Characteristic tryptic peptides and gelling properties of porcine skin gelatin affected by thermal action

Thermal action in extraction process had effects on characteristic tryptic peptides identification and gelling properties of porcine gelatin. SDS-PAGE, HPLC-LTQ/Orbitrap high-resolution mass spectrometry, texture analyser and rheometer were used to evaluate collagen depolymerisation degree, characteristic tryptic peptides and gelling properties of gelatins prepared in various thermal actions. Results showed that with increasing temperature and time, depolymerisation degree enlarged, while gel strength, gelling and melting temperature decreased. Mass spectra showed that 47 and 49 common characteristic tryptic peptides were identified in gelatins extracted at 50 °C and 100 °C with various times, respectively. Moreover, 34 common characteristic tryptic peptides were identified in all gelatin samples. Further comparison between this work and our previous investigations yielded 20 common characteristic tryptic peptides, which stably exist in various thermal actions. These common characteristic tryptic peptides may be very helpful for the accurate authentication of porcine gelatin.     

Cheese ripening in nonconventional conditions: A multiparameter study applied to Protected Geographical Indication Canestrato di Moliterno cheese

A multiparameter study was performed to evaluate the effect of fondaco, a traditional ripening cellar without any artificial temperature and relative humidity control, on the chemical, microbiological, and sensory characteristics of Protected Geographical Indication Canestrato di Moliterno cheese. Ripening in such a nonconventional environment was associated with lower counts of lactococci, lactobacilli, and total viable bacteria, and higher presence of enterococci, in comparison with ripening in a controlled maturation room. Moreover, fondaco cheese underwent accelerated maturation, as demonstrated by faster casein degradation, greater accumulation of free AA, and higher formation of volatile organic compounds. Secondary proteolysis, as assessed by liquid chromatography-mass spectrometry of free AA and low molecular weight peptides, did not show any qualitative difference among cheeses, but fondaco samples evidenced an advanced level of peptidolysis. On the other hand, significant qualitative differences were observed in the free fatty acid profiles and in the sensory characteristics. Principal component analysis showed a clear separation of the fondaco and control cheeses, indicating that ripening in the natural room conferred unique sensory features to the product.     

Fluorescence Correlation Spectroscopy Analysis of Effect of Molecular Crowding on Self-Assembly of β-Annulus Peptide into Artificial Viral Capsid

Recent progress in the de novo design of self-assembling peptides has enabled the construction of peptide-based viral capsids. Previously, we demonstrated that 24-mer β-annulus peptides from tomato bushy stunt virus spontaneously self-assemble into an artificial viral capsid. Here we propose to use the artificial viral capsid through the self-assembly of β-annulus peptide as a simple model to analyze the effect of molecular crowding environment on the formation process of viral capsid. Artificial viral capsids formed by co-assembly of fluorescent-labelled and unmodified β-annulus peptides in dilute aqueous solutions and under molecular crowding conditions were analyzed using fluorescence correlation spectroscopy (FCS). The apparent particle size and the dissociation constant (K_d) of the assemblies decreased with increasing concentration of the molecular crowding agent, i.e., polyethylene glycol (PEG). This is the first successful in situ analysis of self-assembling process of artificial viral capsid under molecular crowding conditions.     

Structural insights into peptide self-assembly using photo-induced crosslinking experiments and discontinuous molecular dynamics

Determining the structure of the (oligomeric) intermediates that form during the self-assembly of amyloidogenic peptides is challenging because of their heterogeneous and dynamic nature. Thus, there is need for methodology to analyze the underlying molecular structure of these transient species. In this work, a combination of fluorescence quenching, photo-induced crosslinking (PIC) and molecular dynamics simulation was used to study the assembly of a synthetic amyloid-forming peptide, Aβ_16-22. A PIC amino acid containing a trifluormethyldiazirine (TFMD) group—Fmoc(TFMD)Phe—was incorporated into the sequence (Aβ*_16–22). Electrospray ionization ion-mobility spectrometry mass-spectrometry (ESI-IMS-MS) analysis of the PIC products confirmed that Aβ*_16–22 forms assemblies with the monomers arranged as anti-parallel, in-register β-strands at all time points during the aggregation assay. The assembly process was also monitored separately using fluorescence quenching to profile the fibril assembly reaction. The molecular picture resulting from discontinuous molecule dynamics simulations showed that Aβ_16-22 assembles through a single-step nucleation into a β-sheet fibril in agreement with these experimental observations. This study provides detailed structural insights into the Aβ_16-22 self-assembly processes, paving the way to explore the self-assembly mechanism of larger, more complex peptides, including those whose aggregation is responsible for human disease.     

