A Combined Molecular Cloning and Mass Spectrometric Method to Identify, Characterize, and Design Frenatin Peptides from the Skin Secretion of Litoria infrafrenata

Amphibian skin secretions are unique sources of bioactive molecules, particularly bioactive peptides. In this study, the skin secretion of the white-lipped tree frog (Litoria infrafrenata) was obtained to identify peptides with putative therapeutic potential. By utilizing skin secretion-derived mRNA, a cDNA library was constructed, a frenatin gene was cloned and its encoded peptides were deduced and confirmed using RP-HPLC, MALDI-TOF and MS/MS. The deduced peptides were identified as frenatin 4.1 (GFLEKLKTGAKDFASAFVNSIKGT) and a post-translationally modified peptide, frenatin 4.2 (GFLEKLKTGAKDFASAFVNSIK.NH2). Antimicrobial activity of the peptides was assessed by determining their minimal inhibitory concentrations (MICs) using standard model microorganisms. Through studying structure–activity relationships, analogues of the two peptides were designed, resulting in synthesis of frenatin 4.1a (GFLEKLKKGAKDFASALVNSIKGT) and frenatin 4.2a (GFLLKLKLGAKLFASAFVNSIK.NH2). Both analogues exhibited improved antimicrobial activities, especially frenatin 4.2a, which displayed significant enhancement of broad spectrum antimicrobial efficiency. The peptide modifications applied in this study, may provide new ideas for the generation of leads for the design of antimicrobial peptides with therapeutic applications.

Gene therapy tools: oligonucleotides and peptides

Genetic mutations can cause a wide range of diseases, e.g. cancer. Gene therapy has the potential to alleviate or even cure these diseases. One of the many gene therapies developed so far is RNA-cleaving deoxyribozymes, short DNA oligonucleotides that specifically bind to and cleave RNA. Since the development of these synthetic catalytic oligonucleotides, the main way of determining their cleavage kinetics has been through the use of a laborious and error prone gel assay to quantify substrate and product at different time-points. We have developed two new methods for this purpose. The first one includes a fluorescent intercalating dye, PicoGreen, which has an increased fluorescence upon binding double-stranded oligonucleotides; during the course of the reaction the fluorescence intensity will decrease as the RNA is cleaved and dissociates from the deoxyribozyme. A second method was developed based on the common denominator of all nucleases, each cleavage event exposes a single phosphate of the oligonucleotide phosphate backbone; the exposed phosphate can simultaneously be released by a phosphatase and directly quantified by a fluorescent phosphate sensor. This method allows for multiple turnover kinetics of diverse types of nucleases, including deoxyribozymes and protein nucleases. The main challenge of gene therapy is often the delivery into the cell. To bypass cellular defenses researchers have used a vast number of methods; one of these are cell-penetrating peptides which can be either covalently coupled to or non-covalently complexed with a cargo to deliver it into a cell. To further evolve cell-penetrating peptides and understand how they work we developed an assay to be able to quickly screen different conditions in a high-throughput manner. A luciferase up- and downregulation experiment was used together with a reduction of the experimental time by 1 day, upscaling from 24- to 96-well plates and the cost was reduced by 95% compared to commercially available assays. In the last paper we evaluated if cell-penetrating peptides could be used to improve the uptake of an LNA oligonucleotide mimic of GRN163L, a telomerase-inhibiting oligonucleotide. The combination of cell-penetrating peptides and our mimic oligonucleotide lead to an IC50 more than 20 times lower than that of GRN163L.

Novel inhibitors of the tRNA-dependent amidotransferase of "Helicobacter pylori": Peptides generated by phage display and dipeptide-like compounds

