Olive cultivar origin is a major cause of polymorphism for Ole e 1 pollen allergen

Background  

Pollens from different olive (Olea europaea L.) cultivars have been shown to differ significantly in their content in Ole e 1 and in their overall allergenicity. This allergen is, in addition, characterized by a high degree of polymorphism in its sequence. The purpose of this study is to evaluate the putative presence of divergences in Ole e 1 sequences from different olive cultivars.     

Solution structure of the phytotoxic protein PcF: The first characterized member of the Phytophthora PcF toxin family

The PcF protein from Phytophthora cactorum is the first member of the “PcF toxin family” from the plant pathogens Phytophthora spp. It is able to induce withering in tomato and strawberry leaves. The lack of sequence similarity with other proteins hampers the identification of the molecular mechanisms responsible for its toxicity. Here, we show that the six cysteines form a disulphide pattern that is exclusive for PcF and essential for the protein withering activity. The NMR solution structure identifies a novel fold among protein effectors: a helix‐loop‐helix motif. The presence of a negatively charged surface suggests that it might act as a site of electrostatic interaction. Interestingly, a good fold match with Ole e 6, a plant protein with allergenic activity, highlighted the spatial superimposition of a stretch of identical residues. This finding suggests a possible biological activity based on molecular mimicry.     

Production of Pharmaceuticals from Papaver Cultivars in Vitro

Iranian (Papaver bracteatum Lindl.) and opium poppy (P. somniferum L.) plantlets obtained from germinated seeds grown on a Murashige and Skoog basal medium (BM) readily manifest alkaloids. Temperature had a profound effect on growth and alkaloid production after 8 weeks in culture. Plantlets of poppy cultivars (cvs.) grew best at 18.5 and 20°C compared to 15 or 25°C. An alkaloid survey study with 24 Iranian and 21 opium poppy cvs. revealed that total morphinan alkaloids ranged from 0 to 6.55 mg/g dw. Prolific axillary branching was achieved from poppy cvs. by maintaining shoots on BM containing 1.0 mg/L N⁶‐benzyladenine and 0.01 mg/L α‐naphthalene acetic acid for an additional 16 weeks. The influence of vessel size on the growth response of established shoot clumps was determined by subculture in a variety of culture vessels for 8 weeks. The tested culture vessels included culture tubes (55 mm³ capacity (cap.)), babyfood jars (143 mm³ cap.), Magenta GA‐7 containers (365 mm³ cap.), and polycarbonate jars (1890 mm³ cap.) employing an in vitro hydroponics system (i.e. an automated plant culture system (APCS)). Highest growth rates occurred employing the APCS. The culture vessel capacity had a significant positive correlation on shoot length, fresh weight, number of leaves, and number of shoots. Shoot length, fresh weight, leaves, and shoots grown in the APCS exhibited increases of 1‐, 21.5‐, 7.8‐, and 8.3‐fold, respectively, compared to shoots grown in culture tubes. Higher culture growth rates that occurred in the larger‐size vessels were correlated with lower alkaloid production (mg alkaloids/g dw). However, the overall total alkaloids/vessel [(mg alkaloid/g dw)×g culture dw] increased because of greater biomass production per vessel. The alkaloid content was found to remain stable for shoots grown over a 6–month evaluation period.     

Site‐specific labeling of synthetic peptide using the chemoselective reaction between N‐methoxyamino acid and isothiocyanate

Site‐specific labeling of synthetic peptides carrying N‐methoxyglycine (MeOGly) by isothiocyanate is demonstrated. A nonapeptide having MeOGly at its N‐terminus was synthesized by the solid‐phase method and reacted with phenylisothiocyanate under various conditions. In acidic solution, the reaction specifically gave a peptide having phenylthiourea structure at its N‐terminus, leaving side chain amino group intact. The synthetic human β‐defensin‐2 carrying MeOGly at its N‐terminus or the side chain amino group of Lys¹⁰ reacted with phenylisothiocyanate or fluorescein isothiocyanate also at the N‐methoxyamino group under the same conditions, demonstrating that this method is generally useful for the site‐specific labeling of linear synthetic peptides as well as disulfide‐containing peptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Effect of structural parameters of peptides on dimer formation and highly oxidized side products in the oxidation of thiols of linear analogues of human β‐defensin 3 by DMSO

