Purification, characterization, and lytic activity against Naegleria fowleri of two amoebicins produced by Bacillus licheniformis A12.

Bacillus licheniformis A12 produces two amoebolytic substances (amoebicins A12-A and A12-B) in liquid media during sporulation. Both substances have been purified and characterized. They are heat- and protease-resistant peptides containing aspartic acid, glutamic acid, serine, proline, and tyrosine in a molar ratio of 5:2:2:2:2. No fatty acids or carbohydrates have been detected. Their molecular weight is 1,430. Purified amoebicins A12-A and A12-B exhibit amoebolytic action against Naegleria fowleri. They also exhibit antibiotic action against yeasts (Saccharomyces heterogenicus and Cryptococcus neoformans) and several fungal species (Aspergillus niger, Microsporum canis, Mucor plumbeus, and Trychophyton mentagrophytes). Their antibacterial spectrum appears to be restricted to Bacillus megaterium, Corynebacterium glutamicum, and Sarcina sp.     

In Silico Design,Synthesis, and In Vitro Evaluation of Novel Amphipathic Short Linear Peptides Against Clinically Relevant Bacterial Biofilms

Microbial biofilms are organized communities of cells that are associated with a wide spectrum of resistant and chronic infections that lead to the treatment failure. Accordingly, there is an urgent demand to create novel effective therapeutic drugs that can inhibit biofilm formation with new mechanisms of action to surmount the current escalating resistance. In this study, in silico hybrid model was utilized to develop three novel short linear peptides (4, 5, and 6) with potential biofilm inhibiting activities (scores?>?1.0). The peptides were composed of cationic and hydrophobic residues. They were synthesized using solid-phase strategy. Synthesized peptides were purified and characterized by reverse-phase high-performance liquid chromatography and matrix-assisted laser desorption/ionization spectroscopy, respectively. They were evaluated using in vitro assay as potential inhibitors of clinically relevant Gram-positive and Gram-negative biofilms. Peptide (4) with five positive charges at physiological pH (4 cationic moieties and W:R?=?1:4) showed activity against biofilms of Gram-positive strains (Staphylococcus epidermidis and Listeria monocytogenes). On the other hand, peptide (5) with six positive charges (5 cationic moieties and W:R?=?2:2) demonstrated activity against Gram-positive (S. epidermidis) and Gram-negative (Escherichia coli) biofilms. Interestingly, peptide (6), with seven positive charges (6 cationic moieties and W:R?=?2:5) revealed higher and broader spectrum of activity against biofilms of Gram-positive (S. epidermidis, S. aureus, L. monocytogenes) and Gram-negative (E. coli).

     

绵羊生殖道抗菌肽

以屠宰场收集的新鲜、健康、雌性绵羊生殖器官为原材料．采用乙酸浸提、透析、Sephadex G-50凝胶过滤层析和反相高效液相色谱(RP-HPLC)等方法分离纯化绵羊生殖道抗菌肽．以G+、G-和真菌为抗菌活性检测指示菌株,利用薄层琼脂糖孔穴扩散法、微量肉汤稀释法进行抗菌活性检测．对分离纯化所得纯品进行分子质量质谱测定、纯度鉴定、N端测序,并对其性质进行研究．结果表明：分离纯化所得两个绵羊生殖道抗菌肽分子质量分别为4820.47 u和4012.5 u,N端部分氨基 酸序列分别为AYVLDEPKP和YDSGA．对G+细菌(S. aureus ATCC2592、Streptococcu ATCC55121)、G-细菌(E． coli ATCC25922)、真菌(C． albicans ATCC2002)均具有良好的抑菌活性．对家兔红细胞无溶血活性,对人血液凝固无影响．目前未见有从绵羊生殖道分离纯化得到抗菌肽的报道,并且这一研究结果进一步证实抗菌肽在多种动物生殖道天然免疫防御方面起着重要作用．     

Immunosuppressive,antimicrobial and insecticidal activities of inhibitor cystine knot peptides produced by teratocytes of the endoparasitoid wasp Cotesia flavipes (Hymenoptera: Braconidae)

