 levels were correlating with GluR2 and D1 levels were correlating with D2 and NR1. Involvement of the glutamatergic, NMDA, dopaminergic and nicotinergic system in modafinil and memory training were herein described for the first time. A brain receptor complex pattern was revealed showing the concerted action following modafinil treatment.

Keywords: Modafinil; Brain receptor; Glutamatergic; Nicotinergic; Dopaminergic; Spatial memory

High clinical accuracy of asymmetric dimethylarginine and symmetric dimethylarginine in patients with ischemic heart disease by B. V. Djordjević; R. Pavlović; V. Ćosić; M. Deljanin-Ilić; T. Ristić; N. Krstić; T. Jevtović-Stoimenov (pp. 2293-2300).

Elevated plasma concentrations of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) were found in various clinical settings including coronary heart disease. To assess ADMA and SDMA diagnostic validity in patients with different stages of ischemic heart disease, we studied these markers in patients having stable angina pectoris (SAP), unstable angina (USAP), and acute myocardial infarction (AMI). The results were compared with the values of healthy individuals. Plasma ADMA and SDMA levels were measured by high-performance liquid chromatography. In all patient groups both markers were significantly elevated in comparison with control ones (p < 0.001). In SAP patients, the median ADMA value was 0.75 (0.31–2.73) μmol/L, and SDMA 1.11 (0.69–0.1.42) μmol/L, in USAP patients, the marker values were 0.94 (0.34–3.13) μmol/L and 1.23 (0.88–4.72) μmol/L, and in AMI patients, 0.98 (0.48–2.01) μmol/L and 1.26 (0.75–2.93) μmol/L, while in healthy subjects they were 0.31 (0.17–0.87) μmol/L and 0.29 (0.20–0.83) μmol/L, respectively. SDMA was found significantly different in SAP and AMI patients (p < 0.05). Diagnostic accuracy was determined by receiver operating characteristic (ROC) curve analysis. The highest area under the ROC (AUC) for ADMA was obtained in AMI patients (0.976), while for SDMA in USAP patients (1.000). There was no significant difference between the AUCs. The greatest sensitivity and specificity were found in the USAP group (95.65 and 96.30 % for ADMA, and 100 % for each characteristic of SDMA). Considering these results, SDMA showed better clinical accuracy in assessing ischemic disease, where it could be used as a valid marker and a therapeutic target.

Keywords: Asymmetric dimethylarginine; Symmetric dimethylarginine; Ischemic heart disease

l-Valine production with minimization of by-products’ synthesis in Corynebacterium glutamicum and Brevibacterium flavum by Xiaohu Hou; Xinde Chen; Yue Zhang; He Qian; Weiguo Zhang (pp. 2301-2311).

Corynebacterium glutamicum ATCC13032 and Brevibacterium flavum JV16 were engineered for l-valine production by over-expressing ilvEBN r C genes at 31 °C in 72 h fermentation. Different strategies were carried out to reduce the by-products’ accumulation in l-valine fermentation and also to increase the availability of precursor for l-valine biosynthesis. The native promoter of ilvA of C. glutamicum was replaced with a weak promoter MPilvA (P-ilvAM1CG) to reduce the biosynthetic rate of l-isoleucine. Effect of different relative dissolved oxygen on l-valine production and by-products’ formation was recorded, indicating that 15 % saturation may be the most appropriate relative dissolved oxygen for l-valine fermentation with almost no l-lactic acid and l-glutamate formed. To minimize l-alanine accumulation, alaT and/or avtA was inactivated in C. glutamicum and B. flavum, respectively. Compared to high concentration of l-alanine accumulated by alaT inactivated strains harboring ilvEBN r C genes, l-alanine concentration was reduced to 0.18 g/L by C. glutamicum ATCC13032MPilvA△avtA pDXW-8-ilvEBN r C, and 0.22 g/L by B. flavum JV16avtA::Cm pDXW-8-ilvEBN r C. Meanwhile, l-valine production and conversion efficiency were enhanced to 31.15 g/L and 0.173 g/g by C. glutamicum ATCC13032MPilvA△avtA pDXW-8-ilvEBN r C, 38.82 g/L and 0.252 g/g by B. flavum JV16avtA::Cm pDXW-8-ilvEBN r C. This study provides combined strategies to improve l-valine yield by minimization of by-products’ production.

Keywords: l-Valine; Corynebacterium glutamicum ; Brevibacterium flavum ; alaT ; avtA

A biomimetic domino reaction for the concise synthesis of capreomycidine and epicapreomycidine by Martin Büschleb; Markus Granitzka; Dietmar Stalke; Christian Ducho (pp. 2313-2328).