Cette thèse présente la découverte de nouveaux inhibiteurs de l’amidotransférase ARNt-dépendante (AdT), et résume les connaissances récentes sur la biosynthèse du Gln-ARNtGln et de l’Asn-ARNtAsn par la voie indirecte chez la bactérie Helicobacter pylori. Dans le cytoplasme des eucaryotes, vingt acides aminés sont liés à leur ARNt correspondant par vingt aminoacyl-ARNt synthétases (aaRSs). Ces enzymes sont très spécifiques, et leur fonction est importante pour le décodage correct du code génétique. Cependant, la plupart des bactéries, dont H. pylori, sont dépourvues d’asparaginyl-ARNt synthétase et/ou de glutaminyl-ARNt synthétase. Pour former le Gln-ARNtGln, H. pylori utilise une GluRS noncanonique nommée GluRS2 qui glutamyle spécifiquement l’ARNtGln ; ensuite, une AdT trimérique, la GatCAB corrige le Glu-ARNtGln mésapparié en le transamidant pour former le Gln-ARNtGln, qui lira correctement les codons glutamine pendant la biosynthèse des protéines sur les ribosomes. La formation de l’Asn-ARNtAsn est similaire à celle du Gln-ARNtGln, et utilise la même GatCAB et une AspRS non-discriminatrice. Depuis des années 2000, la GatCAB est considérée comme une cible prometteuse pour le développement de nouveaux antibiotiques, puisqu’elle est absente du cytoplasme de l’être humain, et qu’elle est encodée dans le génome de plusieurs bactéries pathogènes. Dans le chapitre 3, nous présentons la découverte par la technique du « phage display » de peptides cycliques riches en tryptophane et en proline, et qui inhibent l’activité de la GatCAB de H. pylori. Les peptides P10 (CMPVWKPDC) et P9 (CSAHNWPNC) inhibent cette enzyme de façon compétitive par rapport au substrat Glu-ARNtGln. Leur constante d’inhibition (Ki) est 126 μM pour P10, et 392 μM pour P9. Des modèles moléculaires ont montré qu’ils lient le site actif de la réaction de transmidation catalysée par la GatCAB, grâce à la formation d’une interaction π-π entre le résidu Trp de ces peptides et le résidu Tyr81 de la sous-unité GatB, comme fait le A76 3’-terminal de l’ARNt. Dans une autre étude concernant des petits composés contenant un groupe sulfone, et qui mimiquent l’intermédiaire de la réaction de transamidation, nous avons identifié des composés qui inhibent la GatCAB de H. pylori de façon compétitive par rapport au substrat Glu-ARNtGln. Cinq fois plus petits que les peptides cycliques mentionnés plus haut, ces composés inhibent l’activité de la GatCAB avec des Ki de 139 μM pour le composé 7, et de 214 μM pour le composé 4. Ces inhibiteurs de GatCAB pourraient être utiles pour des études mécanistiques, et pourraient être des molécules de base pour le développement de nouvelles classes d’antibiotiques contre des infections causées par H. pylori.

Lipid-Based Liquid Crystals As Carriers for Antimicrobial Peptides: Phase Behavior and Antimicrobial Effect

International audience

Amélioration des propriétés pharmacocinétiques de peptides par différentes alkylations N-terminales

Modélisations moléculaires réalisés avec le logiciel HyperChem 8.

Biosynthesis and mode of action of ribosomally synthesized and post-translationally modified antimicrobial peptides

One of the greatest sources of biologically active compounds is natural products. Often these compounds serve as platforms for the design and development of novel drugs and therapeutics. The overwhelming amount of genomic information acquired in recent years has revealed that ribosomally synthesized and post-translationally modified natural products are much more widespread than originally anticipated. Identified in nearly all forms of life, these natural products display incredible structural diversity and possess a wide range of biological functions that include antimicrobial, antiviral, anti-inflammatory, antitumor, and antiallodynic activities. The unique pathways taken to biosynthesize these compounds offer exciting opportunities for the bioengineering of these complex molecules. The studies described herein focus on both the mode of action and biosynthesis of antimicrobial peptides. In Chapter 2, it is demonstrated that haloduracin, a recently discovered two-peptide lantibiotic, possesses nanomolar antimicrobial activity against a panel of bacteria strains. The potency of haloduracin rivals that of nisin, an economically and therapeutically relevant lantibiotic, which can be attributed to a similar dual mode of action. Moreover, it was demonstrated that this lantibiotic of alkaliphile origin has better stability at physiological pH than nisin. The molecular target of haloduracin was identified as the cell wall peptidoglycan precursor lipid II. Through the in vitro biosynthesis of haloduracin, several analogues of Halα were prepared and evaluated for their ability to inhibit peptidoglycan biosynthesis as well as bacterial cell growth. In an effort to overcome the limitations of in vitro biosynthesis strategies, a novel strategy was developed resulting in a constitutively active lantibiotic synthetase enzyme. This methodology, described in Chapter 3, enabled the production of fully-modified lacticin 481 products with proteinogenic and non-proteinogenic amino acid substitutions. A number of lacticin 481 analogues were prepared and their antimicrobial activity and ability to bind lipid II was assessed. Moreover, site-directed mutagenesis of the constitutively active synthetase resulted in a kinase-like enzyme with the ability to phosphorylate a number of peptide substrates. The hunt for a lantibiotic synthetase enzyme responsible for installing the presumed dehydro amino acids and a thioether ring in the natural product sublancin, led to the identification and characterization of a unique post-translational modification. The studies described in Chapter 4, demonstrate that sublancin is not a lantibiotic, but rather an unusual S-linked glycopeptide. Its structure was revised based on extensive chemical, biochemical, and spectroscopic characterization. In addition to structural investigation, bioinformatic analysis of the sublancin gene cluster led to the identification of an S-glycosyltransferase predicted to be responsible for the post-translational modification of the sublancin precursor peptide. The unprecedented glycosyltransferase was reconstituted in vitro and demonstrated remarkable substrate promiscuity for both the NDP-sugar co-substrate as well as the precursor peptide itself. An in vitro method was developed for the production of sublancin and analogues which were subsequently evaluated in bioactivity assays. Finally, a number of putative biosynthetic gene clusters were identified that appear to harbor the necessary genes for production of an S-glycopeptide. An additional S-glycosyltransferase with more favorable intrinsic properties including better expression, stability, and solubility was reconstituted in vitro and demonstrated robust catalytic abilities.