The purpose of this study was to examine the effects of structural parameters of peptides on their oxidation by DMSO, including location of cysteine, effect of adjunct group participation, molecular hydrophobicity, steric hindrance or the accessibility of thiol group and peptide conformation, on oxidation rates, dimer formation and associated side products. We designed and synthesized two series of linear cysteine‐containing analogues of human β‐defensin 3 (the C1‐peptides with cysteine at the N‐terminus residue 1, the C29‐peptides with cysteine located at residue 29 in the centre of peptide), which were used for preparation of disulphide‐linked homodimers. HPLC–ESI–MS was used to monitor the oxidation process and to characterize the molecular weights of dimers and side products of high oxidation. The formations of dimers and side products were dependent on the position of cysteines. Hydrophobicity generally rendered the thiol groups less accessible and hence exposed them to slow oxidation to form dimers (or even fail to form dimers during the timescale of observation). Molecular dynamics simulations showed that the exposure of cysteines (and sulphurs) of the C1‐peptides was much larger than for the C29‐peptides. The larger hydrophobic side chains tended to enable clustering of the side chains that sequester cysteine, particularly in the C29‐peptides, which provided a molecular explanation for the observed trends in oxidation rates. Together with molecular modelling, we propose a reaction mechanism to elucidate the oxidation results of these peptides. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Pollen Ole e 1 content variations in olive cultivars of different Portugal regions

Olive is recognized as a crop with great impact in agricultural, socioeconomic, environmental and public health sectors. The last is becoming more important during recent years as consequence of the increase of the pollen allergy in south Europe prompted by the widespread Olea pollen allergic reactions. The aim of the study was to quantify, for the first time, the variations of the Ole e 1 allergen amount in Olea pollen grains from four cultivars in three regions of Portugal. How weather parameters can affect the allergen production was also assessed. The study was conducted in three olive producer areas of Portugal from 2010 to 2013, Santarém (Central), Elvas (Southeast) and Mirandela (Trás-os-Montes region, Northeast). Mature pollen of four different cultivars (Cobrançosa, Arbequina, Picual and Verdeal) was collected during the olive flowering season. Ole e 1 was quantified using specific 2-site antibody ELISA. Pollen of the olive groves at the boundary Olea bioclimatic distribution in the Mirandela registered the higher allergen content for all varieties in each study year. Arbequina was the variety that showed the lower Ole e 1 allergen concentration, whereas the higher content was registered for Cobrançosa. The main meteorological parameters that influenced the allergen Ole e 1 concentration in the pollen grains were the rainfall and temperatures related variables. The knowledge of the allergenicity in different olive cultivars is an important tool in the selection of the most adequate for planting as ornamental crop and to adjust the pollen extracts used for diagnosis or even immunotherapy.

     

The action of fish peptide Orpotrin analogs on microcirculation

In order to investigate the relationship between the primary structure of Orpotrin, a vasoactive peptide previously isolated from the freshwater stingray Potamotrygon gr. orbignyi, and its microcirculatory effects, three Orpotrin analogs were synthesized. The analogs have a truncated N‐terminal with a His residue deletion and two substituted amino acid residues, where one Nle is substituted for one internal Lys residue and the third analog has a substitution of a Pro for an Ala (Orp‐desH¹, Orp‐Nle and Orp‐Pro/Ala, respectively). Only Orp‐desH¹ could induce a lower vasoconstriction effect compared with the natural Orpotrin, indicating that besides the N‐terminal, the positive charge of Lys and the Pro residues located at the center of the amino acid chain is crucial for this vasoconstriction effect. Importantly, the suggestions made with bioactive peptides were based on the molecular modeling and dynamics of peptides, the presence of key amino acids and shared activity in microcirculation, characterized by intravital microscopy. Moreover, this study has demonstrated that even subtle changes in the primary structure of Orpotrin alter the biological effects of this native peptide significantly, which could be of interest for biotechnological applications. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

