Substrate Specificities and Conformational Flexibility of 3-Ketosteroid 9α-Hydroxylases

KshA is the oxygenase component of 3-ketosteroid 9α-hydroxylase, a Rieske oxygenase involved in the bacterial degradation of steroids. Consistent with its role in bile acid catabolism, KshA1 from Rhodococcus rhodochrous DSM43269 had the highest apparent specificity (k_cat/K_m) for steroids with an isopropyl side chain at C17, such as 3-oxo-23,24-bisnorcholesta-1,4-diene-22-oate (1,4-BNC). By contrast, the KshA5 homolog had the highest apparent specificity for substrates with no C17 side chain (k_cat/K_m >10⁵ s⁻¹ m⁻¹ for 4-estrendione, 5α-androstandione, and testosterone). Unexpectedly, substrates such as 4-androstene-3,17-dione (ADD) and 4-BNC displayed strong substrate inhibition (K_iS ∼100 μm). By comparison, the cholesterol-degrading KshA_Mtb from Mycobacterium tuberculosis had the highest specificity for CoA-thioesterified substrates. These specificities are consistent with differences in the catabolism of cholesterol and bile acids, respectively, in actinobacteria. X-ray crystallographic structures of the KshA_Mtb·ADD, KshA1·1,4-BNC-CoA, KshA5·ADD, and KshA5·1,4-BNC-CoA complexes revealed that the enzymes have very similar steroid-binding pockets with the substrate''s C17 oriented toward the active site opening. Comparisons suggest Tyr-245 and Phe-297 are determinants of KshA1 specificity. All enzymes have a flexible 16-residue “mouth loop,” which in some structures completely occluded the substrate-binding pocket from the bulk solvent. Remarkably, the catalytic iron and α-helices harboring its ligands were displaced up to 4.4 Å in the KshA5·substrate complexes as compared with substrate-free KshA, suggesting that Rieske oxygenases may have a dynamic nature similar to cytochrome P450.     

Design, Recombinant Expression, and Antibacterial Activity of the Cecropins–Melittin Hybrid Antimicrobial Peptides

In order to evaluate their antibacterial activities and toxicities, the cecropins–melittin hybrid antimicrobial peptide, CA(1-7)-M(4-11) (CAM) and CB(1-7)-M(4-11) (CBM), were designed by APD2 database. The recombinant hybrid antimicrobial peptides were successfully expressed and purified in Pichia pastoris. Antimicrobial activity assay showed that both of the two hybrid antimicrobial peptides had strong antibacterial abilities against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus subtilis, Bacillus thuringiensis, and Salmonella derby. The potency of CAM and CBM to E. coli 25922 were 0.862 and 0.849, respectively, slightly lower than Amp’s 0.957. The hemolytic assays indicated CAM and CBM had no hemolytic in vivo and in vitro, and so they had a good application prospect.     

Synthetic Antimicrobial Peptides: III—Effect of Cationic Groups of Lysine,Arginine, and Histidine on Antimicrobial Activity of Peptides with a Linear Type of Amphipathicity

Russian Journal of Bioorganic Chemistry - We have studied the antimicrobial and hemolytic activity of synthetic antimicrobial peptides (SAMPs), i.e., Arg9Phe2 (P1-Arg), Lys9Phe2 (P2-Lys), and...     

Neurons and β-Cells of the Pancreas Express Connexin36, Forming Gap Junction Channels that Exhibit Strong Cationic Selectivity

