Genetic manipulation of the γ‐aminobutyric acid (GABA) shunt in rice: overexpression of truncated glutamate decarboxylase (GAD2) and knockdown of γ‐aminobutyric acid transaminase (GABA‐T) lead to sustained and high levels of GABA accumulation in rice kernels

Gamma‐aminobutyric acid (GABA) is a non‐protein amino acid commonly present in all organisms. Because cellular levels of GABA in plants are mainly regulated by synthesis (glutamate decarboxylase, GAD) and catabolism (GABA‐transaminase, GABA‐T), we attempted seed‐specific manipulation of the GABA shunt to achieve stable GABA accumulation in rice. A truncated GAD2 sequence, one of five GAD genes, controlled by the glutelin (GluB‐1) or rice embryo globulin promoters (REG) and GABA‐T‐based trigger sequences in RNA interference (RNAi) cassettes controlled by one of these promoters as well, was introduced into rice (cv. Koshihikari) to establish stable transgenic lines under herbicide selection using pyriminobac. T₁ and T₂ generations of rice lines displayed high GABA concentrations (2–100 mg/100 g grain). In analyses of two selected lines from the T₃ generation, there was a strong correlation between GABA level and the expression of truncated GAD2, whereas the inhibitory effect of GABA‐T expression was relatively weak. In these two lines both with two T‐DNA copies, their starch, amylose, and protein levels were slightly lower than non‐transformed cv. Koshihikari. Free amino acid analysis of mature kernels of these lines demonstrated elevated levels of GABA (75–350 mg/100 g polished rice) and also high levels of several amino acids, such as Ala, Ser, and Val. Because these lines of seeds could sustain their GABA content after harvest (up to 6 months), the strategy in this study could lead to the accumulation GABA and for these to be sustained in the edible parts.     

Conformations of peptides containing a chiral cyclic α, α‐disubstituted α‐amino acid within the sequence of Aib residues

A single chiral cyclic α,α‐disubstituted amino acid, (3S,4S)‐1‐amino‐(3,4‐dimethoxy)cyclopentanecarboxylic acid [(S,S)‐Ac₅c^dOM], was placed at the N‐terminal or C‐terminal positions of achiral α‐aminoisobutyric acid (Aib) peptide segments. The IR and ¹H NMR spectra indicated that the dominant conformations of two peptides Cbz‐[(S,S)‐Ac₅c^dOM]‐(Aib)₄‐OEt ( 1) and Cbz‐(Aib)₄‐[(S,S)‐Ac₅c^dOM]‐OMe (2) in solution were helical structures. X‐ray crystallographic analysis of 1 and 2 revealed that a left‐handed (M) 3₁₀‐helical structure was present in 1 and that a right‐handed (P) 3₁₀‐helical structure was present in 2 in their crystalline states. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Basic conformers in β‐peptides

The conformation of oligomers of β‐amino acids of the general type Ac‐[β‐Xaa]_n‐NHMe (β‐Xaa = β‐Ala, β‐Aib, and β‐Abu; n = 1–4) was systematically examined at different levels of ab initio molecular orbital theory (HF/6‐31G*, HF/3‐21G). The solvent influence was considered employing two quantum‐mechanical self‐consistent reaction field models. The results show a wide variety of possibilities for the formation of characteristic elements of secondary structure in β‐peptides. Most of them can be derived from the monomer units of blocked β‐peptides with n = 1. The stability and geometries of the β‐peptide structures are considerably influenced by the side‐chain positions, by the configurations at the C^α‐ and C^β‐atoms of the β‐amino acid constituents, and especially by environmental effects. Structure peculiarities of β‐peptides, in particular those of various helix alternatives, are discussed in relation to typical elements of secondary structure in α‐peptides. © 1999 John Wiley & Sons, Inc. Biopoly 50: 167–184, 1999     

Microbial production of conjugated γ‐linolenic acid from γ‐linolenic acid by Lactobacillus plantarum AKU 1009a