Leucokinin and Associated Neuropeptides Regulate Multiple Aspects of Physiology and Behavior in Drosophila

Leucokinins (LKs) constitute a family of neuropeptides identified in numerous insects and many other invertebrates. LKs act on G-protein-coupled receptors that display only distant relations to other known receptors. In adult Drosophila, 26 neurons/neurosecretory cells of three main types express LK. The four brain interneurons are of two types, and these are implicated in several important functions in the fly’s behavior and physiology, including feeding, sleep–metabolism interactions, state-dependent memory formation, as well as modulation of gustatory sensitivity and nociception. The 22 neurosecretory cells (abdominal LK neurons, ABLKs) of the abdominal neuromeres co-express LK and a diuretic hormone (DH44), and together, these regulate water and ion homeostasis and associated stress as well as food intake. In Drosophila larvae, LK neurons modulate locomotion, escape responses and aspects of ecdysis behavior. A set of lateral neurosecretory cells, ALKs (anterior LK neurons), in the brain express LK in larvae, but inconsistently so in adults. These ALKs co-express three other neuropeptides and regulate water and ion homeostasis, feeding, and drinking, but the specific role of LK is not yet known. This review summarizes Drosophila data on embryonic lineages of LK neurons, functional roles of individual LK neuron types, interactions with other peptidergic systems, and orchestrating functions of LK.     

Lactones in the Synthesis of Prostaglandins and Prostaglandin Analogs

In the total stereo-controlled synthesis of natural prostaglandins (PGs) and their structural analogs, a vast class of compounds and drugs, known as the lactones, are encountered in a few key steps to build the final molecule, as: δ-lactones, γ-lactones, and 1,9-, 1,11-, and 1,15-macrolactones. After the synthesis of 1,9-PGF_2α and 1,15-PGF_2α lactones, many 1,15-lactones of E₂, E₃, F₂, F₃, A₂, and A₃ were found in the marine mollusc Tethys fimbria and the quest for understanding their biological role stimulated the research on their synthesis. Then 1,9-, 1,11-, and 1,15-PG lactones of the drugs were synthesized as an alternative to the corresponding esters, and the first part of the paper describes the methods used for their synthesis. The efficient Corey procedure for the synthesis of prostaglandins uses the key δ-lactone and γ-lactone intermediates with three or four stereocenters on the cyclopentane fragment to link the PG side chains. The paper describes the most used procedures for the synthesis of the milestone δ-Corey-lactones and γ-Corey-lactones, their improvements, and some new promising methods, such as interesting, new stereo-controlled and catalyzed enantioselective reactions, and methods based on the chemical/enzymatic resolution of the compounds in different steps of the sequences. The many uses of δ-lactones not only for the synthesis of γ-lactones, but also for obtaining 9β-halogen-PGs and halogen-substituted cyclopentane intermediates, as synthons for new 9β-PG analogs and future applications, are also discussed.     

Effect of thermal treatment on the antioxidant activity of egg white hydrolysate and the preparation of novel antioxidant peptides

Heat treatment will affect the nutritional properties and potential bioactivity of food materials. The aim of this work was to evaluate the effect of different thermal treatment (4, 56, 65 and 100 ℃) and in vitro gastrointestinal digestion on the antioxidant activity of egg white hydrolysate. The results demonstrated that egg white hydrolysate treated at 65 ℃ exhibited the highest antioxidant. Remarkably, the simulated digestion significantly increased antioxidant activity of egg white hydrolysate. Furthermore, we identified twenty-four potential antioxidant peptides by performing mass spectrometry and bioinformatic analysis. Six peptides were selected based on the activity prediction score of the online tool. The results showed that P6 (ACPECPK) possessed the most outstanding antioxidant properties and had low cytotoxicity and allergenicity. Bioinformatics technology combined with biochemical assays may offer a way for discovering novel antioxidant peptides from different kinds of food under various heat treatment conditions.     

Mechanistic Studies on Trichoacorenol Synthase from Amycolatopsis benzoatilytica

Isotopic labeling experiments performed with a newly identified bacterial trichoacorenol synthase established a 1,5-hydride shift occurring in the cyclization mechanism. During EI-MS analysis, major fragments of the sesquiterpenoid were shown to arise via cryptic hydrogen movements. Therefore, the interpretation of earlier results regarding the cyclization mechanism obtained by feeding experiments in Trichoderma is revised.     

Helices on Interdomain Interface Couple Catalysis in the ATPPase Domain with Allostery in Plasmodium falciparum GMP Synthetase

GMP synthetase catalyses the conversion of XMP to GMP through a series of reactions that include hydrolysis of Gln to generate ammonia in the glutamine amidotransferase (GATase) domain, activation of XMP to adenyl-XMP intermediate in the ATP pyrophosphatase (ATPPase) domain and reaction of ammonia with the intermediate to generate GMP. The functioning of GMP synthetases entails bidirectional domain crosstalk, which leads to allosteric activation of the GATase domain, synchronization of catalytic events and tunnelling of ammonia. Herein, we have taken recourse to the analysis of structures of GMP synthetases, site-directed mutagenesis and steady-state and transient kinetics on the Plasmodium falciparum enzyme to decipher the molecular basis of catalysis in the ATPPase domain and domain crosstalk. Our results suggest an arrangement at the interdomain interface, of helices with residues that play roles in ATPPase catalysis as well as domain crosstalk enabling the coupling of ATPPase catalysis with GATase activation. Overall, the study enhances our understanding of GMP synthetases, which are drug targets in many infectious pathogens.