Assessment of molecular sequences and enzyme activity of amylase trypsin inhibitors (ATIs) from a selection of ancient and modern grains

Wheat amylase-trypsin inhibitors (ATIs) are a family of wheat proteins, which play an important role in plant defence against pest attacks. ATIs are also of great interest for their impact on human health and recently ATIs have been identified as major stimulators of innate immune cells. In this study, ten selected wheat samples with different ploidy level and year of release were used for the agronomic trial, for in vitro enzymatic assays and for ATIs gene sequencing. Wheat samples were grown under organic farming management during three consecutive cropping years at two growing areas (Italy and USA). The PCA analysis performed on the deduced amino acid sequences of four representative ATIs genes (WMAI, WDAI, WTAI-CM3, CMx) evidenced that the ten wheat varieties can be differentiated on the basis of their ploidy level, but not with respect to ancient or recently developed wheat genotypes. The results from in vitro alpha-amylase and trypsin inhibitory activities showed high variability among the ten wheat genotypes and the contribution of the genotype and the cropping year was significant for both inhibitory activities. The hexaploid wheat genotypes showed the highest inhibitory activities. Einkorn showed a very low or even absent alpha-amylase inhibitory activity and the highest trypsin inhibitory activity. It was not possible to differentiate ancient and recently developed wheat genotypes on the basis of their ATIs activity. The weather conditions differently affected the two inhibitory activities. In both cultivation areas, higher precipitation and lower high mean temperatures correlated with lower alpha-amylase inhibitory activities, while there were different correlations considering trypsin inhibitory activity for the two growing areas. The protein content negatively correlated with both inhibitory activities in USA and Italy. This information can be important in the understanding of plant defence mechanisms in relation to the effect of both genotype and abiotic and biotic stress.

Inhibitory Effects Of A Cured Antibacterial Bonding System On Viability And Metabolic Activity Of Oral Bacteria.

To evaluate the antimicrobial efficacy of Clearfil SE Protect (CP) and Clearfil SE Bond (CB) after curing and rinsed against five individual oral microorganisms as well as a mixture of bacterial culture prepared from the selected test organisms. Bacterial suspensions were prepared from single species of Streptococcus mutans, Streptococcus sobrinus, Streptococcus gordonii, Actinomyces viscosus and Lactobacillus lactis, as well as mixed bacterial suspensions from these organisms. Dentin bonding system discs (6 mm×2 mm) were prepared, cured, washed and placed on the bacterial suspension of single species or multispecies bacteria for 15, 30 and 60 min. MTT, Live/Dead bacterial viability (antibacterial effect), and XTT (metabolic activity) assays were used to test the two dentin system's antibacterial effect. All assays were done in triplicates and each experiment repeated at least three times. Data were submitted to ANOVA and Scheffe's f-test (5%). Greater than 40% bacteria killing was seen within 15 min, and the killing progressed with increasing time of incubation with CP discs. However, a longer (60 min) period of incubation was required by CP to achieve similar antimicrobial effect against mixed bacterial suspension. CB had no significant effect on the viability or metabolic activity of the test microorganisms when compared to the control bacterial culture. CP was significantly effective in reducing the viability and metabolic activity of the test organisms. The results demonstrated the antimicrobial efficacy of CP both on single and multispecies bacterial culture. CP may be beneficial in reducing bacterial infections in cavity preparations in clinical dentistry.