A convenient microwave‐enhanced solid‐phase synthesis of short chain N‐methyl‐rich peptides

Structural modification of the peptide backbone via N‐methylation is a powerful tool to modulate the pharmacokinetic profile and biological activity of peptides. Here we describe a rapid and highly efficient microwave(MW)‐assisted Fmoc/tBu solid‐phase method to prepare short chain N‐methyl‐rich peptides, using Rink amide p‐methylbenzhydrylamine (MBHA) resin as solid‐phase support. This method produces peptides in high yield and purity, and reduces the time required for Fmoc‐N‐methyl amino acid coupling. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

General method for selective labelling of double‐chain cysteine‐rich peptides with a lanthanide chelate via solid‐phase synthesis

The use of lanthanides in preference to radioisotopes as probes for various biological assays has gained enormous popularity. The introduction of lanthanide chelates to peptides/proteins can be carried out either in solution using a commercially available labelling kit or by solid‐phase peptide synthesis using an appropriate lanthanide chelate. Herein, a detailed protocol for the latter is provided for the labelling of peptides or small proteins with diethylenetriamine‐N, N, N″, N″‐tetra‐tert‐butyl acetate‐N′‐acetic acid (DTPA) chelate or other similar chelates on a solid support using a chimeric insulin‐like peptide composed of human insulin‐like peptide 5 (INSL5) A‐chain and relaxin‐3 B‐chain as a model peptide. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Building blocks for the synthesis of post‐translationally modified glycated peptides and proteins

Growing interest in synthetic peptides carrying post‐traslational modifications, in general, and the Amadori modification in particular, raises the need for specific building blocks that can be used in stepwise peptide synthesis. Herein, we report the synthesis of N^α‐Fmoc‐Lys‐OH derivatives containing N^ε‐1‐deoxyfructopyranosyl moiety. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Studying the Structural and Folding Features of Long‐Sequence Trichobrachin Peptides

In this theoretical study, the folding processes of long‐sequence trichobrachin peptides (i.e., TB IIb peptides) were investigated by molecular dynamics methods. The formation of various helical structures (i.e., 3₁₀‐, α‐, and left‐handed α‐helices) was studied with regard to the entire sequence of peptides, as well as to each amino acid. The results pointed out that TB IIb molecules showed a propensity to form helical conformations, and they could be characterized by 3₁₀‐helical structure rather than by α‐helical structure. The formation of local (i.e., i←i+3 and i←i+4) as well as of non‐local (i.e., i←i+n, where n>4; and all i→i+n) H‐bonds was also examined. The results revealed that the occurrence of local, helix‐stabilizing H‐bonds was in agreement with the appearance of helical conformations, and the non‐local H‐bonds did not produce relevant effects on the evolution of helical structures. Based on the data obtained by our structural investigation, differences were observed between the TB IIb peptides, according to the type of amino acid located in the 17th position of their sequences. In summary, the folding processes were explored for TB IIb molecules, and our theoretical study led to the conclusion that these long‐sequence peptaibols showed characteristic structural and folding features.     

Evidence That the Yeast Desaturase Ole1p Exists as a Dimer in Vivo

Desaturase enzymes are composed of two classes, the structurally well characterized soluble class found predominantly in the plastids of higher plants and the more widely distributed but poorly structurally defined integral membrane class. Despite their distinct evolutionary origins, the two classes both require an iron cofactor and molecular oxygen for activity and are inhibited by azide and cyanide, suggesting strong mechanistic similarities. The fact that the soluble desaturase is active as a homodimer prompted us test the hypothesis that an archetypal integral membrane desaturase from Saccharomyces cerevisiae, the Δ⁹-acyl-Co-A desaturase Ole1p, also exhibits a dimeric organization. Ole1p was chosen because it is one of the best characterized integral membrane desaturase and because it retains activity when fused with epitope tags. FLAG-Ole1p was detected by Western blotting of immunoprecipitates in which anti-Myc antibodies were used for capture from yeast extracts co-expressing Ole1p-Myc and Ole1p-FLAG. Interaction was confirmed by two independent bimolecular complementation assays (i.e. the split ubiquitin system and the split luciferase system). Co-expression of active and inactive Ole1p subunits resulted in an ∼75% suppression of the accumulation of palmitoleic acid, demonstrating that the physiologically active form of Ole1p in vivo is the dimer in which both protomers must be functional.     