Aims: Optimal production conditions of conjugated γ‐linolenic acid (CGLA) from γ‐linolenic acid using washed cells of Lactobacillus plantarum AKU 1009a as catalysts were investigated. Methods and Results: Washed cells of Lact. plantarum AKU 1009a exhibiting a high level of CGLA productivity were obtained by cultivation in a nutrient medium supplemented with 0·03% (w/v) α‐linolenic acid as an inducer. Under the optimal reaction conditions with 13 mg ml^?1γ‐linolenic acid as a substrate in 5 ‐ml reaction volume, the washed cells [32% (wet cells, w/v) corresponding to 46 mg ml^?1 dry cells] as the catalysts produced 8·8 mg CGLA per millilitre reaction mixture (68% molar yield) in 27 h. The produced CGLA was a mixture of two isomers, i.e., cis‐6,cis‐9,trans‐11‐octadecatrienoic acid (CGLA1, 40% of total CGLA) and cis‐6,trans‐9,trans‐11‐octadecatrienoic acid (CGLA2, 60% of total CGLA), and accounted for 66% of total fatty acid obtained. The CGLA produced was obtained as free fatty acids adsorbed mostly on the surface of the cells of Lact. plantarum AKU1009a. Conclusion: The practical process of CGLA production from γ‐linolenic acid using washed cells of Lact. plantarum AKU 1009a was successfully established. Significance and Impact of the Study: We presented the first example of microbial production of CGLA. CGLA produced by the process is valuable for evaluating their physiological and nutritional effects, and chemical characteristics.     

Asymmetric Allylation of α‐Ketoester‐Derived N‐Benzoylhydrazones Promoted by Chiral Sulfoxides/N‐Oxides Lewis Bases: Highly Enantioselective Synthesis of Quaternary α‐Substituted α‐Allyl‐α‐Amino Acids

Chiral sulfoxides/N‐oxides (R)‐ 1 and (R,R)‐ 2 are effective chiral promoters in the enantioselective allylation of α‐keto ester N‐benzoylhydrazone derivatives 3a , 3b , 3c , 3d , 3e , 3f , 3g to generate the corresponding N‐benzoylhydrazine derivatives 4a , 4b , 4c , 4d , 4e , 4f , 4g , with enantiomeric excesses as high as 98%. Representative hydrazine derivatives 4a , 4b were subsequently treated with SmI₂, and the resulting amino esters 5a , 5b with LiOH to obtain quaternary α‐substituted α‐allyl α‐amino acids 6a , 6b , whose absolute configuration was assigned as (S), with fundament on chemical correlation and electronic circular dichroism (ECD) data. Chirality 25:529–540, 2013. © 2013 Wiley Periodicals, Inc.     

Synthesis and antimicrobial activity of α‐aminoboronic‐containing peptidomimetics

A library of 175 dipeptidomimetics and tripeptidomimetics containing an α‐amino boronic acid or boronate has been synthesized, and the activity toward Mycobacterium tuberculosis, Candida albicans, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli and Pseudomonas aeruginosa has been screened. Although there is no clear structure–activity relationship, several compounds exhibit promising activity against different pathogens. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Human α1β3γ2L gamma‐aminobutyric acid type A receptors: High‐level production and purification in a functional state

Gamma‐aminobutyric acid type A receptors (GABA_ARs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABA_ARs determine their function and pharmacological profile. GABA_ARs are heteropentamers of subunits, and (α1)₂(β3)₂(γ2L)₁ is a common subtype. Biochemical and biophysical studies of GABA_ARs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high‐level production of active human α1β3 GABA_AR using tetracycline‐inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline‐inducible HEK293‐TetR cell line expressing human (N)–FLAG–α1β3γ2L–(C)–(GGS)₃GK–1D4 GABA_AR. These cells achieved expression levels of 70–90 pmol [³H]muscimol binding sites/15‐cm plate at a specific activity of 15–30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [³H]flunitrazepam to [³H]muscimol binding sites and sensitivity of GABA‐induced currents to benzodiazepines and zinc. The α1β3γ2L GABA_ARs were solubilized in dodecyl‐d ‐maltoside, purified by anti‐FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ～30%. Typical purifications yielded 1.0–1.5 nmoles of [³H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [³H]muscimol binding were maintained in the purified state.     