The non-proteinogenic amino acids capreomycidine and epicapreomycidine are constituents of antibiotically active natural products, but the synthesis of these unusual cyclic guanidine derivatives is challenging. The biosynthesis of capreomycidine has therefore been employed as a guideline to develop a concise biomimetic synthesis of both epimeric amino acids. The resulting domino-guanidinylation-aza-Michael-addition reaction provides the most convenient access to these amino acids in racemic form. Attempts to dissect the domino reaction into two separate transformations for a stereocontrolled version of this synthetic approach have also been made. The synthesized didehydro-arginine derivatives with urethane-protected guanidine moieties did not undergo the aza-Michael-addition anymore. These results may have wider implications for the 1,4-addition of guanidines to α,β-unsaturated carbonyl compounds, particularly to didehydro amino acids.

Keywords: Natural products; Antibiotics; Amino acids; Biomimetic synthesis; Domino reactions; Guanidines

A biomimetic domino reaction for the concise synthesis of capreomycidine and epicapreomycidine by Martin Büschleb; Markus Granitzka; Dietmar Stalke; Christian Ducho (pp. 2313-2328).

Keywords: Natural products; Antibiotics; Amino acids; Biomimetic synthesis; Domino reactions; Guanidines

A photoactivable amino acid based on a novel functional coumarin-6-yl-alanine by Andrea S. C. Fonseca; M. Sameiro T. Gonçalves; Susana P. G. Costa (pp. 2329-2338).

A novel fluorescent amino acid, l-4-chloromethylcoumarin-6-yl-alanine, was obtained from tyrosine by a Pechmann reaction. The assembly of the heterocyclic ring at the tyrosine side chain could be achieved before or after incorporation of tyrosine into a dipeptide, and amino acid and dipeptide ester conjugates were obtained by coupling to a model N-protected alanine. The behaviour of one of the fluorescent conjugates towards irradiation was studied in a photochemical reactor at different wavelengths (254, 300, 350 and 419 nm). The photoreaction course in methanol/HEPES buffer solution (80:20) was followed by HPLC/UV monitoring. It was found that the novel unnatural amino acid could act as a fluorescent label, due to its fluorescence properties, and, more importantly, as a photoactivable unit, due to the short irradiation times necessary to cleave the ester bond between the model amino acid and the coumarin-6-yl-alanine.

Keywords: Coumarin-6-yl-alanine; Coumarin; Fluorescence; Amino acid conjugates; Photolysis

Characterization and dietary regulation of glutamate dehydrogenase in different ploidy fishes by Zhen Liu; Yi Zhou; Shaojun Liu; Huan Zhong; Chun Zhang; Xuewei kang; Yun Liu (pp. 2339-2348).

Glutamate dehydrogenase (GDH) plays a crucial role in amino acid deamination and has been used as an inductor of nutrients metabolism. In this study, we cloned and analyzed the GDH cDNAs in diploids (red crucian carp), triploids and tetraploids and characterized their expression profiles upon dietary treatments. Results showed a high sequence similarity of GDH among the three kinds of ploidy fishes and other vertebrates. Expression analysis revealed that GDH exhibited a distinct spatial pattern of expression in different types of fishes. The triploids and tetraploids had higher levels of expression than diploids in heart, liver, gill, muscle, fore-gut and mid-gut. The GDH expression was also developmentally regulated with a stronger expression around blastula stage. The maternal GDH transcripts were first detected from eggs and their expression dropped down from the gastrula stage to heart beat stage. Adult triploids showed the highest levels of GDH expression in liver during breeding season which may contribute to the good appetite and fast growth. In addition, triploids exhibited high growth rates and excess GDH expression compared with other two types of fishes. The liver GDH enzyme activities were also higher in triploids than red crucian carp and tetraploids. Moreover, GDH expression is regulated by dietary protein levels. Fish fed with either high or low protein diets showed higher levels of GDH expression. In summary, our results demonstrated for the first time that the different ploidy fishes had different patterns of GDH mRNA expression during development, breeding and non-breeding seasons, and as well dietary effects from different protein levels in diet. These data indicate that abundant GDH expression may play an important role in growth rates in triploids.

Keywords: Glutamate dehydrogenase; Red crucian carp; Triploid; Tetraploid; Embryogenesis; Dietary protein level

Gustatory sensation of l- and d-amino acids in humans by Misako Kawai; Yuki Sekine-Hayakawa; Atsushi Okiyama; Yuzo Ninomiya (pp. 2349-2358).

Amino acids are known to elicit complex taste, but most human psychophysical studies on the taste of amino acids have focused on a single basic taste, such as umami (savory) taste, sweetness, or bitterness. In this study, we addressed the potential relationship between the structure and the taste properties of amino acids by measuring the human gustatory intensity and quality in response to aqueous solutions of proteogenic amino acids in comparison to d-enantiomers. Trained subjects tasted aqueous solution of each amino acid and evaluated the intensities of total taste and each basic taste using a category-ratio scale. Each basic taste of amino acids showed the dependency on its hydrophobicity, size, charge, functional groups on the side chain, and chirality of the alpha carbon. In addition, the overall taste of amino acid was found to be the combination of basic tastes according to the partial structure. For example, hydrophilic non-charged middle-sized amino acids elicited sweetness, and l-enantiomeric hydrophilic middle-sized structure was necessary for umami taste. For example, l-serine had mainly sweet and minor umami taste, and d-serine was sweet. We further applied Stevens’ psychophysical function to relate the total-taste intensity and the concentration, and found that the slope values depended on the major quality of taste (e.g., bitter large, sour small).

