Proteomic analysis of stimulated saliva in gastroesophageal reflux disease patients with and without erosive tooth wear: Observational study.

OBJECTIVE: To evaluate the difference in the proteomic profile of stimulated saliva in patients with gastroesophageal reflux disease (GERD) with (GE) and without (GNE) erosive tooth wear (ETW), regarding both human and bacterial proteins. METHODS: Stimulated saliva (SS) was collected from 16 patients (8/group). Samples were centrifuged at 4.500 g for 15 min under refrigeration to remove all debris. The supernatant from each saliva sample was taken and frozen at -80 °C. After extracting the proteins, they were submitted to reverse phase liquid chromatography and mass spectrometry (nLC-ESI-MS/MS). Label-free proteomic quantification was performed using Protein Lynx Global Service (PLGS) software (p < 0.05) for human and bacterial proteins. RESULTS: In total, 67 human proteins were common for GNE and GE groups. GNE group presented, compared to GE group, increase in proteins that confer antimicrobial and acid resistant properties, such as cystatins, histatin and immunoglobulins. However, GNE group had a marked decrease in subunits of hemoglobin (α, ß and delta). Regarding bacterial proteins, for SS, 7 and 10 unique proteins were identified in the GE and GNE groups, respectively. They are related to protein synthesis and energy metabolism and interact with human proteins typically found in saliva and supramolecular complexes of the acquired pellicle. CONCLUSIONS: Our data indicate that the stimulation of the salivary flow increases acid resistant and antimicrobial proteins in saliva, which might protect against ETW. CLINICAL SIGNIFICANCE: This pioneer study showed important differences in the human and bacterial proteome of SS in patients with GERD with or without ETW.

Rinsing solutions containing natural extracts and fluoride prevent enamel erosion in vitro.

BACKGROUND: Polyphenols interact with salivary proteins and thus can improve the pellicle's erosion protective properties. This effect could be exploited to create rinsing solutions with polyphenols as active ingredients for erosion prevention. Different from the current gold standard for erosion protective rinsing solutions, these rinses would not rely on stannous ions. This would offer alternatives for patients with concerns regarding the composition of rinsing solutions and preferring bio-products. OBJECTIVE: To develop an erosion-preventive rinsing solution containing natural polyphenol-rich extracts. METHODOLOGY: Solutions were prepared with polyphenols from either grapeseed extract or cranberry extract, 500 ppm fluoride added, and additionally flavors and sweeteners. Controls were deionized water, 500 ppm fluoride solution, and the gold standard rinse in the field (Sn2+/F-). In total, 135 enamel specimens (n=15/group) were subjected to five cycles of salivary pellicle formation (30 min, 37°C), modification with the solutions (2 min, 25°C), further salivary pellicle formation (60 min, 37°C), and erosive challenge (1 min, 1% citric acid, pH 3.6). Relative surface microhardness (rSMH), surface reflection intensity (rSRI), and amount of calcium release (CaR) were investigated. Data were analyzed with Kruskal-Wallis and Wilcoxon rank sum tests (α=0.05). RESULTS: The polyphenol solutions containing fluoride, as well as additional flavors, protected enamel better than fluoride alone, and similar to the Sn2+/F- solution, when investigating both rSMH and CaR. When measuring rSRI, Sn2+/F- showed the best protection, while the polyphenol solutions were similar to fluoride. CONCLUSION: For two of the three assessed parameters (rSMH and CaR), both developed polyphenol-rich rinsing solutions were able to protect enamel from erosion, improving/potentializing the effect of fluoride and matching the protection offered by the current gold standard rinsing solution.

Plant extracts have dual mechanism on the protection against dentine erosion: action on the dentine substrate and modification of the salivary pellicle.

