Amino Acids (v.50, #6)

We have designed a novel nanobiosensor for in silico detecting proteins based on leucine/Pd-loaded single-walled carbon nanotube matrix. Density functional theory at the B3LYP/6-31G (d) level of theory was realized to analyze the geometrical and electronic structure of the proposed nanobiosensor. The solvent effects were investigated using the Tomasi’s polarized continuum model. Atoms-in-molecules theory was used to study the nature of interactions by calculating the electron density ρ(r) and Laplacian at the bond critical points. Natural bond orbital analysis was performed to achieve a deep understanding of the nature of the interactions. The biosensor has potential application for high sensitive and rapid response to protein due to the chemical adsorption of l-leucine amino acid onto Pd-loaded single-walled carbon nanotube and reactive functional groups that can incorporate in hydrogen binding, hydrophobic interactions and van der Waals forces with the protein surface in detection process.
Keywords: Nanoreceptor; Protein detection; Carbon nanotube; Chemical sensing

The effects of taurine on repeat sprint cycling after low or high cadence exhaustive exercise in females by Mark Waldron; Francesca Knight; Jamie Tallent; Stephen Patterson; Owen Jeffries (663-669).
This study investigated the effects of taurine on repeated sprint exercise, performed after fixed incremental ramp exercise to exhaustion at isokinetic high (90 r/min) or low (50 r/min) cadences. In a double-blind, repeated measures design, nine females completed an incremental ramp test to volitional exhaustion, followed by 2 min active recovery and 6 × 10 s sprints on a cycle ergometer, in one of four conditions: high cadence (90 r/min) + taurine (50 mg/kg body mass); high cadence + placebo (3 mg/kg body mass maltodextrin); low cadence (50 r/min) + taurine; low cadence + placebo. Heart rate (HR) and blood lactate concentration B[La] were measured before and after the ramp test and after the sprints. Taurine lowered HR vs. placebo prior to the ramp test (P = 0.004; d = 2.1). There was an effect of condition on ramp performance (P < 0.001), with higher end-test power (d = 3.7) in taurine conditions. During repeated sprints, there was a condition × time interaction (P = 0.002), with higher peak sprint power in the placebo conditions compared to taurine (sprint 2–6; P < 0.05). B[La] was higher in taurine compared to placebo post-ramp (P = 0.004; d = 4.7). Taurine-lowered pre-exercise HR and improved incremental end-test power output, with subsequent detrimental effects on sprint performance, independent of cadence. Short endurance performance can be acutely enhanced after taurine ingestion but this effect might not be maintained across longer periods of exercise or induce the need for longer recovery periods.
Keywords: Ergogenic aids; Supplementation; Cycling; Amino acids

A family of novel thienylbenzoxazol-5-yl-l-alanines, consisting of an alanine core bearing a benzoxazole at the side chain with a thiophene ring at position 2, substituted with different (hetero)aryl substituents, was synthesised to study the tuning of the photophysical and chemosensory properties of the resulting compounds. These novel heterocyclic alanines 3a–f and a series of structurally related bis-thienylbenzoxazolyl-alanines 3g–j were evaluated for the first time in the recognition of selected metal cations with environmental, medicinal and analytical interest such as Co2+, Cu2+, Zn2+ and Ni2+, in acetonitrile solution, with the heterocycles at the side chain acting simultaneously as the coordinating and reporting units, via fluorescence changes. This behaviour can be explained by the involvement of the electron donor heteroatoms in the recognition event, through complexation of the metal cations. The spectrofluorimetric titrations showed that thienylbenzoxazolyl-alanines 3a–j and 4a,b were non-selective fluorimetric chemosensors for the above-mentioned cations, with the best results being obtained for the interaction of Cu2+ with bis-alanine 3j and deprotected alanines 4a,b. The encouraging photophysical and metal ion sensing properties of these thienylbenzoxazolyl-alanines suggest that they can be used to obtain bioinspired fluorescent reporters for metal ion such as peptides/proteins with chemosensory/probing ability.
Keywords: Benzoxazole; Thiophene; Unnatural amino acids; Metal cations; Fluorescence; Optical chemosensors