Keywords: Amino acid; Taste; Human psychophysics

Taurine plays an important role in the protection of spermatogonia from oxidative stress by Masato Higuchi; Fritzie T. Celino; Sonoko Shimizu-Yamaguchi; Chiemi Miura; Takeshi Miura (pp. 2359-2369).

It has been demonstrated that taurine has various physiological functions in the body. We demonstrated that taurine is abundant in the serum, liver, muscle and testis of the Japanese eel (Anguilla japonica). In the eel testis, taurine is found mainly in spermatogonia and is weakly expressed also in the Sertoli cells. We have further found in the eel testis that taurine is actively accumulated via the sodium/chloride-dependent taurine transporter (TauT; SLC6A6), which is expressed in germ cells. In our current study, the effects of taurine on the anti-oxidant response were examined. Taurine was found to promote the total superoxide dismutase (SOD) activity in the testis. Moreover, our results indicate that taurine does not affect the mRNA levels of copper–zinc (Cu/Zn) SOD or manganese SOD, but promotes the translation of Cu/Zn SOD. Overall, our present data suggest that taurine may modulate Cu/Zn SOD at the translational level and thereby may play an important role in the protection of germ cells from oxidative stress.

Keywords: Taurine; Cu/Zn SOD; Spermatogonia; Oxidative stress; Japanese eel

Proline protects liver from d-galactosamine hepatitis by activating the IL-6/STAT3 survival signaling pathway by Yoko Obayashi; Harumi Arisaka; Shintaro Yoshida; Masato Mori; Michio Takahashi (pp. 2371-2380).

The oral administration of proline, one of the non-essential amino acids, has been shown to effectively protect the liver from d-galactosamine (GalN)-induced liver injury and to improve the survival rate. The aim of this study was to investigate the mechanism of this protective action of proline. We paid particular attention to the effect of proline on inflammatory activation, regenerative response, and the associated signal transduction in the liver. Male Fischer rats received intraperitoneal injections of GalN (1.4 g/kg) with or without the oral administration of proline (2 g/kg) 1 h before GalN treatment. Liver pathology, plasma indices of inflammation, and the level of proliferative marker in the liver were monitored. The hepatic activation of interleukin-6 (IL-6)/signal transducer and activator of transcription (STAT)-3 pathway, which is downstream of tumor necrosis factor (TNF)-α/nuclear factor-κB, was also studied. GalN induced massive inflammatory expansion in the liver, leading to a high death rate (60 %) more than 72 h after the treatment. Proline administration significantly suppressed inflammatory infiltration in the live after 48 h, which was accompanied by depletion of plasma TNF-α, glutamic oxaloacetic transaminase, and glutamic pyruvic transaminase. The mRNA expression of histone H3, a marker of proliferation, was significantly upregulated in the liver of proline-treated animals. Furthermore, IL-6/STAT-3 pathway, an anti-inflammatory and regenerative signaling pathway, was strongly activated prior to these observations, with the upregulated expression of downstream genes. These results suggest that the tissue-protective mechanism of proline involves the early activation of IL-6/STAT-3 pathway in the liver, with subsequent activation of the regenerative response and suppression of massive inflammatory activation.

Keywords: Proline; d-Galactosamine; TNF-α; Liver; IL-6; STAT3

X-ray crystal structure of CMS1MS2: a high proteolytic activity cysteine proteinase from Carica candamarcensis by Marco T. R. Gomes; Raphael D. Teixeira; Míriam T. P. Lopes; Ronaldo A. P. Nagem; Carlos E. Salas (pp. 2381-2391).

CMS1MS2 (CC-Ib) from Carica candamarcensis (Vasconcellea cundinamarcensis) is a cysteine proteinase found as a single polypeptide containing 213 residues of 22,991 Da. The enzyme was purified by three chromatographic steps, two of them involving cationic exchange. Crystals of CMS1MS2 complexed with E-64 were obtained by the hanging drop vapor-diffusion method at 291 K using ammonium sulfate and polyethylene glycol 4000/8000 as precipitant. The complex CMS1MS2-E-64 crystallized in the tetragonal space group P4₁2₁2 with unit-cell parameters; a = b = 73.64, c = 118.79 Å. The structure was determined by Molecular Replacement and refined at 1.87 Å resolution to a final R factor of 16.2 % (R _free = 19.3 %). Based on the model, the structure of CMS1MS2 (PDB 3IOQ) ranks as one of the least basic cysteine isoforms from C. candamarcensis, is structurally closer to papain, caricain, chymopapain and mexicain than to the other cysteine proteinases, while its activity is twice the activity of papain towards BAPNA substrate. Two differences, one in the S2 subsite and another in the S3 subsite of CMS1MS2 may contribute to the enhanced activity relative to papain. In addition, the model provides a structural basis for the sensitivity of CMS1MS2 to inhibition by cystatin, not shown by other enzymes of the group, e.g., glycyl endopeptidase and CMS2MS2.