We examined the permeability of connexin36 (Cx36) homotypic gap junction (GJ) channels, expressed in neurons and β-cells of the pancreas, to dyes differing in molecular mass and net charge. Experiments were performed in HeLa cells stably expressing Cx36 tagged with EGFP by combining a dual whole-cell voltage clamp and fluorescence imaging. To assess the permeability of the single GJ channel (P(γ)), we used a dual-mode excitation of fluorescent dyes that allowed us to measure cell-to-cell dye transfer at levels not resolvable using whole-field excitation solely. We demonstrate that P(γ) of Cx36 for cationic dyes (EAM-1? and EAM-2?) is ~10-fold higher than that for an anionic dye of the same net charge and similar molecular mass, Alexa fluor-350 (AFl-350?). In addition, P(γ) for Lucifer yellow (LY2?) is approximately fourfold smaller than that for AFl-350?, which suggests that the higher negativity of LY2? significantly reduces permeability. The P(γ) of Cx36 for AFl-350 is approximately 358, 138, 23 and four times smaller than the P(γ)s of Cx43, Cx40, Cx45, and Cx57, respectively. In contrast, it is 6.5-fold higher than the P(γ) of mCx30.2, which exhibits a smaller single-channel conductance. Thus, Cx36 GJs are highly cation-selective and should exhibit relatively low permeability to numerous vital negatively charged metabolites and high permeability to K?, a major charge carrier in cell-cell communication.     

Synthesis, Solution Structure, and Phylum Selectivity of a Spider ��-Toxin That Slows Inactivation of Specific Voltage-gated Sodium Channel Subtypes