Lipid interactions of acylated tryptophan‐methylated lactoferricin peptides by solid‐state NMR

Lactoferricin (LfB) is a 25‐residue innate immunity peptide released by pepsin from the N‐terminal region of bovine lactoferrin. A smaller amidated peptide, LfB6 (RRWQWR‐NH₂) retains antimicrobial activity and is thought to constitute the “antimicrobial active‐site” (Tomita, Acta Paediatr Jpn. 1994; 36 : 585–91). Here we report on N‐acylation of 1‐Me‐Trp⁵‐LfB6, Cn‐RRWQ[1‐Me‐W]R‐NH₂, where Cn is an acyl chain having n = 0, 2, 4, 6 or 12 carbons. Tryptophan 5 (Trp⁵) was methylated to enhance membrane binding and to allow for selective deuteration at that position. Peptide/lipid interactions of Cn‐RRWQ[1‐Me‐W ]R‐NH₂ (deuterated 1‐Me‐Trp⁵ underlined), were monitored by solid state ³¹P NMR and ²H NMR. The samples consisted of macroscopically oriented bilayers of mixed neutral (dimyristoylphosphatidylcholine, DMPC) and anionic (dimyristoylphosphatidylglycerol, DMPG) lipids in a 3:1 ratio with Cn‐RRWQ[&1‐Me‐W ]R‐NH₂ peptides added at a 1:25 peptide to lipid ratio. ²H‐NMR spectra reveal that the acylated peptides are well aligned in DMPC:DMPG bilayers. The ²H NMR quadrupolar splittings suggest that the 1‐Me‐Trp is located in a motionally restricted environment, indicating partial alignment at the membrane interface. ³¹P‐NMR spectra reveal that the lipids are predominantly in a bilayer configuration, with little perturbation by the peptides. Methylation alone, in C0‐RRWQ[1‐Me‐W ]R‐NH₂, resulted in a 3–4 fold increase in antimicrobial activity against E. coli. N‐acylation with a C12 fatty acid enhanced activity almost 90 fold. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Interval Estimation of the Effective Population Size from Heterozygote‐Excess in SNP Markers

The effective population size N_e is an important parameter in population genetics and conservation biology. In recent years, there has been great interest in the use of molecular markers to estimate N_e. Although the point estimates from molecular markers in general suffer from a low reliability, the use of single nucleotide polymorphism (SNP) markers over a wide range of genome is expected to remarkably improve the reliability. In this study, expressions were derived for interval estimates of N_e from one published method, the heterozygote‐excess method, when it is applied to SNP markers. The conditional variance theory is applied to the derivation of a confidence interval for N_e under random union of gametes, monogamy and polygyny. Stochastic simulation shows that the obtained confidence interval is slightly conservative, but fairly useful for practical applications. The result is illustrated with real data on SNP markers in a pig strain.     

Incorporation of N‐amidino‐pyroglutamic acid into peptides using intramolecular cyclization of α‐guanidinoglutaric acid