Keywords: Carica candamarcensis ; Crystal structure; Cysteine protease; Latex; Proteinase; Vasconcellea cundinamarcensis

Comparative proteome analysis of high and low cadmium accumulating soybeans under cadmium stress by Zahed Hossain; Makita Hajika; Setsuko Komatsu (pp. 2393-2416).

A comparative proteomic study was performed to unravel the protein networks involved in cadmium stress response in soybean. Ten-day-old seedlings of contrasting cadmium accumulating soybean cultivars—Harosoy (high cadmium accumulator), Fukuyutaka (low cadmium accumulator), and their recombinant inbred line CDH-80 (high cadmium accumulator) were exposed to 100 μM CdCl₂ treatment for 3 days. Root growth was found to be affected under cadmium stress in all. Varietal differences at root protein level were evaluated. NADP-dependent alkenal double bond reductase P1 was found to be more abundant in low cadmium accumulating Fukuyutaka. Leaf proteome analysis revealed that differentially expressed proteins were primarily involved in metabolism and energy production. The results indicate that both high and low cadmium accumulating cultivars and CDH-80 share some common defense strategies to cope with the cadmium stress. High abundance of enzymes involved in glycolysis and TCA cycle might help cadmium challenged cells to produce more energy necessary to meet the high energy demand. Moreover, enhanced expressions of photosynthesis related proteins indicate quick utilization of photoassimilates in energy generation. Increased abundance of glutamine synthetase in all might be involved in phytochelatin mediated detoxification of cadmium ions. In addition, increased abundance of antioxidant enzymes, namely superoxide dismutase, ascorbate peroxidase, catalase, ensures cellular protection from reactive oxygen species mediated damages under cadmium stress. Enhanced expression of molecular chaperones in high cadmium accumulating cultivar might be another additional defense mechanism for refolding of misfolded proteins and to stabilize protein structure and function, thus maintain cellular homeostasis.

Keywords: Cadmium; Soybean; Proteomics; Heavy metal accumulation

Oligomerization of glycine and alanine on metal(II) octacynaomolybdate(IV): role of double metal cyanides in prebiotic chemistry by Anand Kumar; Kamaluddin (pp. 2417-2429).

Condensation reactions of amino acid (glycine and alanine) on the surface of metal(II) octacyanomolybdate(IV) (MOCMo) complexes are investigated using high-performance liquid chromatography (HPLC) and electron spray ionizations–mass spectroscopy (ESI–MS). The series of MOCMo have been synthesized and the effect of outer sphere metal ions present in the MOCMo on the oligomerization of glycine and alanine at different temperature and time found out. Formation of peptides was observed to start after 7 days at 60 °C. Maximum yield of peptides was found after 35 days at 90 °C. It has been found that zinc(II) octacyanomolybdate(IV) and cobalt(II) were the most effective metal cations present in outer sphere of the MOCMo for the production of high yield of oligomerized products. Surface area of MOCMo seems to play dominating parameter for the oligomerization of alanine and glycine. The results of the present study reveal the role of MOCMo in chemical evolution for the oligomerization of biomolecules.

Keywords: Glycine; Alanine; Metal(II) octacyanomolybdate(IV); Prebiotic chemistry; Oligomerization; HPLC; ESI–MS

Improvement of enzymatic stability and intestinal permeability of deuterohemin-peptide conjugates by specific multi-site N-methylation by Qing-Guang Dong; Yong Zhang; Meng-Shu Wang; Jiao Feng; Hai-Hong Zhang; Yong-Ge Wu; Tie-Jun Gu; Xiang-Hui Yu; Chun-Lai Jiang; Yan Chen; Wei Li; Wei Kong (pp. 2431-2441).