Teratocytes are specialized cells released by parasitoid wasps into their hosts. They are known for producing regulatory molecules that aid the development of immature parasitoids. We have recently reported the primary structures of cystine-rich peptides, including some containing inhibitor cystine knot (ICK) motifs, produced by teratocytes of the parasitoid Cotesia flavipes (Hymenoptera: Braconidae). ICKs are known for their stability and diverse biological functions. In this study, we produced four putative ICK peptides from the teratocytes of C. flavipes using solid-phase peptide synthesis or recombinant expression in E. coli, and investigated their functions on host immune modulation as well their potential to impair the development of two lepidopterans after ingestion of the peptides. In addition, the peptides were assayed against pathogens and human cells. The peptides did not influence total hemocyte count but suppressed cellular immunity, detectable as a reduction of hemocyte encapsulation (CftICK-I, CftICK-II, CftICK-III) and spread indexes (CftICK-IV) in the host. None of the peptides influenced the activities of prophenoloxidase and phenoloxidase in the hemolymph of larval Diatraea saccharalis (Lepidoptera: Crambidae). CftICK-I and CftICK-II with previously unknown function showed antifungal activity against Candida albicans but were non-toxic to human cells. CftICK-I, CftICK-II, and CftICK-III increased larval mortality and reduced leaf consumption of D. saccharalis, a permissive host for C. flavipes. The CftICK-III also increased larval mortality and reduced leaf consumption of Spodoptera frugiperda (Lepidoptera: Noctuidae), a non-permissive host for C. flavipes. This study highlights biological functions and biotechnological potential of ICK peptides from the teratocytes of C. flavipes.     

Amino Acid Sequence and Antimicrobial Activity of Chitin-Binding Peptides,Pp-AMP 1 and Pp-AMP 2, from Japanese Bamboo Shoots (Phyllostachys pubescens)

Two novel chitin-binding peptides, designated Pp-AMP 1 and Pp-AMP 2, which had antimicrobial activity against pathogenic bacteria and fungi, were purified from Japanese bamboo shoots (Phyllostachys pubescens) by a simple procedure based on chitin affinity chromatography. They had the common structural features of the plant defensin family, but they could not be grouped in any type of that family. They showed a high degree of homology to mistletoe toxins.     

Isolation and partial characterization of bacteriocins from<Emphasis Type="Italic"> Pediococcus</Emphasis> species

Lactic acid bacteria have received increased attention as a potential food preservative due to their strong antagonistic activity against many food-spoilage and pathogenic organisms. Three Pediococcus species, P. acidilactici NCIM 2292, P. pentosaceous. NCIM 2296 and P. cervisiae NCIM 2171, were evaluated for bacteriocin production. Inhibitory substance were produced during the late growth phase and maximum production occurred at 37 °C after 36–48 h of incubation. Bacteriocins partially purified from these species by cold-acetone precipitation at 0 °C and cell adsorption desorption techniques have a broad inhibitory spectrum against microorganisms, including gram-negative bacteria such as Escherichia coli and Pseudomonas. Proteolytic enzymes inactivated these peptides, but amylase and lipase did not show any effect. The bacteriocins were stable over a wide pH range (3–8) and apparently most active at pH 4.0–5.0. They were heat-stable (1 h at ~80 °C and autoclaving) at pH 5.0. No loss in activity was observed when stored under refrigeration (4–8 °C). Tris-Tricine SDS-PAGE revealed the molecular masses of these peptides to be between 3.5 and 5.0 kDa.     

Effect of trypsin on D1/D2-cytochrom b 559 Photosystem 2 reaction center complex and reaction center from Rhodopseudomonas viridis

