Isolation of antigen-specific, disulphide-rich knob domain peptides from bovine antibodies.

As a novel alternative to established surface display or combinatorial chemistry approaches for the discovery of therapeutic peptides, we present a method for the isolation of small, cysteine-rich domains from bovine antibody ultralong complementarity-determining regions (CDRs). We show for the first time that isolated bovine antibody knob domains can function as autonomous entities by binding antigen outside the confines of the antibody scaffold. This yields antibody fragments so small as to be considered peptides, each stabilised by an intricate, bespoke arrangement of disulphide bonds. For drug discovery, cow immunisations harness the immune system to generate knob domains with affinities in the picomolar to low nanomolar range, orders of magnitude higher than unoptimized peptides from naïve library screening. Using this approach, knob domain peptides that tightly bound Complement component C5 were obtained, at scale, using conventional antibody discovery and peptide purification techniques.

Identification and Characterization of Nematicidal Volatile Organic Compounds from Deep-Sea Virgibacillus dokdonensis MCCC 1A00493.

Root-knot nematode diseases cause severe yield and economic losses each year in global agricultural production. Virgibacillus dokdonensis MCCC 1A00493, a deep-sea bacterium, shows a significant nematicidal activity against Meloidogyne incognita in vitro. However, information about the active substances of V. dokdonensis MCCC 1A00493 is limited. In this study, volatile organic compounds (VOCs) from V. dokdonensis MCCC 1A00493 were isolated and analyzed through solid-phase microextraction and gas chromatography-mass spectrometry. Four VOCs, namely, acetaldehyde, dimethyl disulfide, ethylbenzene, and 2-butanone, were identified, and their nematicidal activities were evaluated. The four VOCs had a variety of active modes on M. incognita juveniles. Acetaldehyde had direct contact killing, fumigation, and attraction activities; dimethyl disulfide had direct contact killing and attraction activities; ethylbenzene had an attraction activity; and 2-butanone had a repellent activity. Only acetaldehyde had a fumigant activity to inhibit egg hatching. Combining this fumigant activity against eggs and juveniles could be an effective strategy to control the different developmental stages of M. incognita. The combination of direct contact and attraction activities could also establish trapping and killing strategies against root-knot nematodes. Considering all nematicidal modes or strategies, we could use V. dokdonensis MCCC 1A00493 to set up an integrated strategy to control root-knot nematodes.

Potentides: New Cysteine-Rich Peptides with Unusual Disulfide Connectivity from Potentilla anserina.

Cysteine-rich peptides (CRPs), which are disulfide-constrained peptides with 3 to 5 disulfide bonds and molecular weights of 2 to 6 kDa, are generally hyperstable and resistant to thermal, chemical, and enzymatic degradation. Herein, the discovery and characterization of a novel suite of CRPs, collectively named potentides pA1-pA16 from the root of the medicinal herb Potentilla anserina L, are described. Through a combination of proteomic and transcriptomic methods, it is shown that 35-residue potentide pA3, which is the most abundant member of potentides, exhibits high stability against heat, acidic, and proteolytic degradation. Transcriptomic analysis revealed that potentide precursor sequences contained four tandem repeats in the mature domain, which is the first report on tandem repeats being found in the Rosaceae family. Disulfide mapping showed that potentide pA3 displayed a novel disulfide connectivity of C1-C3, C2-C6, and C4-C5; a cystine motif that has not been reported in plant CRPs. Transcriptomic data mining and a neighbor-joining clustering analysis revealed 56 potentide homologues and their distribution in the families of Rosaceae and Ranunculaceae in angiosperm. Altogether, these results reveal a new plant CRP structure with an unusual cystine connectivity. Additionally, this study expands the families and structure diversity of CRPs as potentially active peptide pharmaceuticals.

Identification and Allelopathy of Green Garlic (Allium sativum L.) Volatiles on Scavenging of Cucumber (Cucumis sativus L.) Reactive Oxygen Species.

Garlic and formulations containing allicin are used widely as fungicides in modern agriculture. However, limited reports are available on the allelopathic mechanism of green garlic volatile organic compounds (VOCs) and its component allelochemicals. The aim of this study was to investigate VOCs of green garlic and their effect on scavenging of reactive oxygen species (ROS) in cucumber. In this study, green garlic VOCs were collected by HS-SPME, then analyzed by GS-MS. Their biological activity were verified by bioassays. The results showed that diallyl disulfide (DADS) is the main allelochemical of green garlic VOCs and the DADS content released from green garlic is approximately 0.08 mg/g. On this basis, the allelopathic effects of green garlic VOCs in vivo and 1 mmol/L DADS on scavenging of ROS in cucumber seedlings were further studied. Green garlic VOCs and DADS both reduce superoxide anion and increase the accumulation of hydrogen peroxide of cucumber seedlings. They can also regulate active antioxidant enzymes (SOD, CAT, POD), antioxidant substances (MDA, GSH and ASA) and genes (CscAPX, CsGPX, CsMDAR, CsSOD, CsCAT, CsPOD) responding to oxidative stress in cucumber seedlings.