The deuterohemin-peptide conjugate, DhHP-6 (Dh-β-AHTVEK-NH₂), is a microperoxidase mimetic, which has demonstrated substantial benefits in vivo as a scavenger of reactive oxygen species (ROS). In this study, specific multi-site N-methylated derivatives of DhHP-6 were designed and synthesized to improve metabolic stability and intestinal absorption, which are important factors for oral delivery of therapeutic peptides and proteins. The DhHP-6 derivatives were tested for (1) scavenging potential of hydrogen peroxide (H₂O₂); (2) permeability across Caco-2 cell monolayers and everted gut sacs; and (3) enzymatic stability in serum and intestinal homogenate. The results indicated that the activities of the DhHP-6 derivatives were not influenced by N-methylation, and that tri-N-methylation of DhHP-6 could significantly increase intestinal flux, resulting in a two- to threefold higher apparent permeability coefficient. In addition, molecules with N-methylation at selected sites (e.g., Glu residue) showed high resistance against proteolytic degradation in both diluted serum and intestinal preparation, with 50- to 140-fold higher half-life values. These findings suggest that the DhHP-6 derivatives with appropriate N-methylation could retain activity levels equivalent to that of the parent peptide, while showing enhanced intestinal permeability and stability against enzymatic degradation. The tri-N-methylated peptide Dh-β-AH(Me)T(Me)V(Me)EK-NH₂ derived from this study may be developed as a promising candidate for oral administration.

Keywords: DhHP-6; ROS; N-methylated peptide; Permeability; Enzymatic stability

Gene expression profiling of hybridoma cells after bursal-derived bioactive factor BP5 treatment by Xiu L. Feng; Qing T. Liu; Rui B. Cao; Bin Zhou; De Y. Li; Yuan P. Zhang; Ke Liu; Xiao D. Liu; Jian C. Wei; Ya F. Qiu; Xin F. Li; Zhi Y. Ma; Pu Y. Chen (pp. 2443-2456).

Bursa of Fabricius is the acknowledged vital humoral immune system for B cell differentiation and antibody production. To study the molecular mechanism underlying the effect of bursal-derived BP5, we used gene microarray to analyze the genomic expression profiling of BP5-treated hybridoma cells. BP5 exhibited an immunomodulatory effect on antibody production in hybridoma cells and induced alterations in the gene expression profiles related to the immune-related biological processes, such as T cell activation and proliferation, B cell activation, B cell-mediated immunity, and cytokines cytokine production involved in immune response. In addition, 26 biological pathways associated with immunomodulatory functions were regulated in BP5-treated hybridoma cells, in which p53 signal pathway played an important role in antitumor. Among these regulated genes, 12 differentially expressed genes were verified by qRT-PCR. The activation of p53 activity by BP5 was further confirmed by p53 luciferase reporter assay and p53 expression. Our data revealed that bursal-derived BP5 could regulate various immune-related cellular processes, including antitumor factor p53 signal pathway, perhaps partially accounting for the reported immunomodulatory roles and novel antiproliferation on tumor cells functions of bursal-derived bioactive factor BP5.

Keywords: Bursal-derived BP5; Gene microarray; Go analysis; p53 pathway; p53 expression

Wards in the keyway: amino acids with anomalous pK _as in calycins by Ivano Eberini; Cristina Sensi; Michele Bovi; Henriette Molinari; Monica Galliano; Franco Bonomi; Stefania Iametti; Elisabetta Gianazza (pp. 2457-2468).

As a follow-up to our recent analysis of the electrostatics of bovine β-lactoglobulin (Eberini et al. in Amino Acids 42:2019–2030, 2011), we investigated whether the occurrence in the native structure of calycins—the superfamily to which β-lactoglobulin belongs—of amino acids with anomalous pK _as is an infrequent or, on the contrary, a common occurrence, and whether or not a general pattern may be recognized. To this aim, we randomly selected four calycins we had either purified from natural sources or prepared with recombinant DNA technologies during our previous and current structural and functional studies on this family. Their pIs vary over several pH units and their known functions are as diverse as carriers, enzymes, immunomodulators and/or extracellular chaperones. In our survey, we used both in silico prediction methods and in vitro procedures, such as isoelectric focusing, electrophoretic titration curves and spectroscopic techniques. By comparing the results under native conditions (no exposure of the proteins to chaotropic agents) to those after protein unfolding (in the presence of 8 M urea), a shift is observed in the pK _a of at least one amino acid per protein, which results in a measurable change in pI. Three types of amino acids are involved: Cys, Glu, and His, their position varies along the calycin sequence. Although no common mechanism may thus be recognized, we hypothesize that the ‘normalization’ of anomalous pK _as may be the phenomenon that accompanies, and favors, structural rearrangements such as those involved in ligand binding by these proteins. An interesting, if anecdotal, validation to this view comes from the behavior of human retinol binding protein, for which the pI of the folded and liganded protein is intermediate between those of the folded and unliganded and of the unfolded protein forms. Likewise, both solid (from crystallography) and solution state (from CD spectroscopy) data confirm that the protein undergoes structural rearrangement upon retinol binding.

Keywords: Calycins; Electrostatics; Unfolding; Urea; Electrophoresis; Molecular modeling

Brassinolide enhances cold stress tolerance of fruit by regulating plasma membrane proteins and lipids by Boqiang Li; Changfeng Zhang; Baohua Cao; Guozheng Qin; Weihao Wang; Shiping Tian (pp. 2469-2480).