To investigate the effect of some polyphenol-rich plant extracts on the protection of dentine against demineralization, both acting on the dentine and on the salivary pellicle. Dentine specimens (n = 180) were randomly distributed into 6 experimental groups (n = 30/group): Control (deionized water), Açaí extract, Blueberry extract, Green tea extract, Grape seed extract, and Sn2+/F- (mouthrinse containing stannous and fluoride). Each group was further divided into two subgroups (n = 15), according to the site of action of the substance: on the dentine surface (D) or on the salivary pellicle (P). The specimens were submitted to 10 cycles: 30 min incubation in human saliva (P) or only in humid chamber (D), 2 min immersion in experimental substances, 60 min of incubation in saliva (P) or not (D), and 1 min erosive challenge. Dentine surface loss (DSL), amount of degraded collagen (dColl) and total calcium release were analyzed. Green tea, Grape seed and Sn2+/F- showed significant protection, with least DSL and dColl. The Sn2+/F- showed better protection on D than on P, whereas Green tea and Grape seed showed a dual mode of action, with good results on D, and even better on P. Sn2+/F- showed the lowest values of calcium release, not differing only from Grape seed. Sn2+/F- is more effective when acting directly on the dentine surface, while Green tea and Grape seed have a dual mode of action: with a positive effect on the dentine surface itself, but an improved efficacy in the presence of the salivary pellicle. We further elucidate the mechanism of action of different active ingredients on dentine erosion, where Sn2+/F- acts better on the dentine surface, but plant extracts have a dual mode of action, acting on the dentine itself as well as on the salivary pellicle, improving the protection against acid demineralization.

New insights into the protective effect of statherin-derived peptide for different acquired enamel pellicle formation times on the native human enamel surfaces.

OBJECTIVES: This study evaluated the protective impact of acquired enamel pellicle (AEP) engineering with statherin-derived peptide (StatpSpS), considering different AEP formation times. DESIGN: A total of 120 native human enamel specimens were divided into 2 main groups: 1) No AEP engineering and 2) AEP engineering with StatpSpS (pretreatment for 1 min; 37 °C, under agitation). Each group was further divided into 4 subgroups: No pellicle, or 1, 60-and-120 min AEP formation times (human saliva; 37 °C). The specimens were then subjected to an erosive challenge (1% citric acid; pH 3.6; 1 min; 25 °C). This procedure was repeated for 5 cycles. Relative surface reflection intensity (%SRI) was measured and scanning electron microscopy (SEM) of the enamel surface was done. RESULTS: All AEP engineering groups protected against initial dental erosion in comparison with No pellicle (p < 0.001), likewise all groups with AEP, independent of engineering or formation times (p 0.001). Furthermore, engineering with StatpSpS even without the presence of AEP protected the enamel when compared to the No engineering/No pellicle group (p < 0.0001). No difference was observed regarding the protection from the different AEP formation times (p > 0.05). Regarding the SEM analysis, in the "No AEP engineering & No AEP" group, a more severe effect of citric acid was observed, with more enamel prism heads and scratches on the surface when compared with the other groups. CONCLUSIONS: AEP provides almost instant protection at formation times even as short as 1 min, protecting the native enamel against erosion. Treatment with StatpSpS by itself provides similar protection as the AEP.

Acquired enamel pellicle protects gastroesophageal reflux disease patients against erosive tooth wear

Abstract The objective of this study was to compare the protein profile of the acquired enamel pellicle (AEP) formed in vivo in patients with or without gastroesophageal reflux disease (GERD), and with or without erosive tooth wear (ETW). Twenty-four volunteers were divided into 3 groups: 1) GERD and ETW; 2) GERD without ETW; and 3) control (without GERD). The AEP formed 120 min after prophylaxis was collected from the lingual/palatal surfaces. The samples were subjected to mass spectrometry (nLC-ESI-MS/MS) and label-free quantification by Protein Lynx Global Service software. A total of 213 proteins were identified, or 119, 92 and 106 from each group, respectively. Group 2 showed a high number of phosphorylated and calcium-binding proteins. Twenty-three proteins were found in all the groups, including 14-3-3 protein zeta/delta and 1-phosphatidylinositol. Several intracellular proteins that join saliva after the exfoliation of oral mucosa cells might have the potential to bind hydroxyapatite, or participate in forming supramolecular aggregates that bind to precursor proteins in the AEP. Proteins might play a central role in protecting the dental surface against acid dissolution.