Magi 4, now renamed δ-hexatoxin-Mg1a, is a 43-residue neurotoxic peptide from the venom of the hexathelid Japanese funnel-web spider (Macrothele gigas) with homology to δ-hexatoxins from Australian funnel-web spiders. It binds with high affinity to receptor site 3 on insect voltage-gated sodium (Na_V) channels but, unlike δ-hexatoxins, does not compete for the related site 3 in rat brain despite being previously shown to be lethal by intracranial injection. To elucidate differences in Na_V channel selectivity, we have undertaken the first characterization of a peptide toxin on a broad range of mammalian and insect Na_V channel subtypes showing that δ-hexatoxin-Mg1a selectively slows channel inactivation of mammalian Na_V1.1, Na_V1.3, and Na_V1.6 but more importantly shows higher affinity for insect Na_V1 (para) channels. Consequently, δ-hexatoxin-Mg1a induces tonic repetitive firing of nerve impulses in insect neurons accompanied by plateau potentials. In addition, we have chemically synthesized and folded δ-hexatoxin-Mg1a, ascertained the bonding pattern of the four disulfides, and determined its three-dimensional solution structure using NMR spectroscopy. Despite modest sequence homology, we show that key residues important for the activity of scorpion α-toxins and δ-hexatoxins are distributed in a topologically similar manner in δ-hexatoxin-Mg1a. However, subtle differences in the toxin surfaces are important for the novel selectivity of δ-hexatoxin-Mg1a for certain mammalian and insect Na_V channel subtypes. As such, δ-hexatoxin-Mg1a provides us with a specific tool with which to study channel structure and function and determinants for phylum- and tissue-specific activity.Voltage-gated sodium (Na_V)⁴ channels are responsible for the generation and propagation of electrical signals in excitable cells. At least nine different genes encoding distinct Na_V channels isoforms have been identified, and functionally expressed, in mammals (1). They are characterized by their sensitivity to TTX, with Na_V1.5, Na_V1.8, and Na_V1.9 being TTX-insensitive or TTX-resistant, and the remaining subtypes being sensitive to nanomolar concentrations of TTX. In addition, localization of the subtypes also varies, with Na_V1.1–1.3 mostly distributed in the central nervous system, Na_V1.6–1.9 principally located in the peripheral nervous system, and Na_V1.4 and Na_V1.5 found in skeletal and cardiac muscle, respectively. The structural diversity of Na_V channels also coincides with variations in physiological and pharmacological properties (2). In contrast, insects express only one gene (para) that undergoes extensive alternative splicing and RNA editing (3). The para-encoded Na_V channel is exceptionally well conserved across diverse orders of insects, with the level of identity ranging from 87 to 98% (3). This is one reason why insecticides that target insect Na_V channels have broad activity across many insect orders. In contrast, para-type Na_V channels have significantly lower levels of identity with the various types of mammalian Na_V channels with the level of identity typically around 50–60% (3). This explains why a high degree of phylogenetic specificity can be achieved with both Na_V channel toxins and insecticides that target the Na_V channel.At least seven distinct toxin-binding sites have been identified by radioligand binding and electrophysiological studies on vertebrate and insect Na_V channels (4, 5). Toxins interacting with these neurotoxin receptor sites have been instrumental in the study of Na_V channel topology, function, and pharmacology (6). In particular, a wide range of scorpion α-toxins, sea anemone toxins, and spider δ-hexatoxins (formerly δ-atracotoxins (7)) compete for binding to receptor site-3 on the extracellular surface of Na_V channels. These polypeptide toxins all inhibit the fast inactivation of Na_V channels to prolong Na⁺ currents (I_Na), despite huge diversity in primary and tertiary structures (8, 9). Nevertheless, receptor site-3 has not yet been fully characterized but is believed to involve domains DI/S5-S6, DIV/S5-S6, as well as DIV/S3-S4 (9). Most importantly, however, toxin characterization is often limited to studies using whole-cell I_Na or binding studies on neuronal membranes where there are mixed populations of Na_V channel subtypes. For all of these toxins, the precise pattern of Na_V channel subtype selectivity is either unknown or at best is incomplete.Recently, it was found that receptor site-3 was also recognized by a 43-residue spider toxin, originally named Magi 4, from the hexathelid spider Macrothele gigas (Iriomote, Japan). It binds with high affinity to insect Na_V channels but, similar to scorpion α-like toxins, does not compete for the related site-3 in rat brain synaptosomes, despite being lethal by intracranial injection (10). Magi 4 shares significant homology to four δ-hexatoxin (HXTX)-1 family peptides and δ-actinopoditoxin-Mb1a (formerly δ-missulenatoxin-Mb1a; Fig. 1) but no sequence homology to scorpion α-toxins. Neurochemical studies have shown that δ-HXTX-1 toxins compete at nanomolar concentrations with both anti-mammalian (e.g. Aah2 and Lqh2) and anti-insect (e.g. LqhαIT) scorpion toxins for site-3 (11–13). The three-dimensional structures of δ-HXTX-Ar1a and δ-HXTX-Hv1a peptides have been determined (14, 15) and possess core β regions stabilized by four disulfide bonds, placing them in the inhibitory cystine knot (ICK) structural family (16).Open in a separate windowFIGURE 1.Primary and secondary structure of δ-HXTX-Mg1a. A, comparison of the primary sequence of δ-HXTX-Mg1a and δ-HXTX-Mg1b (formerly Magi 14) with currently known members of the δ-HXTX-1 family and δ-AOTX-Mb1a (δ-actinopoditoxin-Mb1a, formerly δ-missulenatoxin-Mb1a). Homologies are shown relative to δ-HXTX-Mg1a; identities are boxed in gray, and conservative substitutions are in gray italic text. Gaps (dashes) have been inserted to maximize alignment. The disulfide bonding pattern for the strictly conserved cysteine residues determined for δ-HXTX-Mg1a (this study), δ-HXTX-Ar1a (55), and δ-HXTX-Hv1a (15) is indicated above the sequences; it is assumed that δ-AOTX-Mb1a (36), δ-HXTX-Hs20.1a (8), and δ-HXTX-Hv1b (56) have the same disulfide bonding pattern. The percentage identity and homology with δ-HXTX-Mg1a is shown to the right of the sequences. B, summary of δ-HXTX-Mg1a NMR data. Sequential NOEs, classified as very weak, weak, medium, and strong, are represented by the thickness of bars. Filled diamonds indicate backbone amide protons that form hydrogen bonds. ³J_NHCα coupling constants are indicated by ↑ (>8 Hz) and ↓ (<5.5 Hz). Secondary structure is shown at the bottom of the figure where rectangles represent β-turns (the type of turn is indicated in the rectangle) and arrows represent β-sheets.The aim of this study was to first determine the solution structure of Magi 4 and second to investigate the ability of Magi 4 to discriminate between different Na_V channels subtypes. Here we report the tertiary structure of Magi 4 by ¹H NMR and show its disulfide bonding pattern and three-dimensional structure are homologous to δ-HXTX-1 toxins. We highlight the key residues in Magi 4 that appear to be topologically similar to those residues known to be part of the pharmacophore for site-3 scorpion α-toxins, despite Magi 4 having a different overall structure to scorpion α-toxins (11). In addition, we provide a detailed characterization of the selectivity and mode of action of Magi 4 on nine cloned mammalian and insect Na_V channel subtypes, including a detailed characterization on insect neurotransmission. Given that the toxin potently slows the inactivation of Na_V channels, it should be renamed δ-hexatoxin-Mg1a (δ-HXTX-Mg1a) in accordance with the rational nomenclature recently proposed for naming spider peptide toxins (7) (see ArachnoServer spider toxin data base).     