How to enhance fruit tolerance to cold stress is an important biological interest. In this paper, we found that mango (Mangifera indica L.) fruit treated with 10 μM brassinolide (BL) showed a higher tolerance to cold temperature of 5 °C. Further, we compared the changes in expression profiles of plasma membrane (PM) proteins and the corresponding gene expressions between BL-treated and control fruit. Fourteen differential proteins were positively identified by mass spectrometry, and were categorized into four groups, including transport, cellular biogenesis, defense and stress response, and unknown function. Among them, four proteins (remorin, abscisic stress ripening-like protein, type II SK2 dehydrin, and temperature-induced lipocalin) and genes encoding these proteins were up-regulated in BL treatment under cold stress. Moreover, we found that PM lipids in BL-treated fruit showed lower phase transition temperature and higher unsaturation degree, leading to higher fluidity under low temperature. These findings ascertain that PM proteins and lipids are involved in BL-mediated responses to cold stress in mango fruit, and provide novel evidence that BL plays an important role in regulating cold stress tolerance in fruit.

Keywords: Brassinosteroids; Chilling injury; Lipids; Mango fruit; Membrane proteins

Dynamic changes in blood flow and oxygen consumption in the portal-drained viscera of growing pigs receiving acute administration of l-arginine by Bie Tan; Xinguo Li; Guoyao Wu; Xiangfeng Kong; Zhiqiang Liu; Tiejun Li; Yulong Yin (pp. 2481-2489).

This study tested the hypothesis that an increase in arginine concentration in the portal vein may affect blood flow and oxygen consumption in the portal-drained viscera (PDV) of swine. Eight barrows (70 kg body weight) were surgically fitted with chronic catheters in the portal vein, ileal vein, and carotid artery. Thirteen days after the surgery, pigs that had been fasted for 12 h were randomly allocated to receive administration of either l-alanine (103 mg/kg body weight, isonitrogenous control) or l-arginine–HCl (61 mg/kg body weight) via the portal vein. Portal vein blood flow (PVBF) was measured with infusion of p-aminohippuric acid into the ileal vein, and blood samples were simultaneously obtained every 0.5 h for 4 h. Compared with the control, arginine infusion increased PVBF at 30–90 min after infusion but decreased PDV oxygen consumption at 60–150 min after infusion (P < 0.05). Plasma concentrations of glutamate at infusion times of 180–240 min and of arginine at infusion times of 60–240 min in arginine-infused pigs were higher than those for the control group (P < 0.05). Plasma concentrations of insulin and glucagon at the infusion times of 30–90 min were higher and of free fatty acids at the infusion times of 60–120 min were lower than those for the control pigs (P < 0.05). These results indicate that increasing arginine concentration in the portal vein enhances PDV blood flow, reduces PDV oxygen consumption, and beneficially alters the metabolic profile in swine, an established animal model for studying human nutrition and metabolism.

Keywords: Arginine; Blood flow; Oxygen consumption; Pigs; Digestion

Involvement of hippocampal CAMKII/CREB signaling in the spatial memory retention induced by creatine by Mauren Assis Souza; Danieli Valnes Magni; Gustavo Petri Guerra; Mauro Schneider Oliveira; Ana Flávia Furian; Letícia Pereira; Silvia Vacari Marquez; Juliano Ferreira; Michele Rechia Fighera; Luiz Fernando Freire Royes (pp. 2491-2503).

Although Creatine (Cr) and Phosphocreatine (PCr) systems play a key role in cellular energy and energy transport in neuronal cells, its implications for learning and memory are still controversial. Thus, we decided to investigate the involvement of cAMP-dependent protein kinase A (PKA), Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and cAMP responsive element binding protein (CREB) in the spatial consolidation after an intrahippocampal injection of Cr. Statistical analysis revealed that Cr (2.5 nmol/hippocampus) (post-training) decreased the latency for escape and the mean number of errors on Barnes maze test. Post-training co-administration of the PKA inhibitor (H-89 25 ρmol/hippocampus) did not alter the facilitatory effect of Cr in this memory test. On the other hand, Cr-induced spatial retention was reverted by co-administration of the CaMKII inhibitor (STO-609 5 nmol/hippocampus). Neurochemical analysis revealed that intrahippocampal injection of Cr, when analyzed after 30 min rather than after 3 h, increased the levels of pCREB and pCaMKII but not pPKA levels. Statistical analysis also revealed that the post-training co-administration of STO-609 but not H-89 reversed the increase of pCREB levels induced by Cr. The results presented in this report suggest that intracellular CaMKII/CREB pathway plays a key role in the Cr-induced spatial retention. Thus, it is plausible to propose that Cr plays a putative role as a neuromodulator in the brain, and that at least some of its effects may be mediated by intracellular CaMKII/CREB pathway.

Keywords: Creatine; Spatial memory; Hippocampus; Protein kinases; CREB

Identification of l-3-hydroxykynurenine O-sulfate in the buccal gland secretion of the parasitic lamprey, Lethenteron japonicum by Shoji Odani; Naoko Ito; Mai Hasegawa; Toshio Uchiumi; Sumihiro Hase (pp. 2505-2512).