Proteolytic enzyme (trypsin) was used to structurally alter the RCs isolated from plant and bacterium as a way of probing the relation between structure (chromophore-apoprotein interactions) and function (photochemical activity). It was found that neither spectral characteristics (absorption spectrum, the 4th derivative of absorption spectrum) nor photochemical activity (pheophytine photoreduction, P680 photooxidation, etc.) were changed dramatically in D₁/D₂/cytochrom b ₅₅₉ PS 2 reaction center complex digested with trypsin. The PS 2 RC treated with trypsin migrates by one green band during electrophoresis with dodecylmaltoside. The peptides with a molecular mass higher than 3–4 kDa were not separated from PS 2 RC. These data indicate that digestion of D1 and D2 proteins does not disturb yet the conformation of peptides or their interactions in so-called core of RC and the native state of pigments. In contrast to that, the RC from Rhodopseudomonas viridis treated with enzyme has changed absorption spectrum and lost photochemical activity. The stability of the bacterial RC increased after exchange of LDAO by dodecylmaltoside.Abbreviations Chl chlorophyll a - Cyt cytochrome - DPC diphenylcarbazide - Dodecylmaltoside dodecyl--D-maltoside - LDAO lauryldimethylamino oxide - Pheo pheophytine - PS 2 Photosystem 2 - RC reaction center - SiMo silicomolybdate - SD sodium dodecyl sulfate     

New Deblocking Aminopeptidases from Pyrococcus horikoshii

It has been reported that one of the hyperthermostable aminopeptidases from Pyrococcus horikoshii exhibits hydrolytic activity toward short peptides and acyl-peptides (deblocking activity). In the genome database of P. horikoshii, two new open reading frames homologous to the hyperthermostable aminopeptidase of P. horikoshii were found. The two new genes for the proteins were cloned, expressed using E. coli, and characterized. The purified proteins gave a single band on SDS-PAGE corresponding to molecular masses of 42 kDa and 41 kDa respectively, and exhibited aminopeptidase activity, including deblocking activity. These enzymes are likely to exist as oligomeric structures at neutral pH. The optimum pHs of the two enzyme activities were in the range of 7.0 to 7.5, and the optimum temperatures for the activities were around 100 °C. The enzymes exhibited low hydrolytic activity for peptide substrates longer than 10 residues. They were activated by cobalt and zinc ions. Their substrate specificities and activation factors are different. It was confirmed that P. horikoshii has three similar aminopeptidases with deblocking activity and that these enzymes appear to play important roles in hydrolyzing small peptides in P. horikoshii cells.     

BH4 peptide derived from Bcl‐xL and Bax‐inhibitor peptide suppresses apoptotic mitochondrial changes in heat stressed bovine oocytes

Mitochondria play an important role in the integration and transmission of cell death signals mediated by the Bcl‐2 family proteins. Experiments were conducted to determine whether the anti‐apoptotic peptides BH4 domain of Bcl‐xL (TAT‐BH4) and Bax inhibitor peptide (BIP) suppresses heat stress (HS) injury in oocytes by reduction of apoptotic‐like events. Cumulus–oocyte complexes (COCs) were matured at 39°C (control) or 41°C (HS) for 21 hr then placed in maturation medium containing 0 or 100 µM BIP in water and 0 or 1 µM TAT‐BH4 in dimethyl sulfoxide (DMSO), or a combination of both peptides (BIP + BH4). Peptide effects on embryo development, DNA fragmentation, mitochondrial membrane potential (ΔΨm), and mitochondrial DNA (mtDNA) copy number were measured. All groups were fertilized and cultured in vitro at 39°C for 8 days. Compared to control, HS‐treated oocytes induced a decrease in embryo development (P < 0.05), increase in proportion of TUNEL‐positive chromatin in oocytes and blastocysts (P < 0.05), and loss of oocyte ΔΨm (P < 0.001). In the presence of BIP or BIP + BH4, development of HS‐treated oocytes into blastocysts was increased (P < 0.05). Conversely, COCs matured with TAT‐BH4 at 41°C showed reduced embryonic development (P < 0.05). Exposure of HS‐treated to each or both peptides resulted in a reduction of TUNEL frequency in oocytes and blastocysts cells derived from these oocytes (P < 0.05). The loss of ΔΨm in HS‐treated oocytes was not restored by exposure to BIP + BH4 and there was no effect in mtDNA copy number. In conclusion, the present results show that HS‐induced apoptosis in bovine oocytes involves Bax and BH4 domain‐dependent pathways. Mol. Reprod. Dev. 76: 637–646, 2009. © 2008 Wiley‐Liss, Inc.     