Design, Synthesis And Biological Evaluation Of Hybrid Bioisoster Derivatives Of N-acylhydrazone And Furoxan Groups With Potential And Selective Anti-trypanosoma Cruzi Activity.

Hybrid bioisoster derivatives from N-acylhydrazones and furoxan groups were designed with the objective of obtaining at least a dual mechanism of action: cruzain inhibition and nitric oxide (NO) releasing activity. Fifteen designed compounds were synthesized varying the substitution in N-acylhydrazone and in furoxan group as well. They had its anti-Trypanosoma cruzi activity in amastigotes forms, NO releasing potential and inhibitory cruzain activity evaluated. The two most active compounds (6, 14) both in the parasite amastigotes and in the enzyme contain the nitro group in para position of the aromatic ring. The permeability screening in Caco-2 cell and cytotoxicity assay in human cells were performed for those most active compounds and both showed to be less cytotoxic than the reference drug, benznidazole. Compound 6 was the most promising, since besides activity it showed good permeability and selectivity index, higher than the reference drug. Thereby the compound 6 was considered as a possible candidate for additional studies.

A Putative Otu Domain-containing Protein 1 Deubiquitinating Enzyme Is Differentially Expressed In Thyroid Cancer And Identifies Less-aggressive Tumours.

This study aimed to identify novel biomarkers for thyroid carcinoma diagnosis and prognosis. We have constructed a human single-chain variable fragment (scFv) antibody library that was selected against tumour thyroid cells using the BRASIL method (biopanning and rapid analysis of selective interactive ligands) and phage display technology. One highly reactive clone, scFv-C1, with specific binding to papillary thyroid tumour proteins was confirmed by ELISA, which was further tested against a tissue microarray that comprised of 229 thyroid tissues, including: 110 carcinomas (38 papillary thyroid carcinomas (PTCs), 42 follicular carcinomas, 30 follicular variants of PTC), 18 normal thyroid tissues, 49 nodular goitres (NG) and 52 follicular adenomas. The scFv-C1 was able to distinguish carcinomas from benign lesions (P=0.0001) and reacted preferentially against T1 and T2 tumour stages (P=0.0108). We have further identified an OTU domain-containing protein 1, DUBA-7 deubiquitinating enzyme as the scFv-binding antigen using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. The strategy of screening and identifying a cell-surface-binding antibody against thyroid tissues was highly effective and resulted in a useful biomarker that recognises malignancy among thyroid nodules and may help identify lower-risk cases that can benefit from less-aggressive management.

Reduced Insulin Clearance And Lower Insulin-degrading Enzyme Expression In The Liver Might Contribute To The Thrifty Phenotype Of Protein-restricted Mice.

Nutrient restriction during the early stages of life usually leads to alterations in glucose homeostasis, mainly insulin secretion and sensitivity, increasing the risk of metabolic disorders in adulthood. Despite growing evidence regarding the importance of insulin clearance during glucose homeostasis in health and disease, no information exists about this process in malnourished animals. Thus, in the present study, we aimed to determine the effect of a nutrient-restricted diet on insulin clearance using a model in which 30-d-old C57BL/6 mice were exposed to a protein-restricted diet for 14 weeks. After this period, we evaluated many metabolic variables and extracted pancreatic islet, liver, gastrocnemius muscle (GCK) and white adipose tissue samples from the control (normal-protein diet) and restricted (low-protein diet, LP) mice. Insulin concentrations were determined using RIA and protein expression and phosphorylation by Western blot analysis. The LP mice exhibited lower body weight, glycaemia, and insulinaemia, increased glucose tolerance and altered insulin dynamics after the glucose challenge. The improved glucose tolerance could partially be explained by an increase in insulin sensitivity through the phosphorylation of the insulin receptor/protein kinase B and AMP-activated protein kinase/acetyl-CoA carboxylase in the liver, whereas the changes in insulin dynamics could be attributed to reduced insulin secretion coupled with reduced insulin clearance and lower insulin-degrading enzyme (IDE) expression in the liver and GCK. In summary, protein-restricted mice not only produce and secrete less insulin, but also remove and degrade less insulin. This phenomenon has the double benefit of sparing insulin while prolonging and potentiating its effects, probably due to the lower expression of IDE in the liver, possibly with long-term consequences.

The Characterization Of The Endoglucanase Cel12a From Gloeophyllum Trabeum Reveals An Enzyme Highly Active On β-glucan.