Gimmicks of gamma‐aminobutyric acid (GABA) in pancreatic β‐cell regeneration through transdifferentiation of pancreatic α‐ to β‐cells

In vivo regeneration of lost or dysfunctional islet β cells can fulfill the promise of improved therapy for diabetic patients. To achieve this, many mitogenic factors have been attempted, including gamma‐aminobutyric acid (GABA). GABA remarkably affects pancreatic islet cells’ (α cells and β cells) function through paracrine and/or autocrine binding to its membrane receptors on these cells. GABA has also been studied for promoting the transformation of α cells to β cells. Nonetheless, the gimmickry of GABA‐induced α‐cell transformation to β cells has two different perspectives. On the one hand, GABA was found to induce α‐cell transformation to β cells in vivo and insulin‐secreting β‐like cells in vitro. On the other hand, GABA treatment showed that it has no α‐ to β‐cell transformation response. Here, we will summarize the physiological effects of GABA on pancreatic islet β cells with an emphasis on its regenerative effects for transdifferentiation of islet α cells to β cells. We will also critically discuss the controversial results about GABA‐mediated transdifferentiation of α cells to β cells.     

Blockade of JAK2 activity suppressed accumulation of β‐catenin in leukemic cells

The Wnt/β‐catenin pathway has been implicated in leukemogenesis. We found β‐catenin abnormally accumulated in both human acute T cell leukemia Jurkat cells and human erythroleukemia HEL cells. β‐Catenin can be significantly down‐regulated by the Janus kinase 2 specific inhibitor AG490 in these two cells. AG490 also reduces the luciferase activity of a reporter plasmid driven by LEF/β‐catenin promoter. Similar results were observed in HEL cells infected with lentivirus containing shRNA against JAK2 gene. After treatment with 50 µM AG490 or shRNA, the mRNA expression levels of β‐catenin, APC, Axin, β‐Trcp, GSK3α, and GSK3β were up‐regulated within 12–16 h. However, only the protein levels of GSK3β and β‐Trcp were found to have increased relative to untreated cells. Knockdown experiments revealed that the AG490‐induced inhibition of β‐catenin can be attenuated by shRNA targeting β‐TrCP. Taken together; these results suggest that β‐Trcp plays a key role in the cross‐talk between JAK/STAT and Wnt/β‐catenin signaling in leukemia cells. J. Cell. Biochem. 111: 402–411, 2010. © 2010 Wiley‐Liss, Inc.     

Synthesis and receptor binding of opioid peptide analogues containing β3‐homo‐amino acids

β‐Amino acids containing hybrid peptides and β‐peptides show great potential as peptidomimetics. In this paper we describe the synthesis and affinity toward the µ‐ and δ‐opioid receptors of β‐peptides, analogues of Leu‐enkephalin, deltorphin I, dermorphin and α,β‐hybrides, analogues of deltorphin I. Substitution of α‐amino acid residues with β³‐homo‐amino acid residues, in general resulted in decrease of affinity to opioid receptors. However, the incorporation β³h‐D ‐Ala in position 2 or β³hPhe in position 3 of deltorphin I resulted in potent and selective ligand for δ‐opioid receptor. The NMR studies of β‐deltorphin I analogue suggest that conformational motions in the central part of the peptide backbone are partially restricted and some conformational preferences can be expected. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

β‐Amino acids containing peptides and click‐cyclized peptide as β‐turn mimics: a comparative study with ‘conventional’ lactam‐ and disulfide‐bridged hexapeptides