Importance of the length of the N- and C-terminal regions of Helicobacter pylori ribosomal protein L1 (RPL1) on its antimicrobial activity

HP (2-20) (AKKVFKRLEKLFSKIQNDK-NH₂) is an antibacterial 19-mer peptide derived from the N-terminal region of Helicobacter pylori ribosomal protein L1 (RPL1). Several truncated peptides were synthesized to investigate the effects of the N- or C-terminal regions of HP (2-20) on antimicrobial activity. The antimicrobial activity of the peptides was measured by their growth inhibitory effect upon Pseudomonas aeruginosa, Salmonella typhimurium, Saccharomyces cerevisae, Trichosporon beigelii and Candida albicans. Antimicrobial activity required a full length N-terminus. None of the peptides exhibited hemolytic activity against human erythrocyte cells. The membrane-disrupting activity of these peptides, using liposomes and 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe, confirmed that the full N-terminal region of HP (2-20) is a prerequisite for antibiotic activity and that this region may facilitate penetration of the cell membrane. Circular dichroism indicated that the -helical structure of the peptides important for antimicrobial activity.     

Preparation and characterization of novel bioactive peptides responsible for angiotensin I‐converting enzyme inhibition from wheat germ

Reported is the preparation of wheat germ (WG) hydrolyzate with potent angiotensin I‐converting enzyme (ACE) inhibitory activity, and the characterization of peptides responsible for ACE inhibition. Successful hydrolyzate with the most potent ACE inhibitory activity was obtained by 0.5 wt.%–8 h Bacillus licheniformis alkaline protease hydrolysis after 3.0 wt.%–3 h α‐amylase treatment of defatted WG (IC₅₀; 0.37 mg protein ml⁻¹). The activity of WG hydrolyzate was markedly increased by ODS and subsequent AG50W purifications (IC₅₀; 0.018 mg protein ml⁻¹). As a result of isolations by high performance liquid chromatographies, 16 peptides with the IC₅₀ value of less than 20 μm , composed of 2–7 amino acid residues were identified from the WG hydrolyzate. Judging from the high content (260 mg in 100 g of AG50W fraction) and powerful ACE inhibitory activity (IC₅₀; 0.48 μm ), Ile‐Val‐Tyr was identified as a main contributor to the ACE inhibition of the hydrolyzate. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Computational structure–activity study directs synthesis of novel antitumor enkephalin analogs

The capability of a Support Vector Machines QSAR model to predict the antiproliferative ability of small peptides was evaluated by screening a virtual library of enkephalin-like analogs modified by incorporation of the (R,S)-(1-adamantyl)glycine (Aaa) residue. From an initial set of 390 compounds, the peptides, Tyr-Aaa-Gly-Phe-Met (2), Tyr-Aaa-Gly-Phe-Phe (3), Phe-Aaa-Gly-Phe-Phe (4) and Phe-Aaa-Gly-Phe-Met (5) were selected, synthesized and their antitumor activity was tested and compared to that of Met-enkephalin (1). The antiproliferative activity correlated with the computational prediction and with the foldamer-forming ability of the studied peptides. The most active compounds were the hydrophobic peptides, Phe-Aaa-Gly-Phe-Phe (4) and Phe-Aaa-Gly-Phe-Met (5), having a greater propensity to adopt folded structures than the other peptides.     

Metalloproteases Secreted by <Emphasis Type="Italic">Actinobacillus suis</Emphasis>

Actinobacillus suis secretes metalloproteases into its medium. These secreted proteins, when concentrated by precipitation with 70% (NH₄)₂SO₄ or methanol, displayed proteolytic activity at >200 kDa molecular mass bands in 10% polyacrylamide gels copolymerized with bovine casein (1%). They showed activity in a broad pH range (from pH 5 to pH 10) and were inhibited by 20 mM EDTA or EGTA, but could be reactivated by calcium. They were found heat stable at 40°C, 50°C, 60°C, and 70°C, but their activity diminished at 80°C or higher. They degraded pig and bovine IgG and cross-reacted with a polyclonal serum against a high molecular mass secreted protease from A. pleuropneumoniae. Extracellular proteases could play a role in diseases caused by A. suis.     

