Indiana University-Purdue University Indianapolis (IUPUI) / Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, β-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. Mutants with deletions in two PEA transferase genes were significantly more susceptible to β-defensins, and the triple mutant was significantly more susceptible to both α- and β-defensins, but not LL-37; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface, suggesting these three genes contribute to the addition of positively charged moieties on the cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdtA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin may be more important than defensin resistance to H. ducreyi pathogenesis.

Pin1 is a Prolyl Isomerase that catalyzes cis-trans isomerization of peptides with pSer/Thr-Pro motifs in many cell signaling proteins. This conformational switch is implicated in diseases. Pin1 activity is considered a target for therapeutic applications. Pin1 targets motifs by its N-terminal WW-binding domain. A C-terminal PPIase domain is responsible for catalysis. To understand how Pin1 coordinates its enzymatic activities, it is necessary to probe how the domains behave in the presence of substrates. Here, we used novel (Histone H1 and Sic1) and other existing peptides to characterize the dynamics of Pin1 and impact of substrate binding on inter-domain interactions. Pin1- peptide complexes have been used to show that peptide addition causes a conformational change in the two domains. 15N-relaxation data suggest that the flexibility of these domains depends on the substrate peptide We have constructed a hypothesis about which substrate residues may be important for conferring tight binding and inter-domain interactions.

Lentil, black soybean and black turtle have been proved to be phenolic-rich legume varieties and possess higher antioxidant activity. In this study, the three legume varieties were subjected to broad range of processing conditions, and the effects on phenolic contents, antioxidant capacity and individual phenolic acid were investigated. The results showed all processing methods could decrease the total phenolic content, and steaming processing could preserve more phenolics and antioxidant activity than boiling processing. Phenolic acids mainly existed in nonree form and the content of individual free phenolic acids was dependent on the thermal process applied. When in vitro gastrointestinal simulation digestion was applied to the thermally processed beans, it was found that the properties of hydrolysates including total phenolic content, antioxidant activity, degree of hydrolysis, and ACE (angiotensin converting enzyme) inhibitory activity were all affected by thermal conditions employed. There was a weak correlation between the degree of hydrolysis and ACE inhibition. In the current study, for each legume variety, cooking conditions which yielded the highest phenolic content and antioxidant activity were selected. Phenolics of the raw and cooked seeds from each legume variety were extracted, semi-purified (XAD-7) and further fractionated (Sephadex LH-20). The results showed cooking had great effects on yield, phenolic content, antioxidant capacity, and individual phenolic compounds. The phenolic content and antioxidant activity could be enriched tremendously in the semi-purified extracts and some fractions. Some phenolic compounds which were absent in raw material could be found after cooking in the fractions and some phenolic compounds which were present in raw material disappeared after cooking. Among crude phenolic extracts, semi-purified extracts and fractions, only crude extracts showed ACE inhibition. In addition, protein isolates from the legumes varieties were treated with in vitro GI (gastrointestinal) digestion and then separated by ultrafiltration, DEAE anion exchange chromatography and gel permeation chromatography. After ultrafiltration, the lowest molecular weight fraction (< 3kD) had the highest ACE inhibition and the three legume varieties showed different peptide distribution, ACE inhibition, and antioxidant profile in the hydrolysates. Gel filtration chromatography further revealed that the most potent ACE inhibitors were peptides of 2-5 amino acids long.

Self-assembly is an autonomous process where components organize themselves into structures via noncovalent interactions without human intervention. Ultrashort amphiphilic peptides are typical self-assembly molecules with specific sequence motifs which consist of three to seven amino acids. Due to their amphiphilic structure which carries a dominant hydrophobic tail and a polar head group, these peptides can self-assemble to construct nanofibrous scaffolds system to form hydrogels, organogels or aerogels. The nanofibrous scaffolds formed by amphiphilic peptides are very similar to the fiber structure found in collagen which plays an essential role in extracellular matrix showing the potential of applying these peptide scaffolds together in culturing native human cells. Thus the derivate of amphiphilic peptides depsipeptide in which we replaced one amide bond with an ester bond is also worthwhile to explore a novel penitential material for Tissue Engineering. At the same time, because of the perfect biocompatibility of amphiphilic peptides made up of natural l-amino acids and also the excellent gelation properties providing a solution for zinc dendrite growth in Zn batteries, it will be also meaningful to combine the rationally designed peptide gelation system to Zn batteries. This dissertation describes how to characterize and use ultrashort amphiphilic depsipeptide for tissue engineering and use ultrashort amphiphilic peptide for the electrolyte of Zn batteries. The first chapter provides us with an introduction to self-assembly material, 3D bioprinting, and Zn batteries. The second chapter introduces a novel method to synthesize the depsipeptide fully based on solid phase peptide synthesis (SPPS) and also shows the different properties, especially the gelation behavior by clarifying its mechanism via doing the characterization of depsipeptide. At the end of the second chapter, depsipeptide is proved to be a potential material in 3D bioprinting. The third chapter reveals how we synthesized and characterized the amphiphilic peptide and applied it to the Zn batteries. The cycling stability got promoted compared with bard Zn batteries in symmetrical Zn-Zn cells while the formation of Zn dendrite was also suppressed. The promising results suggest peptide gelation systems are promising electrolytes for use in Zn batteries.