The increasing interest in click chemistry and its use to stabilize turn structures led us to compare the propensity for β‐turn stabilization of different analogs designed as mimics of the β‐turn structure found in tendamistat. The β‐turn conformation of linear β‐amino acid‐containing peptides and triazole‐cyclized analogs were compared to ‘conventional’ lactam‐ and disulfide‐bridged hexapeptide analogs. Their 3D structures and their propensity to fold in β‐turns in solution, and for those not structured in solution in the presence of α‐amylase, were analyzed by NMR spectroscopy and by restrained molecular dynamics with energy minimization. The linear tetrapeptide Ac‐Ser‐Trp‐Arg‐Tyr‐NH₂ and both the amide bond‐cyclized, c[Pro‐Ser‐Trp‐Arg‐Tyr‐D ‐Ala] and the disulfide‐bridged, Ac‐c[Cys‐Ser‐Trp‐Arg‐Tyr‐Cys]‐NH₂ hexapeptides adopt dominantly in solution a β‐turn conformation closely related to the one observed in tendamistat. On the contrary, the β‐amino acid‐containing peptides such as Ac‐(R)‐β³‐hSer‐(S)‐Trp‐(S)‐β³‐hArg‐(S)‐β³‐hTyr‐NH₂, and the triazole cyclic peptide, c[Lys‐Ser‐Trp‐Arg‐Tyr‐βtA]‐NH₂, both specifically designed to mimic this β‐turn, do not adopt stable structures in solution and do not show any characteristics of β‐turn conformation. However, these unstructured peptides specifically interact in the active site of α‐amylase, as shown by TrNOESY and saturation transfer difference NMR experiments performed in the presence of the enzyme, and are displaced by acarbose, a specific α‐amylase inhibitor. Thus, in contrast to amide‐cyclized or disulfide‐bridged hexapeptides, β‐amino acid‐containing peptides and click‐cyclized peptides may not be regarded as β‐turn stabilizers, but can be considered as potential β‐turn inducers. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

5α‐Androst‐16‐en‐3α‐ol β‐D‐Glucuronide,Precursor of 5α‐Androst‐16‐en‐3α‐ol in Human Sweat

5α‐Androst‐16‐en‐3α‐ol (α‐androstenol) is an important contributor to human axilla sweat odor. It is assumed that α‐andostenol is excreted from the apocrine glands via a H₂O‐soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H₂O‐soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α‐androstenol, β‐androstenol sulfates, 5α‐androsta‐5,16‐dien‐3β‐ol (β‐androstadienol) sulfate, α‐androstenol β‐glucuronide, α‐androstenol α‐glucuronide, β‐androstadienol β‐glucuronide, and α‐androstenol β‐glucuronide furanose. The occurrence of α‐androstenol β‐glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative‐ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α‐androstenol was observed after incubation of the sterile human sweat or α‐androstenol β‐glucuronide with a commercial glucuronidase enzyme, the urine‐isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have β‐glucuronidase activities. We demonstrated that if α‐ and β‐androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H₂O‐soluble precursor of α‐androstenol in apocrine secretion should be a β‐glucuronide.     

Synthetic studies toward labionin,a new α,α‐disubstituted amino acid from type III lantibiotic labyrinthopeptin A2

The labyrinthopeptins are a new class of lantibiotics containing two identical quaternary α,α‐disubstituted amino acids, named labionin (Lab). The synthetic formation of this unique structural feature represents the key step in the total synthesis of these polycyclic peptides. In this report we describe the synthesis of an orthogonally protected α,α‐disubstituted amino acid building block serving as labionin precursor for the future assembly of labyrinthopeptin A2 and of other labyrinthopeptin derivatives. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of anticancer heptapeptides containing a unique lipophilic β2,2‐amino acid building block