Diallyl Disulfide Induces Apoptosis and Autophagy in Human Osteosarcoma MG-63 Cells through the PI3K/Akt/mTOR Pathway.

Diallyl disulfide (DADs), a natural organic compound, is extracted from garlic and scallion and has anti-tumor effects against various tumors. This study investigated the anti-tumor activity of DADs in human osteosarcoma cells and the mechanisms. MG-63 cells were exposed to DADs (0, 20, 40, 60, 80, and 100 µM) for different lengths of time (24, 48, and 72 h). The CCK8 assay results showed that DADs inhibited osteosarcoma cell viability in a dose-and time-dependent manner. FITC-Annexin V/propidium iodide staining and flow cytometry demonstrated that the apoptotic ratio increased and the cell cycle was arrested at the G2/M phase as the DADs concentration was increased. A Western blot analysis was employed to detect the levels of caspase-3, Bax, Bcl-2, LC3-II/LC3-I, and p62 as well as suppression of the mTOR pathway. High expression of LC3-II protein revealed that DADs induced formation of autophagosome. Furthermore, DADs-induced apoptosis was weakened after adding 3-methyladenine, demonstrating that the DADs treatment resulted in autophagy-mediated death of MG-63 cells. In addition, DADs depressed p-mTOR kinase activity, and the inhibited PI3K/Akt/mTOR pathway increased DADs-induced apoptosis and autophagy. In conclusion, our results reveal that DADs induced G2/M arrest, apoptosis, and autophagic death of human osteosarcoma cells by inhibiting the PI3K/Akt/mTOR signaling pathway.

Biochemical and Anti-Triple Negative Metastatic Breast Tumor Cell Properties of Psammaplins.

Breast tumors reprogram their cellular metabolism, nutrient uptake, and utilization-associated biochemical processes. These processes become further transformed as genetically predisposed metastatic breast tumor cells colonize specific organs. Breast tumor cells often metastasize to the brain, bone, lung and liver. Massagué and colleagues isolated organotropic subclones and established organ-specific gene signatures associated with lung-, bone-, and brain-specific metastatic triple-negative breast cancer (TNBC) MDA-MB-231 cells. Using these genetically characterized metastatic subclones specific to lung (LM4175), bone (BoM1833), and brain (BrM-2a), we evaluated marine natural products for the ability to differentially suppress metastatic breast cancer cells in a target organ-dependent manner. Psammaplin-based histone deacetylase (HDAC) inhibitors were found to differentially inhibit HDAC activity, induce activation of hypoxia-inducible factor-1 (HIF-1), and disrupt organotropic metastatic TNBC subclone growth. Further, psammaplins distinctly suppressed the outgrowth of BoM1833 tumor spheroids in 3D-culture systems. Similar results were observed with the prototypical HDAC inhibitor trichostatin A (TSA). These organotropic tumor cell-based studies suggest the potential application of HDAC inhibitors that may yield new directions for anti-metastatic breast tumor research and drug discovery.

Disulfides with Anti-inflammatory Activity from the Brown Alga Dictyopteris membranacea.

Six new (1, 2, and 4-7) and two previously reported (3 and 8) disulfides, along with 4-butyl-2,6-cycloheptadienone, γ-tocopherol, and δ-tocopherol, were isolated from an organic extract of the brown alga Dictyopteris membranacea, collected at Gerolimenas Bay, Greece. The structure elucidation of the isolated natural products was based on analysis of their spectroscopic data. Compounds 1, 3-6, and 8 were evaluated for their antibacterial and anti-inflammatory activities. None of the compounds displayed antibacterial activity against two resistant strains of Staphylococcus aureus and one strain of Escherichia coli. In contrast, metabolite 5 was able to cause strong inhibition of NO production with an IC50 value of 3.8 µM using an LPS stimulation assay.

Tumor Protein (TP)-p53 Members as Regulators of Autophagy in Tumor Cells upon Marine Drug Exposure.