Rinsing solutions containing natural extracts and fluoride prevent enamel erosion in vitro

Abstract Polyphenols interact with salivary proteins and thus can improve the pellicle's erosion protective properties. This effect could be exploited to create rinsing solutions with polyphenols as active ingredients for erosion prevention. Different from the current gold standard for erosion protective rinsing solutions, these rinses would not rely on stannous ions. This would offer alternatives for patients with concerns regarding the composition of rinsing solutions and preferring bio-products. Objective To develop an erosion-preventive rinsing solution containing natural polyphenol-rich extracts. Methodology Solutions were prepared with polyphenols from either grapeseed extract or cranberry extract, 500 ppm fluoride added, and additionally flavors and sweeteners. Controls were deionized water, 500 ppm fluoride solution, and the gold standard rinse in the field (Sn2+/F-). In total, 135 enamel specimens (n=15/group) were subjected to five cycles of salivary pellicle formation (30 min, 37°C), modification with the solutions (2 min, 25°C), further salivary pellicle formation (60 min, 37°C), and erosive challenge (1 min, 1% citric acid, pH 3.6). Relative surface microhardness (rSMH), surface reflection intensity (rSRI), and amount of calcium release (CaR) were investigated. Data were analyzed with Kruskal-Wallis and Wilcoxon rank sum tests (α=0.05). Results The polyphenol solutions containing fluoride, as well as additional flavors, protected enamel better than fluoride alone, and similar to the Sn2+/F- solution, when investigating both rSMH and CaR. When measuring rSRI, Sn2+/F- showed the best protection, while the polyphenol solutions were similar to fluoride. Conclusion For two of the three assessed parameters (rSMH and CaR), both developed polyphenol-rich rinsing solutions were able to protect enamel from erosion, improving/potentializing the effect of fluoride and matching the protection offered by the current gold standard rinsing solution.

Synergistic effect between plant extracts and fluoride to protect against enamel erosion: An in vitro study.

Polyphenol-rich solutions, such as plant extracts and teas, can modify the salivary pellicle and improve the protection against dental erosion. In this study, we further explored how these polyphenol-rich plant extracts solutions behave in the presence of fluoride. We distributed enamel specimens into 9 groups (n = 15): Control_No_F- (Deionized water); Control_F- (500 ppm F-), Grape_Seed_No_F- (Grape seed extract), Grape_Seed_F- (Grape seed extract + 500 ppm F-), Grapefruit_Seed_No_F- (Grapefruit seed extract), Grapefruit_Seed_F- (Grapefruit seed extract + 500 ppm F-), Blueberry_No_F- (Blueberry extract), Blueberry_F- (Blueberry extract + 500 ppm F-), and Sn2+/F-_Rinse (commercial solution containing 800 ppm Sn2+ and 500 ppm F-). The specimens were submitted to 5 cycles (1 cycle per day), and each cycle consisted of: salivary pellicle formation (human saliva, 30 min, 37°C), modification of the pellicle (2 min, 25°C), pellicle formation (60 min, 37°C), and an erosive challenge (1 min, citric acid). Between cycles, the specimens were kept in a humid chamber. Relative surface hardness (rSH), relative surface reflection intensity (rSRI) and calcium released to the acid were analysed, using general linear models, and Kruskal-Wallis with post-hoc Dunn's tests. We observed that the presence of fluoride in synergy with the extract solutions provided better protection than the groups containing extract or fluoride only. For rSH, we observed a significant main effect of extracts (F(4,117) = 9.20; p<0.001) and fluoride (F(1,117) = 511.55; p<0.001), with a significant interaction (F(3,117) = 6.71; p<0.001). Grape_Seed_F- showed the best protection, better than fluoride, and Sn2+/F-_Rinse. Calcium results also showed greater protection for the groups containing fluoride, whereas for rSRI, despite a significant interaction between extract and fluoride (F(3,117) = 226.05; p<0.001), the differences between the groups were not as clearly observed. We conclude that polyphenols from plant extracts, when combined with fluoride, improve the protective effect of salivary pellicles against enamel erosion.