We report a series of synthetic anticancer heptapeptides (H‐KKWβ^2,2WKK‐NH₂) containing eight different central lipophilic β^2,2‐amino acid building blocks, which have demonstrated high efficiency when used as scaffolds in small cationic antimicrobial peptides and peptidomimetics. The most potent peptides in the present study had IC₅₀ values of 9–23 µm against human Burkitt's lymphoma and murine B‐cell lymphoma and were all nonhaemolytic (EC₅₀ > 200 µm ). The most promising peptide 10e also demonstrated low toxicity against human embryonic lung fibroblast cells and peripheral blood mononuclear cells and exceptional proteolytic stability. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

TAPP analogs containing β3‐homo‐amino acids: synthesis and receptor binding

β‐Amino acids containing α,β‐hybrid peptides show great potential as peptidomimetics. In this paper, we describe the synthesis and affinity to μ‐opioid and δ‐opioid receptors of α,β‐hybrids, analogs of the tetrapeptide Tyr‐ d ‐Ala‐Phe‐Phe‐NH₂ (TAPP). Each amino acid was replaced with an l ‐ or d ‐β³‐h‐amino acid. All α,β‐hybrids of TAPP analogs were synthesized in solution and tested for affinity to μ‐opioid and δ‐opioid receptors. The analog Tyr‐β³h‐ d ‐Ala‐Phe‐PheNH₂ was found to be as active as the native tetrapeptide. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Comparative study of toxic effects of zearalenone and its two major metabolites α‐zearalenol and β‐zearalenol on cultured human Caco‐2 cells

Zearalenone (ZEN) is a fusarotoxin converted predominantly into α‐zearalenol (α‐Zol) and β‐zearalenol (β‐Zol) by hepatic hydroxysteroid dehydrogenases. The feeding of naturally contaminated grains with ZEN was associated with hyperestrogenic and adverse effects on humans and animals. There is a lack of information on the attribution of the toxic effects of these toxins. One wonders if these effects are due to the parent molecule (ZEN) or to its major metabolites (α‐Zol and β‐Zol). Using human Caco‐2 cells, we looked for the molecular mechanisms of toxicity of ZEN, α‐Zol, and β‐Zol. Toxicity effects were studied by MTT viability assay and oxidative stress induction by measuring malondialdehyde (MDA) generation. To check whether the oxidative stress induction was associated to DNA lesions, we looked for DNA fragmentation by means of the Comet and the diphenylamine assays. To specify cell death pathway, we investigated caspase‐3 activation, confirmed by poly(ADP‐ribose) polymerase cleavage and by Bcl‐2 depletion. Our results clearly demonstrated that ZEN as well as its two metabolites presented variable toxic effects. They induced cell death and an increase in MDA generation. These effects were associated to DNA fragmentation as well as caspase‐3 activation. The observed toxic effects seem to be relieved by the metabolism of ZEN into α‐Zol and β‐Zol. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:233–243, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20284     

Conformation of poly(γ‐glutamic acid) in aqueous solution

Local conformation and overall conformation of poly(γ‐DL‐glutamic acid) (PγDLGA) and poly(γ‐L‐glutamic acid) (PγLGA) in aqueous solution was studied as a function of degree of ionization ε by ¹H‐NMR, circular dichroism, and potentiometric titration. It was clarified that their local conformation is represented by random coil over an entire ε range and their overall conformation is represented by expanded random‐coil in a range of ε > ε^*, where ε^* is about 0.3, 0.35, 0.45, and 0.5 for added‐salt concentration of 0.02M, 0.05M, 0.1M, and 0.2M, respectively. In a range of ε < ε^*, however, ε dependence of their overall conformation is significantly differentiated from each other. PγDLGA tends to aggregate intramolecularly and/or intermolecularly with decreasing ε, but PγLGA still behaves as expanded random‐coil. It is speculated that spatial arrangement of adjacent carboxyl groups along the backbone chain essentially affects the overall conformation of PγGA in acidic media. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 191–198, 2016.