Inhibitory Effects and Mechanism of the Combined Use of α-Helical Peptides HPRP-A1/HPRP-A2 and Chlorhexidine Acetate Against Bacterial and Fungal Biofilms

International Journal of Peptide Research and Therapeutics - Drug resistance is an increasing problem in the treatment of vaginitis bacterial vaginosis and vulvovaginal candidiasis caused by...     

Antibacterial and Anticancer activity of ε -poly-L-lysine (ε -PL) produced by a marine Bacillus subtilis sp

A marine Bacillus subtilis SDNS was isolated from sea water in Alexandria and identified using 16S rDNA sequence analysis. The bacterium produced a compound active against a number of gram negativeve bacteria. Moreover, the anticancer activity of this bacterium was tested against three different human cell lines (Hela S3, HepG2 and CaCo). The highest inhibition activity was recorded against Hela S3 cell line (77.2%), while almost no activity was recorded towards CaCo cell line. HPLC and TLC analyses supported evidence that Bacillus subtilis SDNS product is ?;-poly-L-lysine. To achieve maximum production, Plackett-Burman experimental design was applied. A 1.5 fold increase was observed when Bacillus subtilis SDNS was grown in optimized medium composed of g/l: (NH(4) )(2) SO(4) , 15; K(2) HPO(4) , 0.3; KH(2) PO(4) , 2; MgSO(4) · 7 H(2) O, 1; ZnSO(4) · 7 H(2) O, 0; FeSO(4) · 7 H(2) O, 0.03; glucose, 25; yeast extract, 1, pH 6.8. Under optimized culture condition, a product value of 76.3 mg/l could be obtained. According to available literature, this is the first announcement for the production of ?;-poly-L-lysine (?;-PL) by a member of genus Bacillus. (? 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).     

Alcadein Cleavages by Amyloid ��-Precursor Protein (APP) ��- and ��-Secretases Generate Small Peptides, p3-Alcs, Indicating Alzheimer Disease-related ��-Secretase Dysfunction