Targeting autophagic pathways might play a critical role in designing novel chemotherapeutic approaches in the treatment of human cancers, and the prevention of tumor-derived chemoresistance. Marine compounds were found to decrease tumor cell growth in vitro and in vivo. Some of them were shown to induce autophagic flux in tumor cells. In this study, we observed that the selected marine life-derived compounds (Chromomycin A2, Psammaplin A, and Ilimaquinone) induce expression of several autophagic signaling intermediates in human squamous cell carcinoma, glioblastoma, and colorectal carcinoma cells in vitro through a transcriptional regulation by tumor protein (TP)-p53 family members. These conclusions were supported by specific qPCR expression analysis, luciferase reporter promoter assay, and chromatin immunoprecipitation of promoter sequences bound to the TP53 family proteins, and silencing of the TP53 members in tumor cells.

Top-down Targeted Metabolomics Reveals a Sulfur-Containing Metabolite with Inhibitory Activity against Angiotensin-Converting Enzyme in Asparagus officinalis.

The discovery of bioactive natural compounds containing sulfur, which is crucial for inhibitory activity against angiotensin-converting enzyme (ACE), is a challenging task in metabolomics. Herein, a new S-containing metabolite, asparaptine (1), was discovered in the spears of Asparagus officinalis by targeted metabolomics using mass spectrometry for S-containing metabolites. The contribution ratio (2.2%) to the IC50 value in the crude extract showed that asparaptine (1) is a new ACE inhibitor.

Investigating the responses of Cronobacter sakazakii to garlic-drived organosulfur compounds: a systematic study of pathogenic-bacterium injury by use of high-throughput whole-transcriptome sequencing and confocal micro-raman spectroscopy.

We present the results of a study using high-throughput whole-transcriptome sequencing (RNA-seq) and vibrational spectroscopy to characterize and fingerprint pathogenic-bacterium injury under conditions of unfavorable stress. Two garlic-derived organosulfur compounds were found to be highly effective antimicrobial compounds against Cronobacter sakazakii, a leading pathogen associated with invasive infection of infants and causing meningitis, necrotizing entercolitis, and bacteremia. RNA-seq shows changes in gene expression patterns and transcriptomic response, while confocal micro-Raman spectroscopy characterizes macromolecular changes in the bacterial cell resulting from this chemical stress. RNA-seq analyses showed that the bacterial response to ajoene differed from the response to diallyl sulfide. Specifically, ajoene caused downregulation of motility-related genes, while diallyl sulfide treatment caused an increased expression of cell wall synthesis genes. Confocal micro-Raman spectroscopy revealed that the two compounds appear to have the same phase I antimicrobial mechanism of binding to thiol-containing proteins/enzymes in bacterial cells generating a disulfide stretching band but different phase II antimicrobial mechanisms, showing alterations in the secondary structures of proteins in two different ways. Diallyl sulfide primarily altered the α-helix and ß-sheet, as reflected in changes in amide I, while ajoene altered the structures containing phenylalanine and tyrosine. Bayesian probability analysis validated the ability of principal component analysis to differentiate treated and control C. sakazakii cells. Scanning electron microscopy confirmed cell injury, showing significant morphological variations in cells following treatments by these two compounds. Findings from this study aid in the development of effective intervention strategies to reduce the risk of C. sakazakii contamination in the food production environment and on food contact surfaces, reducing the risks to susceptible consumers.

Enhancement of intestinal IgA production by Ajoene in mice.

We investigated the effects of ajoene on intestinal IgA production. Ajoene (1.35, 4.5, and 13.5 µg/kg/d) was administered to mice for 4 weeks. The fecal IgA level in the 13.5 µg/kg/d group increased after 3 weeks. The intestinal IgA level also increased in a dose-dependent manner upon ajoene administration. An oil-macerated garlic extract, with 1500 µg/g of ajoene, enhanced the intestinal IgA production.

Cyclic sulfoxides garlicnins B2, B3, B4, C2, and C3 from Allium sativum.

Several novel sulfides, called garlicnins B2 (1), B3 (2), B4 (3), C2 (4), and C3 (5), were isolated from acetone extracts of garlic, Allium sativum L. and characterized. These garlicnins are capable of suppressing M2 macrophage activation and they have a novel skeleton of cyclic sulfoxide. The structures of the former 3 and latter of 2 were deduced to be 2-(sulfenic acid)-5-(allyl)-3,4-dimethyltetrahydrothiophene-S-oxides and 2-(allyldithiine)-5-(propenylsulfoxide)-3,4-dimethyltetrahydrothiophene-S-oxides, respectively. The mechanism of the proposed production of these compounds is discussed. The identification of these novel sulfoxides from garlic accumulates a great deal of new chemistry in the Allium sulfide field, and future pharmacological investigations of these compounds will aid the development of natural, healthy foods and anti-cancer agents that may prevent or combat disease.