Pellicle modification with natural bioproducts: Influence on tooth color under erosive conditions.

Salivary pellicle was modified with bioproducts and we assessed the change in tooth color and the protection of enamel to erosion. Human enamel specimens were assigned to one of three solutions: grape seed extract or black tea (bioproducts), or deionized water (negative control); after which one half the specimens underwent erosive challenges. The specimens underwent 15 cycles involving salivary pellicle formation (10 min, 37°C), incubation in solution (2 min, 25°C), subsequent pellicle formation (90 min, 37°C). Half of the specimens was kept in a humid chamber and the other half was submitted to erosion (2 min, 1% citric acid). After 15 such cycles, the pellicle was removed. Tooth color and the surface reflection intensity were assessed after every five cycles and after pellicle removal. For non-eroded specimens, the exposure to bioproducts promoted significantly greater color change than the deionized water, with increases in yellow appearance. After pellicle removal, the color was similar in all non-eroded specimens. The bioproducts increased the surface reflection intensity over cycles. For the erosion-exposed specimens, erosion itself resulted in color change. Black tea and deionized water resulted in increased yellow appearance. Exposure to the bioproducts resulted in higher relative surface reflection intensity values over time, but only grape seed extract resulted in higher relative surface reflection intensity value at the time of pellicle removal. The bioproducts caused transient staining effect, which was reduced after pellicle removal. For enamel submitted to erosion, grape seed extract resulted in less color change and better protection of enamel against erosion than black tea or water.

Acquired Pellicle Engineering Using a Combination of Organic (Sugarcane Cystatin) and Inorganic (Sodium Fluoride) Components against Dental Erosion.

This study evaluated the combination of a sugarcane cystatin (CaneCPI-5) and sodium fluoride (NaF) in acquired pellicle engineering for the prevention of dental erosion in vitro. Seventy-five human enamel specimens were prepared and divided into 5 treatment groups (n = 15/group): Deionized water (Control); Elmex™ (SnCl2/NaF/AmF); 0.1 mg/mL CaneCPI-5; 500 ppm NaF; and CaneCPI-5+NaF (Combination). The specimens were individually treated (200 µL; 2 min; 37°C), then incubated in human saliva (200 µL; 1 h, at 37°C) for acquired pellicle formation. Afterward, the specimens were submitted to an erosive challenge (1% citric acid [CR], pH 3.6, 10 mL, 2 min, 25 °C). This sequence was conducted 5 times. Percentage of surface microhardness change (%SMC), relative surface reflection intensity (rSRI), and calcium released to the CR were measured and analyzed by one-way ANOVA followed by Tukey's test (p < 0.05). In general, all the treatments (SnCl2/NaF/AmF, CaneCPI-5, NaF, and Combination) significantly protected the enamel when compared the control group. Regarding %SMC and rSRI, the Combination was the most effective treatment, reducing the %SMC significantly (p < 0.01) when compared to all the other treatments, although this difference was not significant in the CR analysis. All treatments demonstrated a protective effect on enamel against dental erosion; however, the combination of CaneCPI-5 with NaF showed a greater protection.

Salivary Pellicle Modification with Grape-seed Extract: In Vitro Study on the Effect on Bacterial Adhesion and Biofilm Formation.