N‐terminal modification of peptides by unnatural amino acids significantly affects their enzymatic stability, conformational properties and biological activity. Application of N‐amidino‐amino acids, positively charged under physiological conditions, can change peptide conformation and its affinity to the corresponding receptor. In this article, we describe synthesis of short peptides, containing a new building block—N‐amidino‐pyroglutamic acid. Although direct guanidinylation of pyroglutamic acid and oxidation of N‐amidino‐proline using RuO₄ did not produce positive results, N‐amidino‐Glp‐Phe‐OH was synthesized on Wang polymer by cyclization of α‐guanidinoglutaric acid residue. In the course of synthesis, it was found that literature procedure of selective Boc deprotection using TMSOTf/TEA reagent is accompanied by concomitant side reaction of triethylamine alkylation by polymer linker fragment. It should be mentioned that independently from cyclization time and coupling agent (DIC or HCTU), the lactam formation was incomplete. Separation of the cyclic product from the linear precursor was achieved by HPLC in ammonium formate buffer at pH 6. HPLC analysis showed N‐amidino‐Glp‐Phe‐OH stability at acidic and physiological pH and fast ring opening in water solution at pH 9. The suggested method of N‐amidino‐Glp residue formation can be applied in the case of short peptide chains, whereas synthesis of longer ones will require fragment condensation approach. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Toward engineering efficient peptidomimetics. Screening conformational landscape of two modified dehydroaminoacids

Effective peptidomimetics should posses structural rigidity and appropriate interaction pattern leading to potential spatial and electronic matching to the target receptor site. Rational design of such small bioactive molecules could push chemical synthesis and molecular modeling toward faster progress in medicinal chemistry. Conformational properties of N‐t‐butoxycarbonyl‐glycine‐(E/Z)‐dehydrophenylalanine N′,N′‐dimethylamides (Boc‐Gly‐(E/Z)‐ΔPhe‐NMe₂) in chloroform were studied by NMR and IR spectroscopy. The experimental findings were supported by extensive calculations at DFT(B3LYP, M06‐2X) and MP2 levels of theory and the β‐turn tendency for both isomers of the studied dipeptide were determined in vacuum and in solution. The theoretical data and experimental IR results were used as an additional information for the NMR‐based determination of the detailed solution conformations of the peptides. The obtained results reveal that N‐methylation of C‐terminal amide group changes dramatically the conformational properties of studied dehydropeptides. Theoretical conformational analysis reveals that the tendency to adopt β‐turn conformations is much weaker for the N‐methylated Z isomer (Boc‐Gly‐(Z)‐ΔPhe‐NMe₂), both in vacuum and in polar environment. On the contrary, N‐methylated E isomer (Boc‐Gly‐(E)‐ΔPhe‐NMe₂) can easily adopt β‐turn conformation, but the backbone torsion angles (φ₁, ψ₁, φ₂, ψ₂) are off the limits for common β‐turn types. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 28–40, 2014.     

Assessing performance of single‐sample molecular genetic methods to estimate effective population size: empirical evidence from the endangered Gochu Asturcelta pig breed

Estimating effective population size (N_e) using linkage disequilibrium (LD) information (N_e(LD)) has the operational advantage of using a single sample. However, N_e(LD) estimates assume discrete generations and its performance are constrained by demographic issues. However, such concerns have received little empirical attention so far. The pedigree of the endangered Gochu Asturcelta pig breed includes individuals classified into discrete filial generations and individuals with generations overlap. Up to 780 individuals were typed with a set of 17 microsatellites. Performance of N_e(LD) was compared with N_e estimates obtained using genealogical information, molecular coancestry (N_e(M)) and a temporal (two‐sample) method (N_e(JR)). Molecular‐based estimates of N_e exceeded those obtained using pedigree data. Estimates of N_e(LD) for filial generations F3 and F4 (17.0 and 17.3, respectively) were lower and steadier than those obtained using yearly or biannual samplings. N_e(LD) estimated for samples including generations overlap could only be compared with those obtained for the discrete filial generations when sampling span approached a generation interval and demographic correction for bias was applied. Single‐sample N_e(M) estimates were lower than their N_e(LD) counterparts. N_e(M) estimates are likely to partially reflect the number of founders rather than population size. In any case, estimates of LD and molecular coancestry tend to covary and, therefore, N_e(M) and N_e(LD) can hardly be considered independent. Demographically adjusted estimates of N_e(JR) and N_e(LD) took comparable values when: (1) the two samples used for the former were separated by one equivalent to discrete generations in the pedigree and (2) sampling span used for the latter approached a generation interval. Overall, the empirical evidence given in this study suggested that the advantage of using single‐sample methods to obtain molecular‐based estimates of N_e is not clear in operational terms. Estimates of N_e obtained using methods based in molecular information should be interpreted with caution.     