Ajoene, a sulfur-rich molecule from garlic, inhibits genes controlled by quorum sensing.

In relation to emerging multiresistant bacteria, development of antimicrobials and new treatment strategies of infections should be expected to become a high-priority research area. Quorum sensing (QS), a communication system used by pathogenic bacteria like Pseudomonas aeruginosa to synchronize the expression of specific genes involved in pathogenicity, is a possible drug target. Previous in vitro and in vivo studies revealed a significant inhibition of P. aeruginosa QS by crude garlic extract. By bioassay-guided fractionation of garlic extracts, we determined the primary QS inhibitor present in garlic to be ajoene, a sulfur-containing compound with potential as an antipathogenic drug. By comprehensive in vitro and in vivo studies, the effect of synthetic ajoene toward P. aeruginosa was elucidated. DNA microarray studies of ajoene-treated P. aeruginosa cultures revealed a concentration-dependent attenuation of a few but central QS-controlled virulence factors, including rhamnolipid. Furthermore, ajoene treatment of in vitro biofilms demonstrated a clear synergistic, antimicrobial effect with tobramycin on biofilm killing and a cease in lytic necrosis of polymorphonuclear leukocytes. Furthermore, in a mouse model of pulmonary infection, a significant clearing of infecting P. aeruginosa was detected in ajoene-treated mice compared to a nontreated control group. This study adds to the list of examples demonstrating the potential of QS-interfering compounds in the treatment of bacterial infections.

Effect of allyl sulfides from garlic essential oil on intracellular ca2+ levels in renal tubular cells.

Diallyl sulfide (1), diallyl disulfide (2), and diallyl trisulfide (3), which are major organosulfur compounds of garlic (Allium sativum), are recognized as a group of potential chemopreventive compounds. In this study, the early signaling effects of 3 were examined on Madin-Darby canine kidney (MDCK) cells loaded with the Ca(2+)-sensitive dye fura-2. It was found that 3 caused an immediate and sustained increase of [Ca(2+)](i) in a concentration-dependent manner (EC(50) = 40 µM). Compound 3 also induced a [Ca(2+)](i) elevation when extracellular Ca(2+) was removed, but the magnitude was reduced by 45%. In Ca(2+)-free medium, the 3-induced [Ca(2+)](i) level was abolished by depleting stored Ca(2+) with 1 µM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor). Elevation of [Ca(2+)](i) caused by 3 in the Ca(2+)-containing medium was not affected by modulation of protein kinase C activity. The 3-induced Ca(2+) influx was inhibited by nifedipine and nicardipine (1 µM). U73122, an inhibitor of phospholipase C, abolished ATP (but not the 3-induced [Ca(2+)](i) level). These findings suggest that 3 induced a significant [Ca(2+)](i) elevation in MDCK renal tubular cells by stimulating both extracellular Ca(2+) influx and thapsigargin-sensitive intracellular Ca(2+) release via as yet unidentified mechanisms. Furthermore, the order of the allyl sulfide-induced [Ca(2+)](i) elevation and cell viability was 1 < 2 < 3. The differential effect of allyl sulfides on Ca(2+) signaling and cell death appears to correlate with the number of sulfur atoms in the structure of these allyl sulfides.

Investigation of volatiles emitted from freshly cut onions (Allium cepa L.) by real time proton-transfer reaction-mass spectrometry (PTR-MS).

Volatile organic compounds (VOCs) in cut onions (Allium cepa L.) were continuously measured by PTR-MS during the first 120 min after cutting. The headspace composition changed rapidly due to the very reactive volatile sulfurous compounds emitted from onion tissue after cell disruption. Mass spectral signals corresponding to propanethial S-oxide (the lachrymatory factor) and breakdown products of this compound dominated 0-10 min after cutting. Subsequently, propanethiol and dipropyl disulfide predominantly appeared, together with traces of thiosulfinates. The concentrations of these compounds reached a maximum at 60 min after cutting. Propanethiol was present in highest concentrations and had an odor activity value 20 times higher than dipropyl disulfide. Thus, propanethiol is suggested to be the main source of the characteristic onion odor. Monitoring the rapid changes of VOCs in the headspace of cut onion necessitates a high time resolution, and PTR-MS is demonstrated to be a very suitable method for monitoring the headspace of freshly cut onions directly after cutting without extraction or pre-concentration.

Studies on purification of allicin by molecular distillation.