PURPOSE: Grape-seed extract (GSE) contains polyphenols that readily adhere to proteins and modify the acquired enamel pellicle (AEP). The first step in biofilm formation is bacterial adhesion to the AEP-covered enamel. The aim of this in vitro study was to test whether AEP modification with GSE, fluoride (F-), or their combination (GSE+F-) modulates bacterial adhesion, biofilm metabolism and composition, or cariogenic demineralisation of the enamel. MATERIALS AND METHODS: The study comprised 3 parts: 1) single-strain Streptococcus gordonii species, 2) a five-species biofilm model, or 3) biofilm (re-)formation using the five-species biofilm model after removal of initial biofilm with toothbrushing. Human whole-mouth stimulated saliva was used to form an AEP on human enamel specimens. The AEP was incubated in water (control), or modified with GSE, F-, or GSE+F-. Bacterial adhesion, biofilm diversity, metabolic activity, biofilm mass, and cariogenic demineralisation (surface hardness) of enamel were assessed after incubation in bacterial broths after 4 h or 22 h. Differences between groups were analysed with one-way ANOVA and post-hoc Bonferroni tests. RESULTS: GSE and GSE+F- statistically significantly decreased single-strain S. gordonii adhesion, but had no relevant influence when the five-species biofilm model was used. In the biofilm (re-)formation model, GSE reduced bacterial adhesion compared to GSE+F-, while F- caused less cariogenic demineralisation than was found in the control group. CONCLUSION: AEP modified with GSE retards S. gordonii adhesion, but it does not influence the formation, metabolism and composition of a cariogenic multi-species biofilm.

Acquired pellicle engineering with proteins/peptides: Mechanism of action on native human enamel surface.

OBJECTIVE: This study investigated the mechanism of action of different proteins/peptides (separately or in combination), focusing on how they act directly on the native enamel surface and on modifying the salivary pellicle. METHODS: A total of 170 native human enamel specimens were prepared and submitted to different treatments (2 h; 37 °C): with deionized water, CaneCPI-5, Hemoglobin, Statherin, or a combination of all three proteins/peptides. The groups were subdivided into treatment acting on the enamel surface (NoP - absence of salivary pellicle), and treatment modifying the salivary pellicle (P). Treatment was made (2 h; 37 °C) in all specimens, and later, for P, the specimens were incubated in human saliva (2 h; 37 °C). In both cases, the specimens were immersed in 1% citric acid (pH 3.6; 2 min; 25 °C). Calcium released from enamel (CaR) and its relative surface reflection intensity (%SRI) was measured after 5 cycles. Between-group differences were verified with two-way ANOVA, with "presence of pellicle" and "treatment" as factors (α = 0.05). RESULTS: The presence of pellicle provided better protection regarding %SRI (p < 0.01), but not regarding CaR (p = 0.201). In relation to treatment, when compared to the control group, all proteins/peptides provided significantly better protection (p < 0.01 for %SRI and Car). The combination of all three proteins/peptides demonstrated the best protective effect (p < 0.01 for %SRI). CONCLUSION: Depending on the protein or peptide, its erosion-inhibiting effect derives from their interaction with the enamel surface or from modifying the pellicle, so a combination of proteins and peptides provides the best protection. CLINICAL SIGNIFICANCE: The present study opens a new direction for a possible treatment with a combination of proteins for native human enamel, which can act directly on the enamel surface as well on the modification of the salivary pellicle, for the prevention of dental erosion.

Salivary pellicle modification with polyphenol-rich teas and natural extracts to improve protection against dental erosion.