Alcadeins (Alcs) constitute a family of neuronal type I membrane proteins, designated Alc_α, Alc_β, and Alc_γ. The Alcs express in neurons dominantly and largely colocalize with the Alzheimer amyloid precursor protein (APP) in the brain. Alcs and APP show an identical function as a cargo receptor of kinesin-1. Moreover, proteolytic processing of Alc proteins appears highly similar to that of APP. We found that APP α-secretases ADAM 10 and ADAM 17 primarily cleave Alc proteins and trigger the subsequent secondary intramembranous cleavage of Alc C-terminal fragments by a presenilin-dependent γ-secretase complex, thereby generating “APP p3-like” and non-aggregative Alc peptides (p3-Alcs). We determined the complete amino acid sequence of p3-Alc_α, p3-Alc_β, and p3-Alc_γ, whose major species comprise 35, 37, and 31 amino acids, respectively, in human cerebrospinal fluid. We demonstrate here that variant p3-Alc C termini are modulated by FAD-linked presenilin 1 mutations increasing minor β-amyloid species Aβ42, and these mutations alter the level of minor p3-Alc species. However, the magnitudes of C-terminal alteration of p3-Alc_α, p3-Alc_β, and p3-Alc_γ were not equivalent, suggesting that one type of γ-secretase dysfunction does not appear in the phenotype equivalently in the cleavage of type I membrane proteins. Because these C-terminal alterations are detectable in human cerebrospinal fluid, the use of a substrate panel, including Alcs and APP, may be effective to detect γ-secretase dysfunction in the prepathogenic state of Alzheimer disease subjects.     

Cell Adhesion, the Backbone of the Synapse: ��Vertebrate�� and ��Invertebrate�� Perspectives