Investigation of α/γ hybrid peptide self‐assembled structures with antimicrobial and antibiofilm properties

The present work describes the synthesis and characterization of α/γ hybrid peptides, Boc‐Phe‐γ⁴‐Phe‐Val‐OMe, P1 ; Boc‐Ala‐γ⁴‐Phe‐Val‐OMe, P2 ; and Boc‐Leu‐γ⁴‐Phe‐Val‐OMe, P3 together with the formation of self‐assembled structures formed by these hybrid peptides in dimethyl sulfoxide (DMSO)/water (1:1). The self‐assembled structures were characterized by infrared (IR) spectroscopy, circular dichroism (CD), and scanning electron microscopy (SEM). Further, α/γ hybrid peptide self‐assembled structures were evaluated for antibacterial properties. Among all, the self‐assembled peptide P1 exhibited the antimicrobial activity against Escherichia coli and Klebsiella pneumoniae, while self‐assembled peptide P3 inhibited the biofilms of Salmonella typhimurium and Pseudomonas aeruginosa. In this study, we have shown the significance of self‐assembled structures formed from completely hydrophobic α/γ hybrid peptides in exploring the antibacterial properties together with biofilm inhibition.     

Immunocytochemical identification of α-endorphin-like material in neurones of the brain and corpus cardiacum of the blowfly,Calliphora vomitoria (Diptera)

Summary A group of the 24–26 paraldehyde fuchsin-positive median neurosecretory cells (MNC) in the pars intercerebralis of the brain of the blowfly, Calliphora vomitoria, has shown immunoreactivity towards three different antibodies to -endorphin, a peptide that corresponds to the amino acid sequence present between residues 61 and 76 of the precursor molecule, -lipotropin (-LPH). The immunoreactive material could be followed in axons within the median bundle, the tract through which neurosecretory material from the MNC is passed down to the corpus cardiacum (CC). The -endorphin-immunoreactive material was observed leaving the CC in the cardiac-recurrent nerve, dorsal to the proventriculus, in the direction of the abdomen. The cells that contain the -endorphin-like material are different from those of the MNC that contain insulin-, pancreatic polypeptide-, and gastrin/CCK-like peptides. This finding demonstrates the considerable complexity and peptidergic nature of the MNC and constitutes further evidence that morphinomimetic-like peptides are present in the nervous system of invertebrates.     

Two antimicrobial and nematicidal peptides derived from sequences encoded Picea sitchensis

Two antimicrobial peptides (piceain 1 and 2) derived from sequences encoded Picea sitchensis are identified. Their amino acid sequences are KSLRPRCWIKIKFRCKSLKF and RPRCWIKIKFRCKSLKF, respectively. One intra‐molecular disulfide bridge is formed by these two half‐cysteines in both piceain 1 and 2. Antimicrobial activities of synthesized piceains against several kinds of microorganisms were tested. They showed antimicrobial activities against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and fungus Candida albicans but little antimicrobial activity against Bacillus subtilis. The results of nematicidal test showed they exerted strong nematicidal activities against Caenorhabditis elegans, following exposure for 5 h at concentrations as low as 10 µg/ml. They had weak hemolytic abilities against human and rabbit red cells. At the concentration of 250 µg/ml, they induced red cell hemolysis of less than 5%. Circular dichroism spectra of the two antimicrobial peptides were investigated in several solutions. Their main secondary structure components are β‐sheet and random. The current work provides a novel family of antimicrobial and nematicidal peptides with unique disulfided loop containing nine amino acid residues. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Breakdown of different peptides byPrevotella (Bacteroides) ruminicola and mixed microorganisms from the sheep rumen

Several di-, tri-, and oligopeptides were incubated individually in vitro with rumen fluid from two sheep receiving a mixed grass hay/concentrate diet and with washed cells ofPrevotella (formerlyBacteroides)ruminicola M384 andP. ruminicola B₁4. The rates of breakdown of most peptides were similar in the rumen fluid from the two sheep. Acidic and proline-containing peptides tended to be more slowly degraded than neutral or basic peptides. The dipeptide at the N-terminus of higher peptides was observed as an early product of hydrolysis, confirming that a dipeptidyl aminopeptidase type of activity was present. The relative rates of breakdown of dipeptides byP. ruminicola were different from that of rumen fluid, but the hydrolysis of higher peptides followed a similar pattern, and dipeptides from the N-terminus were detected as early products.     