OBJECTIVE: To investigate the modification of the salivary pellicle with different polyphenol-rich teas and natural extracts for the protection against dental erosion. METHODS: We performed two experiments: one with teas (Green tea, Black tea, Peppermint tea, Rosehip tea, negative control [NC]) and other with natural extracts (Grape seed, Grapefruit seed, Cranberry, Propolis, NC), where NC was deionized water. A total of 150 enamel specimens were used (n = 15/group). Both experiments followed the same design, consisting of 5 cycles of: salivary pellicle formation (30 min, 37 °C), modification with the solutions (30 min, 25 °C), further salivary pellicle formation (60 min, 37 °C) and erosive challenge (1 min, 1% citric acid, pH 3.6). Relative surface microhardness (rSMH), relative surface reflection intensity (rSRI) and amount of calcium release (CaR) were evaluated. Data were analysed with Kruskal-Wallis and Wilcoxon rank sum tests with Bonferroni correction (α = 0.05). RESULTS: Regarding teas, Black and Green teas showed the best protection against dental erosion, presenting higher rSMH and lower CaR than NC. Peppermint tea was not different to NC and Rosehip tea caused erosion, showing the highest CaR and greatest loss of SMH and SRI. Regarding natural extracts, Grape seed and Grapefruit seed extracts presented the best protective effect, with significantly higher rSMH and lower CaR. Cranberry caused significantly more demineralization; and Propolis did not differ from NC. CONCLUSION: Green tea, Black tea, Grape seed extract and Grapefruit seed extract were able to modify the salivary pellicle and improve its protective effect against enamel erosion, but Rosehip tea and Cranberry extract caused erosion. CLINICAL RELEVANCE: Some some bio-products, such as teas and natural extracts, improve the protective effect of the salivary pellicle against enamel erosion. More studies should be performed in order to test the viability of their use as active ingredients for oral care products.

The effect of red wine in modifying the salivary pellicle and modulating dental erosion kinetics.

This study investigated the potential of red wine in modulating dental erosion kinetics in the presence or absence of salivary pellicle. Polished human enamel specimens were used in two conditions; presence or absence of acquired enamel pellicle; and subdivided according to exposure: red wine, orange juice, apple juice, or citric acid. The specimens were incubated in clarified whole human saliva (presence of acquired enamel pellicle) or in a humid chamber (absence of acquired enamel pellicle) for 2 h at 37°C, then in the test substances for 1 min, at 25°C, under shaking. This was repeated four times. Surface hardness was measured initially and after each cycle and surface reflection intensity was measured initially and after all cycles. In the presence of acquired enamel pellicle, red wine caused the least surface hardness loss, followed by orange juice, apple juice, and citric acid. Statistically significantly less surface reflection intensity loss was observed for red wine and orange juice than for apple juice and citric acid. In the absence of acquired enamel pellicle, red wine and orange juice caused less surface hardness loss than apple juice and citric acid. Orange juice showed the least surface reflection intensity loss, followed by red wine, citric acid, and apple juice. The polyphenol composition of these drinks can notably modulate the erosion kinetics.

Proteomics of acquired pellicle in gastroesophageal reflux disease patients with or without erosive tooth wear.

OBJECTIVES: This in vivo study compared the protein profile of the acquired enamel pellicle (AEP) in volunteers 1) with gastroesophageal reflux disease (GERD) and erosive tooth wear (ETW) (BEWE ≥ 9; GE group); 2) with GERD without ETW (BEWE = 0; GNE group) and 3) control (without GERD and BEWE = 0; C group). MATERIALS AND METHODS: Twenty-four subjects (8/group) participated. AEP was formed during 120 min and collected. After protein extraction, the samples were submitted to reverse phase liquid chromatography coupled to mass spectrometry. Label-free proteomic quantification was performed using Protein Lynx Global Service software. RESULTS: In total, 458 proteins were identified. Seventy-six proteins were common to all the groups. The proteomic profile of the AEP was quite different among the distinct groups. The numbers of proteins exclusively found in the C, GE and GNE groups were 113, 110 and 81, respectively. Most of the proteins exclusively identified in the C and GNE groups bind metals, while those in the GE group are mainly membrane proteins. Many proteins were found exclusively in the reflux groups. In the quantitative analyses, when the GNE group was compared with the GE group, the proteins with the highest decreases were Lysozyme C, Antileukoproteinase, Cathepsin G, Neutrophil defensins and Basic salivary proline-rich proteins, while those with the highest increases were subunits of Hemoglobin, Albumin and isoforms of Cystatin. CONCLUSION: Profound alterations in the proteomic profile of the AEP were seen in GNE compared with GE volunteers, which might play a role in the resistance to ETW seen in the first. CLINICAL SIGNIFICANCE: This pioneer study compared the proteomic profile of the AEP of patients with GERD with or without ETW. Increased proteins in those without ETW might be protective and are good candidates to be added to dental products to protect against erosion caused by intrinsic acids.