In this investigation, venom from the giant Neotropical hunting ant Dinoponera australis (Order: Hymenoptera) has been harvested and subjected to chromatographic separations for the purpose of elucidating possible peptides that display neuroactivity by bioassay guided venom fractionation (BGVF). The venom of this arcane solitary predator paralyzes small invertebrate prey and causes highly exaggerated pain in large vertebrates. The hypothesis that the venom has a peptide component highly effective in modulating neuronal conduction by depolarization of cellular membranes has been tested and subsequent biochemical characterization has been performed to elucidate the primary structure. The data suggests that the modulation of neuronal conduction appears to result from the formation of a de novo pore that allows non-selective ion movements in a concentration dependent manner. The venom contains a variety of proteinaceous candidates and one particular peptide from the venom, -Dinoponeratoxin Da-1837, has been observed to cause very fast, large and sustained depolarization in two types of normally quiescent peripheral neurons (primary cultures of trigeminal and dorsal root ganglia) in whole cell patch clamp recordings. The profound depolarization is due to non-selective cationic flux which is irreversible at high concentrations. Preliminary studies suggest that the peptide also has a minor inhibitory effect on voltage-gated sodium channels, which does not contribute to the depolarization. Membrane assays with microsomes, fluorescent probes and lipid bilayers confirmed peptide-induced non-selective and concentration dependent permeabilization of the membrane. The primary structure of the peptide was determined by iterations of product ion scans in multiple configurations utilizing high resolution tandem mass spectrometry, commonly referred to as MS-MS data dependent acquisition. -DpTx Da-1837 is an eighteen residue peptide that is highly hydrophobic, positively charged at physiological pH and has one atypical post translational modification, i.e. C-terminal peptidyl-lysine. The authentication of the toxin was confirmed by the successful solid phase synthesis of an analog that showed neither biochemical nor physiological variation from the properties of the peptide isolated from Dinoponera australis. The conclusion of this study was the creation of derivative analogs that provide the platform for the first fundamental step in drug discovery: establishing the structure-function relationship. Although the purpose of these cytolytic peptides in venom may be to capture prey or discourage predation, the discoveries of new molecules that affect cell viability by interactions with the cellular envelope provide the genesis for studies of targeted cell death. As a novel anti-microbial agent or as a potent tumor suppressor, the development of peptide derivatives could also help direct the development of new therapeutic interventions.

Retrocyclin-101 (RC101) and Protegrin-1 (PG1) are two important antimicrobial peptides that can be used as therapeutic agents against bacterial and/or viral infections, especially those caused by the HIV-1 or sexually-transmitted bacteria. Because of their antimicrobial activity and complex secondary structures, they have not yet been produced in microbial systems and their chemical synthesis is prohibitively expensive. Therefore, we created chloroplast transformation vectors with the RC101 or PG1 coding sequence, fused with GFP to confer stability, furin or Factor Xa cleavage site to liberate the mature peptide from their fusion proteins and a His-tag to aid in their purification. Stable integration of RC-101 into the tobacco chloroplast genome and homoplasmy were confirmed by Southern blots. RC-101 and PG1 accumulated up to 32-38% and 17~26% of the total soluble protein. Both RC-101 and PG1 were cleaved from GFP by corresponding proteases in vitro and Factor Xa like protease activity was observed within chloroplasts. Confocal microscopy studies showed location of GFP fluorescence within chloroplasts. Organic extraction resulted in 10.6 fold higher yield of RC 101 than purification by affinity chromatography using His-tag. In planta bioassays with Erwinia carotovora confirmed the antibacterial activity of RC101 and PG1 expressed in chloroplasts. RC101 transplastomic plants were resistant to TMV infections, confirming antiviral activity. Because RC101 and PG1 have not yet been produced in other cell culture or microbial systems, chloroplasts can be used as bioreactors for producing these proteins. Adequate yield of purified antimicrobial peptides from transplastomic plants should facilitate further pre-clinical studies

No description available.