The basidiomycete fungus Gloeophyllum trabeum causes a typical brown rot and is known to use reactive oxygen species in the degradation of cellulose. The extracellular Cel12A is one of the few endo-1,4-β-glucanase produced by G. trabeum. Here we cloned cel12A and heterologously expressed it in Aspergillus niger. The identity of the resulting recombinant protein was confirmed by mass spectrometry. We used the purified GtCel12A to determine its substrate specificity and basic biochemical properties. The G. trabeum Cel12A showed highest activity on β-glucan, followed by lichenan, carboxymethylcellulose, phosphoric acid swollen cellulose, microcrystalline cellulose, and filter paper. The optimal pH and temperature for enzymatic activity were, respectively, 4.5 and 50 °C on β-glucan. Under these conditions specific activity was 239.2 ± 9.1 U mg(-1) and the half-life of the enzyme was 84.6 ± 3.5 hours. Thermofluor studies revealed that the enzyme was most thermal stable at pH 3. Using β-glucan as a substrate, the Km was 3.2 ± 0.5 mg mL(-1) and the Vmax was 0.41 ± 0.02 µmol min(-1). Analysis of the effects of GtCel12A on oat spelt and filter paper by scanning electron microscopy revealed the morphological changes taking place during the process.

Immobilization Of Papain On Chitin And Chitosan And Recycling Of Soluble Enzyme For Deflocculation Of Saccharomyces Cerevisiae From Bioethanol Distilleries.

Yeast flocculation (Saccharomyces cerevisiae) is one of the most important problems in fuel ethanol production. Yeast flocculation causes operational difficulties and increase in the ethanol cost. Proteolytic enzymes can solve this problem since it does not depend on these changes. The recycling of soluble papain and the immobilization of this enzyme on chitin or chitosan were studied. Some cross-linking agents were evaluated in the action of proteolytic activity of papain. The glutaraldehyde (0.1-10% w·v(-1)), polyethyleneimine (0.5% v·v(-1)), and tripolyphosphate (1-10% w·v(-1)) inactivated the enzyme in this range, respectively. Glutaraldehyde inhibited all treatments of papain immobilization. The chitosan cross-linked with TPP in 5 h of reaction showed the yield of active immobilized enzyme of 15.7% and 6.07% in chitosan treated with 0.1% PEI. Although these immobilizations have been possible, these levels have not been enough to cause deflocculation of yeast cells. Free enzyme was efficient for yeast deflocculation in dosages of 3 to 4 g·L(-1). Recycling of soluble papain by centrifugation was effective for 14 cycles with yeast suspension in time perfectly compatible to industrial conditions. The reuse of proteases applied after yeast suspension by additional yeast centrifugation could be an alternative to cost reduction of these enzymes.

Análise do perfil de humor e da enzima alfa-amilase salivar em indivíduos fisicamente ativos = : Analysis of profile of mood states and salivary alpha amylase enzyme in physically active individuals

Universidade Estadual de Campinas . Faculdade de Educação Física

Maximum inhibitory dilution of mouthwashes containing chlorhexidine and polyhexamethylene biguanide against salivary staphylococcus aureus

OBJECTIVE: The aim of the present study was to determine the in vitro maximum inhibitory dilution (MID) of two chlorhexidinebased oral mouthwashes (CHX): Noplak®, Periogard®, and one polyhexamethylene biguanide-based mouthwash (PHMB): Sanifill Premium® against 28 field Staphylococcus aureus strains using the agar dilution method. MATERIALS AND METHODS: For each product, decimal dilutions ranging from 1/10 to 1/655,360 were prepared in distilled water and added to Mueller Hinton Agar culture medium. After homogenization, the culture medium was poured onto Petri dishes. Strains were inoculated using a Steers multipoint inoculator and dishes were incubated at 37ºC for 24hours. For reading, MID was considered as the maximum dilution of the mouthwash still capable of inhibiting microbial growth. RESULTS: Sanifill Premium® inhibited the growth of all strains at 1/40 dilution and of 1 strain at 1/80 dilution. Noplak® inhibited the growth of 23 strains at 1/640 dilution and of all 28 strains at 1/320 dilution. Periogard® showed inhibited growth of 7 strains at 1/640 dilution and of all 28 strains at 1/320 dilution. Data were submitted to Kruskal-Wallis statistical test, showing significant differences between the mouthwashes evaluated (p<0.05). No significant difference was found between Noplak® and Periogard® (p>0.05). Sanifill Premium® was the least effective (p<0.05). CONCLUSION: It was concluded that CHX-based mouthwashes present better antimicrobial activity against S. Aureus than the PHMB-based mouthwash.