In vitro salivary pellicles from adults and children have different protective effects against erosion.

OBJECTIVES: We aimed at analyzing the protective effects of salivary pellicles, formed with saliva from adults or children, on enamel from permanent or deciduous teeth. MATERIALS AND METHODS: Ninety human enamel specimens (45 permanent premolars and 45 deciduous canines) were ground, and the outer 200 µm of enamel was removed. We divided the teeth into three further subgroups: no salivary pellicle (control), adult salivary pellicle (AP), and child salivary pellicle (CP). We collected stimulated saliva from adults and children and placed 160 µl of either saliva on enamel specimens from AP and CP, respectively. Control specimens received no saliva. Specimens were stored at 37 °C for 2 h and then submitted to an erosive challenge (10 mL; 1 % citric acid; pH 3.6; 25 °C, 1 min). Pellicle formation and erosion was repeated for a total of 4 cycles. After every cycle, relative surface reflection intensity (rSRI) and surface microhardness (rSMH) were calculated. RESULTS: On permanent enamel, AP presented significantly better protective effects, with less rSMH loss (p < 0.001) and less rSRI loss (p < 0.001). On deciduous enamel, CP presented significantly better protective effects than AP and control (p < 0.05), for both measured parameters. CONCLUSION: We conclude that pellicles from adults and children promote different erosion protective effects, where adult pellicle provides better protection for permanent enamel, and child pellicle promotes better protection on deciduous enamel. CLINICAL RELEVANCE: The present results provide a better understanding toward the protective effect of salivary pellicle against dental erosion and brings light to one more factor involved in the erosion of deciduous teeth.

Expression of defensins in non-infected araneomorph spiders.

Defensins are a major family of antimicrobial peptides found throughout the phylogenetic tree. From the spider species: Cupiennius salei, Phoneutria reidyi, Polybetes pythagoricus, Tegenaria atrica, and Meta menardi, defensins belonging to the 'ancestral' class of invertebrate defensins were cloned and sequenced. The deduced amino acid sequences contain the characteristic six cysteines of this class of defensins and reveal precursors of 60 or 61 amino acid residues. The mature peptides consist of 37 amino acid residues, showing up to 70% identities with tick and scorpion defensins. In C. salei, defensin mRNA was found to be constitutively expressed in hemocytes, ovaries, subesophageal nerve mass, hepatopancreas, and muscle tissue. This is the first report presenting and comparing antimicrobial peptides belonging to the family of defensins from spiders.

Ctenidins: antimicrobial glycine-rich peptides from the hemocytes of the spider Cupiennius salei.

Three novel glycine-rich peptides, named ctenidin 1-3, with activity against the Gram-negative bacterium E. coli, were isolated and characterized from hemocytes of the spider Cupiennius salei. Ctenidins have a high glycine content (>70%), similarly to other glycine-rich peptides, the acanthoscurrins, from another spider, Acanthoscurria gomesiana. A combination of mass spectrometry, Edman degradation, and cDNA cloning revealed the presence of three isoforms of ctenidin, at least two of them originating from simple, intronless genes. The full-length sequences of the ctenidins consist of a 19 amino acid residues signal peptide followed by the mature peptides of 109, 119, or 120 amino acid residues. The mature peptides are post-translationally modified by the cleavage of one or two C-terminal cationic amino acid residue(s) and amidation of the newly created mature C-terminus. Tissue expression analysis revealed that ctenidins are constitutively expressed in hemocytes and to a small extent also in the subesophageal nerve mass.