Identifying N‐terminal peptides by a combination of the edman procedures with a bromine isotope tag: Application to the silicateins

Silicateins are proteins found within spicules of siliceous sponges. They are analogs of proteinases cathepsins; they catalyze the transformation of silicic acid esters into biogenic silica (SiO₂·nH₂O), and are believed to take part in the processes of silicification in marine and freshwater sponges. Earlier studies by Kalyuzhnaya et al. revealed that the Baikal Sponge Lubomirskia baicalensis Pallas, 1773 (L. baicalensis) contains a gene 1988 bp long, which hosts four sequences that encode four mRNAs giving rise to silicateins α1, α2, α3 and α4 (SILα1, SILα2, SILα3, SILα4) whose predicted amino acid sequences are similar to those of the predicted sequences of marine sponge silicateins. However, the sequences of mature silicateins of L. baicalensis remained unknown, since their N‐terminal peptides were not identified. We found the sequences of these N‐terminal peptides using a combination of the Edman procedure, which involved reaction with phenylisothiocyanate, treatment with trifluoroacetic acid and trypsinolysis followed by treatment with 4‐bromine‐phenylisothiocyanate performed directly within polyacrylamide gel bands, and subsequent mass spectrometry. The N‐terminal peptides are YAESIDWR (SILα1), YVDSIDWR (SILα2 and α4), and YADSLDWR (SILα3). All mature silicateins of L. baicalensis had a length 217 amino acid residues.     

Antimicrobial activity and structure of a consensus human β-defensin and its comparison to a novel putative hBD10

The spread of multidrug resistant bacteria owing to the intensive use of antibiotics is challenging current antibiotic therapies, and making the discovery and evaluation of new antimicrobial agents a high priority. The evaluation of novel peptide sequences of predicted antimicrobial peptides from different sources is valuable approach to identify alternative antibiotic leads. Two strategies were pursued in this study to evaluate novel antimicrobial peptides from the human β-defensin family (hBD). In the first, a 32-residue peptide was designed based on the alignment of all available hBD primary structures, while in the second a putative 35-residue peptide, hBD10, was mined from the gene DEFB110. Both hBDconsensus and hBD10 were chemically synthesized, folded and purified. They showed antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Mycobacterium tuberculosis, but were not hemolytic on human red blood cells. The NMR-based solution structure of hBDconsensus revealed that it adopts a classical β-defensin fold and disulfide connectivities. Even though the mass spectrum of hBD10 confirmed the formation of three disulfide bonds, it showed limited dispersion in ¹H NMR spectra and structural studies were not pursued. The evaluation of different β-defensin structures may identify new antimicrobial agents effective against multidrug-resistant bacterial strains.     

Debittering effect of Actinomucor elegans peptidases on soybean protein hydrolysates

Effects of the enzymes in Actinomucor elegans extract and the enzyme Alcalase 2.4L on debittering the soybean protein hydrolysates were investigated. When the protein was treated only with the latter, a strong bitterness formed; but it decreased if the protein was treated with both the enzymes. The more the enzymes were used, weaker was the bitterness tasted. SDS-PAGE profile and ESI-MS spectrum of the hydrolysates evidenced that the Alcalase could convert the protein into peptides rapidly, while the enzymes in the A. elegans extract were able to further degrade some peptides which were difficult or unable to be hydrolyzed by the Alcalase. Further systematic analysis of the peptidases showed that the Alcalase exhibited a significant endopeptidase activity towards NBZ-Phe-pNA substrate (p < 0.01), whereas many exopeptidases in the A. elegans extract had the carboxypeptidase activity towards N-CBZ-Ile-Leu (p < 0.01). It is concluded that those exopeptidases presented in the A. elegans extract can benefit by decreasing the bitterness of the soybean protein hydroysate. They are also capable of being used with the Alcalase in a single-step enzymatic reaction to prepare the bitterless protein hydrolysate, which may be an efficient application for food industry.