BACKGROUND: With recent improvements in living standards, people have been giving more consideration to the healthcare effects of foods. In this respect, allicin, which is the most important organosulfur compound in garlic and plays a key role in physiological function, has been receiving much attention. RESULTS: Allicin obtained from garlic by supercritical CO(2) extraction was purified by molecular distillation (MD). The effects of operating conditions such as absolute pressure (AP), distillation temperature (DT) and feed flow rate (FFR) on allicin purity and yield were studied. The optimal AP, DT and FFR levels in first-stage MD were found to be 200 Pa, 50 °C and 15 mL min(-1) respectively. After three stages of MD the contents of allicin, diallyl disulfide (DADS) and diallyl trisulfide (DATS) were 68.04, 9.19 and 5.91% (w/w) respectively. CONCLUSION: This study has provided a safe and effective method for the purification of allicin.

Removal of odorant dimethyl disulfide under alkaline and neutral conditions in biotrickling filters.

The aim of this paper was to evaluate the performance of biotrickling filters (BTFs) for treating low concentrations of dimethyl disulfide (DMDS), using different bacterial consortia adapted to consume reduced sulfur compounds under alkaline (pH ≈ 10) or neutral (pH ≈ 7) conditions. Solubility experiments indicated that the partition of DMDS in neutral and alkaline mineral media was similar to the value with distilled water. Respirometric assays showed that oxygen consumption was around ten times faster in the neutrophilic as compared with the alkaliphilic consortium. Batch experiments demonstrated that sulfate was the main product of the DMDS degradation. Two laboratory-scale BTFs were implemented for the continuous treatment of DMDS in both neutral and alkaline conditions. Elimination capacities of up to 17 and 24 g(DMDS) m(-3) h(-1) were achieved for the alkaliphilic and neutrophilic reactors with 100% removal efficiency after an initial adaptation and biomass build-up.

Identification of disulfide bonds in protein proteolytic degradation products using de novo-protein unique sequence tags approach.

Disulfide bonds are a form of post-translational modification that often determines protein structure(s) and function(s). In this work, we report a mass spectrometry method for identification of disulfides in degradation products of proteins, specifically endogenous peptides in the human blood plasma peptidome. LC-Fourier transform tandem mass spectrometry (FT MS/MS) was used for acquiring mass spectra that were de novo sequenced and then searched against the IPI human protein database. Through the use of unique sequence tags (UStags), we unambiguously correlated the spectra to specific database proteins. Examination of the UStags' prefix and/or suffix sequences that contain cysteine(s) in conjunction with sequences of the UStags-specified database proteins is shown to enable the unambigious determination of disulfide bonds. Using this method, we identified the intermolecular and intramolecular disulfides in human blood plasma peptidome peptides that have molecular weights of up to approximately 10 kDa.

Cloning, overexpression, folding and purification of a biosynthetically derived three disulfide scorpion toxin (BTK-2) from Mesobuthus tamulus.

BTK-2, a 32 residue scorpion toxin initially identified in the venom of red Indian scorpion Mesobuthus tamulus was cloned, overexpressed and purified using Cytochrome b(5) fusion protein system developed in our laboratory. The synthetic gene coding for the peptide was designed taking into account optimal codon usage by Escherichia coli. High expression levels of the fusion protein enabled facile purification of this peptide. The presence of disulfide bonded isomers, occurring as distinctly populated states even in the fusion protein, were separated by gel filtration chromatography. The target peptide was liberated from the host protein by Tev protease cleavage and subsequent purification was achieved using RP-HPLC methods. Reverse phase HPLC clearly showed the presence of at least two isomeric forms of the peptide that were significantly populated. The oxidative folding of BTK-2 was achieved under ambient conditions during the course of purification. Structural characterization of the two forms, by solution homonuclear and heteronuclear NMR methods, has shown that these two forms exhibit significantly different structural properties, and represent the natively folded and a "misfolded" form of the peptide. The formation of properly folded BTK-2 as a major fraction without the use of in vitro oxidative refolding methods clearly indicate the versatility of the Cytochrome b(5) fusion protein system for the efficient production of peptides for high resolution NMR studies.

New sesquiterpene coumarins from the roots of Ferula flabelliloba.

Three new sesquiterpene coumarins, namely, farnesiferone B (1), flabellilobin A (2) and flabellilobin B (3), together with nine known compounds, ligupersin A, 7-epi-gamma-eudesmol, persicasulfide A, conferdione, umbelliprenin, conferone, feselol, lehmferin and farnesiferol B were isolated from the roots of Ferula flabelliloba Rech. f. & Aell. (Apiaceae). The structures of these compounds were elucidated by various 1- and 2-D NMR techniques as well as HREIMS.