The protein–protein interaction network and clinical significance of heat-shock proteins in esophageal squamous cell carcinoma by Hong Sun; Xinyi Cai; Haofeng Zhou; Xiaoqi Li; Zepeng Du; Haiying Zou; Jianyi Wu; Lei Xie; Yinwei Cheng; Wenming Xie; Xiaomei Lu; Liyan Xu; Longqi Chen; Enmin Li; Bingli Wu (685-697).
Heat-shock proteins (HSPs), one of the evolutionarily conserved protein families, are widely found in various organisms, and play important physiological functions. Nevertheless, HSPs have not been systematically analyzed in esophageal squamous cell carcinoma (ESCC). In this study, we applied the protein–protein interaction (PPI) network methodology to explore the characteristics of HSPs, and integrate their expression in ESCC. First, differentially expressed HSPs in ESCC were identified from our previous RNA-seq data. By constructing a specific PPI network, we found differentially expressed HSPs interacted with hundreds of neighboring proteins. Subcellular localization analyses demonstrated that HSPs and their interacting proteins distributed in multiple layers, from membrane to nucleus. Functional enrichment annotation analyses revealed known and potential functions for HSPs. KEGG pathway analyses identified four significant enrichment pathways. Moreover, three HSPs (DNAJC5B, HSPA1B, and HSPH1) could serve as promising targets for prognostic prediction in ESCC, suggesting these HSPs might play a significant role in the development of ESCC. These multiple bioinformatics analyses have provided a comprehensive view of the roles of heat-shock proteins in esophageal squamous cell carcinoma.
Keywords: Heat-shock protein; Protein–protein interaction network; Esophageal squamous cell carcinoma

New aspects of antiproliferative activity of 4-hydroxybenzyl isothiocyanate, a natural H2S-donor by Halina Jurkowska; Maria Wróbel; Dominika Szlęzak; Ewa Jasek-Gajda (699-709).
The effect of 4-hydroxybenzyl isothiocyanate (HBITC), a natural H2S-donor from white mustard seeds (Sinapis alba), on the proliferation of human neuroblastoma (SH-SY5Y) and glioblastoma (U87MG) cells was studied and some aspects of the mechanism of its activity were suggested. The inhibition of both SH-SY5Y and U87MG cell proliferation was associated with an increase in the thiosulfate level, the number of cells with the inactive form of Bcl-2 protein, and with a decrease of mitochondrial membrane potential. Interestingly, HBITC results in downregulation of p53 protein and upregulation of p21 protein levels in SH-SY5Y cells. In the presence of elevated levels of H2S and thiosulfate, the sulfhydryl groups of p53 protein as well as Bcl-2 protein could be modified via HBITC-induced S-sulfuration or by oxidative stress. It seems that the induction of p21 protein level is mediated in SH-SY5Y cells by p53-independent mechanisms. In addition, HBITC-treatment caused downregulation of the level of mitochondrial rhodanese and 3-mercaptopyruvate sulfurtransferase, and consequently increased the level of the reactive oxygen species in SH-SY5Y cells.
Keywords: 4-Hydroxybenzyl isothiocyanate; Hydrogen sulfide; Thiosulfate; Apoptosis; Sulfurtransferases; Cancer cells

Nucleoprotein from the unique human infecting Orthobunyavirus of Simbu serogroup (Oropouche virus) forms higher order oligomers in complex with nucleic acids in vitro by Juliana Londoño Murillo; Aline Diniz Cabral; Mabel Uehara; Viviam Moura da Silva; Juliete Vitorino dos Santos; João Renato Carvalho Muniz; Leandro Farias Estrozi; Daphna Fenel; Wanius Garcia; Márcia Aparecida Sperança (711-721).
Oropouche virus (OROV) is the unique known human pathogen belonging to serogroup Simbu of Orthobunyavirus genus and Bunyaviridae family. OROV is transmitted by wild mosquitoes species to sloths, rodents, monkeys and birds in sylvatic environment, and by midges (Culicoides paraensis and Culex quinquefasciatus) to man causing explosive outbreaks in urban locations. OROV infection causes dengue fever-like symptoms and in few cases, can cause clinical symptoms of aseptic meningitis. OROV contains a tripartite negative RNA genome encapsidated by the viral nucleocapsid protein (NP), which is essential for viral genome encapsidation, transcription and replication. Here, we reported the first study on the structural properties of a recombinant NP from human pathogen Oropouche virus (OROV–rNP). OROV–rNP was successfully expressed in E. coli in soluble form and purified using affinity and size-exclusion chromatographies. Purified OROV–rNP was analyzed using a series of biophysical tools and molecular modeling. The results showed that OROV–rNP formed stable oligomers in solution coupled with endogenous E. coli nucleic acids (RNA) of different sizes. Finally, electron microscopy revealed a total of eleven OROV–rNP oligomer classes with tetramers (42%) and pentamers (43%) the two main populations and minor amounts of other bigger oligomeric states, such as hexamers, heptamers or octamers. The different RNA sizes and nucleotide composition may explain the diversity of oligomer classes observed. Besides, structural differences among bunyaviruses NP can be used to help in the development of tools for specific diagnosis and epidemiological studies of this group of viruses.
Keywords: Oropouche; Viral protein; Nucleoprotein–RNA complex; Arboviruses