A study of the anti‐plasmodium activity of angiotensin II analogs

Controlling the dissemination of malaria requires the development of new drugs against its etiological agent, a protozoan of the Plasmodium genus. Angiotensin II and its analog peptides exhibit activity against the development of immature and mature sporozoites of Plasmodium gallinaceum. In this study, we report the synthesis and characterization of angiotensin II linear and cyclic analogs with anti‐plasmodium activity. The peptides were synthesized by a conventional solid‐phase method on Merrifield's resin using the t‐Boc strategy, purified by RP‐HPLC and characterized by liquid chromatography/ESI (+) MS (LC‐ESI(+)/MS), amino acid analysis, and capillary electrophoresis. Anti‐plasmodium activity was measured in vitro by fluorescence microscopy using propidium iodine uptake as an indicator of cellular damage. The activities of the linear and cyclic peptides are not significantly different (p < 0.05). Kinetics studies indicate that the effects of these peptides on plasmodium viability overtime exhibit a sigmoidal profile and that the system stabilizes after a period of 1 h for all peptides examined. The results were rationalized by partial least‐square analysis, assessing the position‐wise contribution of each amino acid. The highest contribution of polar amino acids and a Lys residue proximal to the C‐terminus, as well as that of hydrophobic amino acids in the N‐terminus, suggests that the mechanism underlying the anti‐malarial activity of these peptides is attributed to its amphiphilic character. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

A critique of avian CHD‐based molecular sexing protocols illustrated by a Z‐chromosome polymorphism detected in auklets

The sexes of non‐ratite birds can be determined routinely by PCR amplification of the CHD‐Z and CHD‐W genes. CHD‐based molecular sexing of four species of auklets revealed the presence of a polymorphism in the Z chromosome. No deviation from a 1:1 sex ratio was observed among the chicks, though the analyses were of limited power. Polymorphism in the CHD‐Z gene has not been reported previously in any bird, but if undetected it could lead to the incorrect assignment of sex. We discuss the potential difficulties caused by a polymorphism such as that identified in auklets and the merits of alternative CHD‐based sexing protocols and primers.     

Sequences of Tolypins,Insecticidal Efrapeptin‐Type Peptaibiotics from Species of the Fungal Genus Tolypocladium

A peptide mixture named tolypin, originally isolated from species of the fungal genus Tolypocladium, was structurally characterised and sequences compared to those reported for efrapeptins isolated from strains of Tolypocladium inflatum. Chiral amino acid analysis, direct infusion, and online HPLC electrospray ionization tandem mass spectrometry provided composition, molecular weights of peptides, and series of diagnostic fragment ions. Sequences deduced from ESI‐MS revealed that tolypins C?G are identical to efrapeptins C?G. The results were corroborated by ESI‐MS and HPLC of an authentic efrapeptin sample from Eli Lilly Research Laboratories (USA). Comparison of the HPLC elution profiles of efrapeptin and tolypin indicated a pronounced microheterogeneity of the former. A high‐resolution HPLC of authentic efrapeptin has not been published before. Close relationship and partial identity of sequences of tolypins and efrapeptins, which had previously been postulated, were definitely proven. The geographical origin of the two most important T. inflatum strains used for sequencing of efrapeptins/tolypins could unambiguously be clarified. A new minor compound, designated tolypin H1, was sequenced. High proportions of helicogenic Aib (α‐aminoisobutyric acid) and l ‐isovaline, N‐terminal acetyl‐l ‐pipecolic acid and the unusual, amide‐bound C‐terminal residue, named (S)‐2‐amino‐1‐(1,5‐diazabicyclo[4.3.0]non‐5‐ene‐5‐ylium)‐4‐methylpentane corresponding to 1‐[(2S)‐2‐amino‐4‐methylpentyl]‐2,3,4,6,7,8‐hexahydropyrrolo[1,2‐a]pyrimidin‐1‐ium, define these peptides as linear, cationic peptaibiotics.