Parasitic lampreys are known to secrete proteins having anticoagulant and vasodilator activities from the buccal glands during feeding on their host’s blood. However, small molecules in the secretion have never been explored in detail. We examined the secretion of Japanese liver lamprey (Lethenteron japonicum) for small molecules and found an intensely fluorescent substance upon gel filtration. After purification by anion-exchange chromatography and reversed-phase HPLC, structure of the compound was determined to be l-3-hydroxykynurenine O-sulfate by NMR- and UV-spectrometry, complemented with enzymatic and chemical degradation. In vertebrates, the sulfate ester of 3-hydroxykynurenine is a compound that has been regarded as a urinary metabolite of tryptophan but not reported from normal tissues to date. Although the function of this molecule in the buccal glands remains to be elucidated, it is remarkable that the same substance was described in 1960s from two species of blood-sucking insects, Rhodnius prolixus and Triatoma infestans, suggesting its potential role in blood-feeding.

Keywords: 3-Hydroxykynurenine; 3-Hydroxykynurenine O-sulfate; Lamprey; Buccal gland; Lethenteron japonicum

A comparative proteomics analysis in roots of soybean to compatible symbiotic bacteria under flooding stress by Amana Khatoon; Shafiq Rehman; Afshin Salavati; Setsuko Komatsu (pp. 2513-2525).

A proteomics approach was used to evaluate the effects of flooding stress on early symbiotic interaction between soybean roots and soil bacteria, Bradyrhizobium japonicum. Three-day-old soybean was inoculated with B. japonicum followed by flooding. The number of root hairs in seedlings, without or with flooding stress, was increased after 3 days of inoculation. Proteins were extracted from roots and separated by two-dimensional polyacrylamide gel electrophoresis. Out of 219 protein spots, 14 and 8 proteins were increased and decreased, respectively, by inoculation under flooding compared with without flooding. These proteins were compared in untreated and flooded seedlings. Increased level of 6 proteins in flooded seedlings compared with untreated seedlings was suppressed by inoculation in seedlings under flooding. They were related to disease/defense, protein synthesis, energy, and metabolism. Differential abundance of glucan endo-1,3-beta-glucosidase, phosphoglycerate kinase, and triosephosphate isomerase, based on their localization in middle and tip of root, indicated that they might be related to increase in number of root hairs. These results suggest that disease/defense, energy, and metabolism-related proteins may be particularly subjected to regulation in flooded soybean seedlings, when inoculated with B. japonicum and that this regulation may lead to increase in number of root hair during early symbiotic differentiation.

Keywords: Soybean; Flooding; Proteomics; Root; Symbiosis

Biochemical property and membrane-peptide interactions of de novo antimicrobial peptides designed by helix-forming units by Qing-Quan Ma; Na Dong; An-Shan Shan; Yin-Feng Lv; Yu-Zhi Li; Zhi-Hui Chen; Bao-Jing Cheng; Zhong-Yu Li (pp. 2527-2536).

Typical peptides composed of Phe, Ile, and Arg residues have not been reported, and the effect of the helix-forming unit (HFU) composed of the tripeptide core on biological activity remains unclear. In this study, multimers of the 3-residue HFU were designed to investigate the structure–function relationships. The in vitro biological activities of the peptides were determined. We used synthetic lipid vesicles and intact bacteria to assess the interactions of the peptides with cell membranes. The well-studied peptide melittin was chosen as a control peptide. The results showed that the antimicrobial and hemolytic activities of the peptides increased with the number of HFUs. HFU3 had optimal cell selectivity as determined by the therapeutic index. HFU3 and HFU4 exhibited strong resistance to salts, pH, and heat. CD spectra revealed that the peptides except HFU2 displayed α-helix-rich secondary structures in the presence of SDS or trifluoroethanol (TFE). The peptides interacted weakly with zwitterionic phospholipids (mimicking mammalian membranes) but strongly with negatively charged phospholipids (mimicking bacterial membranes), which corresponds well with the data for the biological activities. There was a correlation between the cell selectivity of the peptides and their high binding affinity with negatively charged phospholipids. Cell membrane permeability experiments suggest that the peptides targeted the cell membrane, and HFU3 showed higher permeabilization of the inner membrane but lower permeabilization of the outer membrane than melittin. These findings provide the new insights to design antimicrobial peptides with antimicrobial potency by trimers.

Keywords: Antimicrobial peptides; Amino acids; Helix; Cell membrane; Residue

Synthesis of a diaminopropanoic acid-based nucleoamino acid and assembly of cationic nucleopeptides for biomedical applications by Giovanni N. Roviello; Domenica Musumeci; Enrico M. Bucci; Carlo Pedone (pp. 2537-2543).