Synapses are asymmetric intercellular junctions that mediate neuronal communication. The number, type, and connectivity patterns of synapses determine the formation, maintenance, and function of neural circuitries. The complexity and specificity of synaptogenesis relies upon modulation of adhesive properties, which regulate contact initiation, synapse formation, maturation, and functional plasticity. Disruption of adhesion may result in structural and functional imbalance that may lead to neurodevelopmental diseases, such as autism, or neurodegeneration, such as Alzheimer''s disease. Therefore, understanding the roles of different adhesion protein families in synapse formation is crucial for unraveling the biology of neuronal circuit formation, as well as the pathogenesis of some brain disorders. The present review summarizes some of the knowledge that has been acquired in vertebrate and invertebrate genetic model organisms.Synapses are asymmetric, intercellular junctions that are the basic structural units of neuronal transmission. The correct development of synaptic specializations and the establishment of appropriate connectivity patterns are crucial for the assembly of functional neuronal circuits. Improper synapse formation and function may cause neurodevelopmental disorders, such as mental retardation (MsR) and autism spectrum disorders (ASD) (McAllister 2007; Sudhof 2008), and likely play a role in neurodegenerative disorders, such as Alzheimer''s disease (AD) (Haass and Selkoe 2007).At chemical synapses (reviewed in Sudhof 2004; Zhai and Bellen 2004; Waites et al. 2005; McAllister 2007; Jin and Garner 2008), the presynaptic compartment contains synaptic vesicles (SV), organized in functionally distinct subcellular pools. A subset of SVs docks to the presynaptic membrane around protein-dense release sites, named active zones (AZ). Upon the arrival of an action potential at the terminal, the docked and “primed” SVs fuse with the plasma membrane and release neurotransmitter molecules into the synaptic cleft. Depending on the type of synapse (i.e., excitatory vs. inhibitory synapses), neurotransmitters ultimately activate an appropriate set of postsynaptic receptors that are accurately apposed to the AZ.Synapse formation occurs in several steps (Fig. 1) (reviewed in Eaton and Davis 2003; Goda and Davis 2003; Waites et al. 2005; Garner et al. 2006; Gerrow and El-Husseini 2006; McAllister 2007). Spatiotemporal signals guide axons through heterogeneous cellular environments to contact appropriate postsynaptic targets. At their destination, axonal growth cones initiate synaptogenesis through adhesive interactions with target cells. In the mammalian central nervous system (CNS), immature postsynaptic dendritic spines initially protrude as thin, actin-rich filopodia on the surface of dendrites. Similarly, at the Drosophila neuromuscular junction (NMJ), myopodia develop from the muscles (Ritzenthaler et al. 2000). The stabilization of intercellular contacts and their elaboration into mature, functional synapses involves cytoskeletal arrangements and recruitment of pre- and postsynaptic components to contact sites in spines and boutons. Conversely, retraction of contacts results in synaptic elimination. Both stabilization and retraction sculpt a functional neuronal circuitry.Open in a separate windowFigure 1.(A–C) Different stages of synapse formation. (A) Target selection, (B) Synapse assembly, (C) Synapse maturation and stabilization. (D–F) The role of cell adhesion molecules in synapse formation is exemplified by the paradigm of N-cadherin and catenins in regulation of the morphology and strength of dendritic spine heads. (D) At an early stage the dendritic spines are elongated from motile structures “seeking” their synaptic partners. (E) The contacts between the presynaptic and postsynaptic compartments are stabilized by recruitment of additional cell adhesion molecules. Adhesional interactions activate downstream pathways that remodel the cytoskeleton and organize pre- and postsynaptic apparatuses. (F) Cell adhesion complexes, stabilized by increased synaptic activity, promote the expansion of the dendritic spine head and the maturation/ stabilization of the synapse. Retraction and expansion is dependent on synaptic plasticity.In addition to the plastic nature of synapse formation, the vast heterogeneity of synapses (in terms of target selection, morphology, and type of neurotransmitter released) greatly enhances the complexity of synaptogenesis (reviewed in Craig and Boudin 2001; Craig et al. 2006; Gerrow and El-Husseini 2006). The complexity and specificity of synaptogenesis relies upon the modulation of adhesion between the pre- and postsynaptic components (reviewed in Craig et al. 2006; Gerrow and El-Husseini 2006; Piechotta et al. 2006; Dalva et al. 2007; Shapiro et al. 2007; Yamada and Nelson 2007; Gottmann 2008). Cell adhesive interactions enable cell–cell recognition via extracellular domains and also mediate intracellular signaling cascades that affect synapse morphology and organize scaffolding complexes. Thus, cell adhesion molecules (CAMs) coordinate multiple synaptogenic steps.However, in vitro and in vivo studies of vertebrate CAMs are often at odds with each other. Indeed, there are no examples of mutants for synaptic CAMs that exhibit prominent defects in synapse formation. This apparent “resilience” of synapses is probably caused by functional redundancy or compensatory effects among different CAMs (Piechotta et al. 2006). Hence, studies using simpler organisms less riddled by redundancy, such as Caenorhabditis elegans and Drosophila, have aided in our understanding of the role that these molecules play in organizing synapses.In this survey, we discuss the roles of the best characterized CAM families of proteins involved in synaptogenesis. Our focus is to highlight the complex principles that govern the molecular basis of synapse formation and function from a comparative perspective. We will present results from cell culture studies as well as in vivo analyses in vertebrate systems and refer to invertebrate studies, mainly performed in Drosophila and C. elegans, when they have provided important insights into the role of particular CAM protein families. However, we do not discuss secreted factors, for which we refer the reader to numerous excellent reviews (as for example Washbourne et al. 2004; Salinas 2005; Piechotta et al. 2006; Shapiro et al. 2006; Dalva 2007; Yamada and Nelson 2007; Biederer and Stagi 2008; Salinas and Zou 2008).     

Synthesis,Computational Insights,and Anticancer Activity of Novel Indole–Schiff Base Derivatives

Russian Journal of Bioorganic Chemistry - Synthetic and naturally available compounds with indole moiety are known to show significant biological activity. This paper describes computational...     

Conformational Flexibility and Absolute Stereochemistry of (3R)-3-hydroxy-4-aryl-β-lactams Investigated by Chiroptical Properties and TD-DFT Calculations