Insulinotropic, glucose-lowering, and beta-cell anti-apoptotic actions of peptides related to esculentin-1a(1-21).NH2 by Vishal Musale; Yasser H. A. Abdel-Wahab; Peter R. Flatt; J. Michael Conlon; Maria Luisa Mangoni (723-734).
Long-standing Type 2 diabetes is associated with loss of both β‐cell function and β‐cell mass. Peptides derived from the frog-skin host-defense peptide esculentin-1 have been shown to exhibit potent, broad-spectrum antimicrobial activity. The aim of the present study is to determine whether such peptides also show insulinotropic and β-cell protective activities. Esculentin-1a(1-21).NH2, esculentin-1b(1-18).NH2, and esculentin-1a(1-14).NH2 produced concentration-dependent stimulations of insulin release from BRIN-BD11 rat clonal β-cells, 1.1B4 human-derived pancreatic β-cells, and isolated mouse islets with no cytotoxicity at concentrations of up to 3 μM. The mechanism of insulinotropic action involved membrane depolarization and an increase in intracellular Ca2+ concentrations. The analogue [D-Lys14, D-Ser17]esculentin-1a(1-21).NH2 (Esc(1-21)-1c) was less potent in vitro than the all L-amino acid containing peptides and esculentin-1a(9-21) was inactive indicating that helicity is an important determinant of insulinotropic activity. However, intraperitoneal injection of Esc(1-21)-1c (75 nmol/kg body weight) together with a glucose load (18 mmol/kg body weight) in C57BL6 mice improved glucose tolerance with a concomitant increase in insulin secretion, whereas administration of esculentin-1a(1-21).NH2, esculentin-1b(1-18).NH2, and esculentin-1a(1-14) was without significant effect on plasma glucose levels. Esc(1-21)-1c (1 µM) protected BRIN-BD11 cells against cytokine-induced apoptosis (P < 0.01) and augmented proliferation of the cells (P < 0.01) to a similar extent as glucagon-like peptide-1. The data demonstrate that the multifunctional peptide Esc(1-21)-1c, as well as showing therapeutic potential as an anti-infective and wound-healing agent, may constitute a template for development of compounds for treatment of patients with Type 2 diabetes.
Keywords: Esculentin-1a(1-21); Type 2 diabetes; Amphibian skin peptide; Insulin release; β-cell proliferation; Anti-apoptotic peptide

Polymorphism at donkey β-lactoglobulin II locus: identification and characterization of a new genetic variant with a very low expression by Andrea Criscione; Vincenzo Cunsolo; Serena Tumino; Antonella Di Francesco; Salvatore Bordonaro; Vera Muccilli; Rosaria Saletti; Donata Marletta (735-746).
In the last years, donkey milk had evidenced a renewed interest as a potential functional food and a breast milk substitute. In this light, the study of the protein composition assumes an important role. In particular, β-lactoglobulin (β-LG), which is considered as one of the main allergenic milk protein, in donkey species consists of two molecular forms, namely β-LG I and β-LG II. In the present research, a genetic analysis coupled with a proteomic approach showed the presence of a new allele, here named F, which is apparently associated with a null or a severely reduced expression of β-LG II protein. The new β-LG II F genetic variant shows a theoretical average mass (M av) of 18,310.64 Da, a value practically corresponding with that of the variant D (∆mass < 0.07 Da), but differs from β-LG II D for two amino acid substitutions: Thr100 (variant F) → Ala100 (variant D) and Thr118 (variant F) → Met118 (variant D). Proteomic investigation of the whey protein fraction of an individual milk sample, homozygous FF at β-LG II locus, allowed to identify, as very minor component, the new β-LG II F genetic variant. By MS/MS analysis of enzymatic digests, the sequence of the β-LG II F was characterized, and the predicted genomic data confirmed.
Keywords: Polymorphism; LGB2 gene; Donkey milk; Allergy; Mass spectrometry; SNPs