In this work, we report a synthetic approach to a Fmoc-protected nucleoamino acid, based on l-diaminopropanoic acid, carrying the DNA nucleobase on the alpha-amino group by means of an amide bond, suitable for the solid-phase synthesis of novel nucleopeptides of potential interest in biomedicine. After ESI–MS and NMR characterization this building block was used for the assembly of a thymine-functionalized nucleopeptide, composed of nucleobase-containing l-diaminopropanoic acid moieties and underivatized l-lysine residues alternated in the backbone. Circular dichroism studies performed on the cationic nucleopeptide and adenine-containing DNA and RNA molecules suggested that the thymine-containing peptide is able to interact with both DNA and RNA. In particular, a significant conformational variation in the RNA structure was suggested by CD studies. Human serum stability assays were also conducted on the cationic nucleopeptide, which was found to be highly resistant to enzymatic degradation.

Keywords: Nucleopeptide; DNA; RNA; CD

Leucine accelerates blood ethanol oxidation by enhancing the activity of ethanol metabolic enzymes in the livers of SHRSP rats by Hitoshi Murakami; Michiko Ito; Yuji Furukawa; Michio Komai (pp. 2545-2551).

Chronic ethanol consumption induces liver diseases, such as alcoholic hepatitis and cirrhosis. The enhancement of alcohol oxidation is important in the prevention of these liver diseases. Chronic supplementation with branched chain amino acids (BCAAs) prevents liver cirrhosis. Therefore, BCAAs may be associated with enhanced ethanol oxidation. To evaluate this hypothesis, we investigated the effect of the administration of individual BCAAs on ethanol oxidation and changes in alcohol-metabolizing enzyme activities following acute alcohol intake in rats. Blood ethanol concentrations and the activities of alcohol-metabolizing enzymes, such as alcohol dehydrogenase (ADH) and low and high Km aldehyde dehydrogenase (ALDH), were measured in the liver following acute ethanol administration in rats; the ethanol was administered 30 min after the treatment with amino acids [such as leucine (Leu), isoleucine (Ile), valine (Val) or alanine (Ala)]. Leu significantly decreased the blood ethanol concentration 1 h after ethanol administration compared to the water-treated control (C) [C 0.46 ± 0.09, Leu 0.18 ± 0.04, Ile 0.27 ± 0.09, Val 0.46 ± 0.1, Ala 0.43 ± 0.06, mean ± SEM (g/l), P < 0.05]. In addition, leucine significantly stimulated ADH activity 30 min after ethanol intake [C 0.042 ± 0.014, Leu 0.090 ± 0.016, Ile 0.042 ± 0.008, Val 0.022 ± 0.010, Ala 0.070 ± 0.016, mean ± SEM (unit/mg protein), P < 0.05] and low Km ALDH activity 15 min after ethanol intake [C 0.51 ± 0.63, Leu 3.72 ± 0.66, Ile 1.26 ± 0.89, Val: ND, Ala 1.86 ± 1.57, mean ± SEM (unit/mg protein), P < 0.05]. However, leucine and its metabolite α-keto-isocaproic acid did not enhance ethanol clearance in isolated rat hepatocytes. These results indicate that leucine accelerates ethanol oxidation by indirectly enhancing ADH and low Km ALDH activities in the liver.

Keywords: Leucine; Ethanol oxidation; Alcohol metabolic enzymes

Genetic incorporation of d-lysine into diketoreductase in Escherichia coli cells by Zhizhi Liu; Xin Yang; Denghuan Yi; Shuzhen Wang; Yijun Chen (pp. 2553-2559).

Pyrococcus horikoshii lysyl-tRNA synthetase/tRNA orthogonal pair exhibited high selectivity towards d-lysine in the presence of excess amount of d-lysine. Based on the observation, this orthogonal pair was employed to encode d-lysine, and d-lysine was site-specifically incorporated into the diketoreductase in E. coli cells.

Keywords: Genetic incorporation; d-Lysine; tRNA/aminoacyl-tRNA synthetase orthogonal pair; Stereoselectivity

Glutamine stimulates mTORC1 independent of the cell content of essential amino acids by Martina Chiu; Saverio Tardito; Amelia Barilli; Massimiliano G. Bianchi; Valeria Dall’Asta; Ovidio Bussolati (pp. 2561-2567).

Glutamine and leucine are important mTORC1 modulators, although their roles are not precisely defined. In HepG2 and HeLa cells glutamine-free incubation lowers mTORC1 activity, although cell leucine is not decreased. mTORC1 activity, suppressed by amino acid-free incubation, is completely rescued only if essential amino acids (EAA) and glutamine are simultaneously restored, although cell leucine is higher in the absence than in the presence of glutamine. Thus, glutamine stimulates mTORC1 independent of cell leucine, suggesting the existence of two distinct stimulatory signals from either glutamine or EAA.

Keywords: Glutamine; Essential amino acids; Leucine; mTOR; S6K1

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.43, #6)

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.43, #6)