No description available.

The structural requirements for the binding of dynorphin to the kappa opioid receptor are of profound clinical interest in the search for a powerful non-addictive analgesic. These requirements are thought to be met by the membrane-mediated conformation of the opioid peptide dynorphin A-(1-13}, Tyr¹-Gly²-Gly³-Phe⁴-Leu⁵-Arg⁶-Arg⁷-Ile⁸-Arg⁹-Pro¹⁰-Lys¹¹-Leu¹²-Lys¹³. Schwyzer [𝘉𝘪𝘰𝘤𝘩𝘦𝘮𝘪𝘴𝘵𝘳𝘺 25: 4281-4286 (1986)] has proposed an essentially α-helical membrane-mediated conformation of the tridecapeptide. In the present study, the hydrophobic moment, the helix probability and a four -state secondary structure prediction were computed. They signified, in agreement with circular dichroism (CD) studies on phospholipid-bound dynorphin A-(1-13)-tridecapeptide, negligible helical content of the peptide. CD studies demonstrated that the aqueous-membraneous interphase can be mimicked by methanol. The 500 and 620 MHz ¹H nuclear magnetic resonance (NMR) spectra of dynorphin A-(1-13) in methanolic solution were sequence-specifically assigned with the aid of correlated spectroscopy (COSY), double-quantum filtered phase-sensitive COSY, relayed COSY (RELAY) and nuclear Overhauser enhancement spectroscopy (NOESY). 2-D CAMELSPIN/ROESY experiments indicated that at least the part of the molecule from Arg⁷ to Arg⁹ was in an extended or β-strand conformation, which was in line with deuterium exchange and temperature dependence studies of the amide protons. ¹³C_α spin-lattice relaxation rate constants indicated a non-rigid backbone conformation. Transferred nuclear Overhauser effect studies on aqueous systems containing dynorphin A-(1-13) in the presence of dimyristoyl-phosphatidylcholine bilayers indicated a folded conformation from Tyr¹ to Leu⁵. The findings were incorporated into a tentative molecular model, which also indicated a non-helical, non-extended conformation for the rest of the molecule in the presence of corresponding distance-restrained negative charges. / Thesis / Master of Science (MSc)

The mechanisms which cause post exercise hypotension (a phenomenon of prolonged, decreased resting blood pressure following physical exertion) are unknown. Atrial natriuretic peptide (ANP) is known to exert potent natriuretic and vasodilatory properties which play an integral role in fluid regulation and blood pressure control. Elevations in plasma ANP concentration have been shown to occur during dynamic endurance exercise, and to a lesser extent during heavy resistance exercise. The purposes of this investigation were to 1) examine the effects of resistance and endurance exercise on the release of ANP, 2) examine the effects of resistance and endurance exercise on post exercise blood pressure and 3) evaluate the potential correlations of ANP release with any observed changes. Thirteen males (24.3±2.4yrs.) performed 15 min of unilateral leg press (65% 1 RM) and, one week later ~15 min (based on summed cardiac cycles of the resistance trial) of cycle ergometry (65% V0₂ ₚₑₐₖ). Blood pressure was measured using an intra-arterial catheter during exercise and for 1 h post exercise. Arterial blood was drawn at rest, 5, 10 and 15 min of exercise and 1 1/2, 3, 5, 10, 15, 30, 45 and 60 min post exercise for subsequent analysis of hematocrit and αANP. No differences occurred in blood pressure responses between trials, but significant decrements in blood pressure occurred post exercise compared to pre exercise. Systolic pressure was ~20mmHg lower from 10 min post exercise until measurements terminated at 60 min post exercise. Mean pressure was also significantly attenuated by ~7 mmHg from 30 min post exercise onwards. Only slight (non significant) elevations in αANP concentration were detected immediately following exercise with no elevation present by 5 min post exercise. It was concluded that post exercise hypotension occurs with acute bouts of either resistance or endurance exercise and that αANP does not appear to be directly related to this hypotensive effect. This study was supported by the Natural Sciences and Engineering Research Council of Canada / Thesis / Master of Science (MSc)