The effect of conformational flexibility on the chiroptical properties of a series of synthetic (3R)-3-hydroxy-4-aryl-β-lactams of known stereochemistry (1-6) was investigated by means of electronic circular dichroism (ECD) measurements and time-dependent density functional theory (TD-DFT) calculations. The application of the β-lactam sector rules allowed a correct stereochemical characterization of these compounds, with the exception of a thienyl-substituted derivative (cis-). TD-DFT calculations yielded accurate predictions of experimental ECD spectra and [α](D) values, allowing us to assign the correct absolute configuration to all the investigated compounds. A detailed analysis of the β-lactam ring equilibrium geometry on optimized conformers identified regular patterns for the arrangement of atoms around the amide chromophore, confirming the validity of the β-lactam sector rules. However, relevant variations in theoretical chiroptical properties were found for compounds bearing a heterocyclic substituent at C4 or a phenyl substituent at C3, whose conformers deviate from these regular geometric patterns. This behavior explains the failure of the β-lactam sector rules in cis-. This study showed the importance of conformational flexibility for the determination of chiroptical properties and highlighted the strengths and weaknesses of the different methods for the stereochemical characterization of chiral molecules in solution. Chirality 24:741-750, 2012. ? 2012 Wiley Periodicals, Inc.     

Conformational stability of LYLA1, a synthetic chimera of human lysozyme and bovine α-lactalbumin

LYLA1 is a chimeric protein mainly consisting of residues originating from human lysozyme but in which the central part (Ca²⁺-binding site and helix C) of bovine α-lactalbumin has been inserted. The equilibrium unfolding of this hybrid protein has been examined by circular dichroism and tryptophan fluorescence techniques. The reversible denaturation process induced by temperature or by addition of chemical denaturant is three-state in the case of apo-LYLA1 and two-state in the presence of Ca²⁺. The Ca²⁺-bound form of the chimera exhibits higher stability than both wild-type lysozyme and α-lactalbumin. The stability of the apo-form, however, is intermediate between that of the parent molecules. Unfolding of apo-LYLA1 involves an intermediate state that becomes populated to a different extent under various experimental conditions. Combination of circular dichroism with bis-ANS fluorescence experiments has permitted us to characterize the acid state of LYLA1 as a molten globule. Furthermore our results strongly suggest the presence of multiple denatured states depending on external conditions. Received: 24 April 1996 / Accepted: 4 September 1996     

Brazilian Kefir-Fermented Sheep’s Milk,a Source of Antimicrobial and Antioxidant Peptides

Fermented milks are a source of bioactive peptides and may be considered as functional foods. Among these, sheep’s milk fermented with kefir has not been widely studied and its most relevant properties need to be more thoroughly characterized. This research study is set out to investigate and evaluate the antioxidant and antimicrobial properties of peptides from fermented sheep’s milk in Brazil when produced by using kefir. For this, the chemical and microbiological composition of the sheep’s milk before and after the fermentation was evaluated. The changes in the fermented milk and the peptides extracted before the fermentation and in the fermented milk during its shelf life were verified. The antimicrobial and antioxidant activities of the peptides from the fermented milk were evaluated and identified according to the literature. The physicochemical properties and mineral profile of the fermented milk were like those of fresh milk. The peptide extract presented antimicrobial activity and it was detected that 13 of the 46 peptides were able to inhibit the growth of pathogenic microorganisms. A high antioxidant activity was observed in the peptides extracted from fermented milk (3.125 mg/mL) on the 28th day of storage. Two fractions displayed efficient radical scavenging properties by DPPH and ABTS methods. At least 11 peptides distributed in the different fractions were identified by tandem mass spectrometry. This sheep’s milk fermented by Brazilian kefir grains, which has antioxidant and antimicrobial activities and probiotic microorganisms, is a good candidate for further investigation as a source for bioactive peptides. The fermentation process was thus a means by which to produce potential bioactive peptides.     

Rapid and Efficient Synthesis of Peptides Containing α,α-dialkylamino acids employing KOBt

Summary Several Fmoc-α,α-dialkylamino acids and their acid chlorides have been prepared, isolated and characterised. The synthesis of peptides containing sterically hindered dialkylamino acids has been accomplished using acid chloride/KOBt in dichloromethane. The yields as well as the purity of the peptides were satistactory.