Arginase inhibition prevents the development of hypertension and improves insulin resistance in obese rats by Kelly J. Peyton; Xiao-ming Liu; Ahmad R. Shebib; Fruzsina K. Johnson; Robert A. Johnson; William Durante (747-754).
This study investigated the temporal activation of arginase in obese Zucker rats (ZR) and determined if arginase inhibition prevents the development of hypertension and improves insulin resistance in these animals. Arginase activity, plasma arginine and nitric oxide (NO) concentration, blood pressure, and insulin resistance were measured in lean and obese animals. There was a chronological increase in vascular and plasma arginase activity in obese ZR beginning at 8 weeks of age. The increase in arginase activity in obese animals was associated with a decrease in insulin sensitivity and circulating levels of arginine and NO. The rise in arginase activity also preceded the increase in blood pressure in obese ZR detected at 12 weeks of age. Chronic treatment of 8-week-old obese animals with an arginase inhibitor or l-arginine for 4 weeks prevented the development of hypertension and improved plasma concentrations of arginine and NO. Arginase inhibition also improved insulin sensitivity in obese ZR while l-arginine supplementation had no effect. In conclusion, arginase inhibition prevents the development of hypertension and improves insulin sensitivity while l-arginine administration only mitigates hypertension in obese animals. Arginase represents a promising therapeutic target in ameliorating obesity-associated vascular and metabolic dysfunction.
Keywords: Arginine; Arginase; Obesity; Hypertension

Proanthocyanidin-containing polyphenol extracts from fruits prevent the inhibitory effect of hydrogen sulfide on human colonocyte oxygen consumption by Mireille Andriamihaja; Annaïg Lan; Martin Beaumont; Marta Grauso; Martin Gotteland; Edgar Pastene; Maria Jose Cires; Catalina Carrasco-Pozo; Daniel Tomé; François Blachier (755-763).
Hydrogen sulfide (H2S), a metabolic end product synthesized by the microbiota from l-cysteine, has been shown to act at low micromolar concentration as a mineral oxidative substrate in colonocytes while acting as an inhibitor of oxygen consumption at higher luminal concentrations (65 µM and above). From the previous works showing that polyphenols can bind volatile sulfur compounds, we hypothesized that different dietary proanthocyanidin-containing polyphenol (PACs) plant extracts might modulate the inhibitory effect of H2S on colonocyte respiration. Using the model of human HT-29 Glc–/+ cell colonocytes, we show here that pre-incubation of 65 µM of the H2S donor NaHS with the different polyphenol extracts markedly reduced the inhibitory effect of NaHS on colonocyte oxygen consumption. Our studies on HT-29 Glc–/+ cell respiration performed in the absence or the presence of PACs reveal rapid binding of H2S with the sulfide-oxidizing unit and slower binding of H2S to the cytochrome c oxidase (complex IV of the respiratory chain). Despite acute inhibition of colonocyte respiration, no measurable effect of NaHS on paracellular permeability was recorded after 24 h treatment using the Caco-2 colonocyte monolayer model. The results are discussed in the context of the binding of excessive bacterial metabolites by unabsorbed dietary compounds and of the capacity of colonocytes to adapt to changing luminal environment.
Keywords: Hydrogen sulfide; Proanthocyanidins; Polyphenols; Colonocyte; Oxygen consumption

Taurine supplementation induces long-term beneficial effects on glucose homeostasis in ob/ob mice by Patricia Cristine Borck; Jean Franciesco Vettorazzi; Renato Chaves Souto Branco; Thiago Martins Batista; Junia Carolina Santos-Silva; Vanessa Yumi Nakanishi; Antonio Carlos Boschero; Rosane Aparecida Ribeiro; Everardo Magalhães Carneiro (765-774).
The sulfur-containing amino acid, taurine (Tau), regulates glucose and lipid homeostasis under normal, pre- and diabetic conditions. Here, we aimed to verify whether Tau supplementation exerts its beneficial effects against obesity, hyperglycemia and alterations in islet functions, in leptin-deficient obese (ob/ob), over a long period of treatment. From weaning until 12 months of age, female ob/ob mice received, or not, 5% Tau in drinking water (obTau group). After this period, a reduction in hypertriglyceridemia and an improvement in glucose tolerance and insulin sensitivity were observed in obTau mice. In addition, the daily metabolic flexibility was restored in obTau mice. In the gastrocnemius muscle of obTau mice, the activation of AMP-activated protein kinase (AMPK) was increased, while total AMPK protein content was reduced. Finally, isolated islets from obTau mice expressed high amounts of pyruvate carboxylase (PC) protein and lower glucose-induced insulin secretion. Taking these evidences together Tau supplementation had long-term positive actions on glucose tolerance and insulin sensitivity, associated with a reduction in glucose-stimulated insulin secretion, in ob/ob mice. The improvement in insulin actions in obTau mice was due, at least in part, to increased activation of AMPK in skeletal muscle, while the increased content of the PC enzyme in pancreatic islets may help to preserve glucose responsiveness in obTau islets, possibly contributing to islet cell survive.
Keywords: Aging; AMP-activated protein; Insulin secretion; Metabolic flexibility; ob/ob mice; Pyruvate carboxylase