Short communication: Characterization of the milk protein expression profiles in dairy buffaloes with and without subclinical mastitis.

The aim of this study was to characterize the proteins present in milk whey from buffaloes with and without subclinical mastitis using a proteomic approach to identify differentially expressed proteins as potential biomarkers for this disease. Whey from Murrah buffaloes with subclinical mastitis was compared with whey from healthy animals using liquid chromatography-tandem mass spectrometry. The annotated protein databases for Bubalus bubalis and Bos taurus were used in the analysis, and the gene annotations from the buffalo and bovine reference assemblies were also used. After integrating gene annotations from both buffaloes and bovines, a total of 1,033 proteins were identified, of which 156 were differentially expressed. Eighteen biological processes were annotated with Gene Ontology. Cathelicidin-3 was identified as a potential biomarker for subclinical mastitis. These results are important to the characterization of mastitis in the buffalo mammary gland and may aid in the development of tools for early diagnosis.

Biochemical analysis of antimicrobial peptides in two different Capsicum genotypes after fruit infection by Colletotrichum gloeosporioides.

There are several phytosanitary problems that have been causing serious damage to the Capsicum crops, including anthracnose. Upon attack by certain pathogens, various protein molecules are produced, which are known as proteins related to pathogenesis (PR proteins), including antimicrobial peptides such as protease inhibitors, defensins and lipid transfer proteins (LTPs). The objective of this work is to identify antimicrobial proteins and/or peptides of two genotypes from Capsicum annuum fruits infected with Colletotrichum gloeosporioides The fungus was inoculated into Capsicum fruits by the deposition of a spore suspension (106 conidia ml-1), and after 24 and 48 h intervals, the fruits were removed from the humid chamber and subjected to a protein extraction process. Protein analysis of the extracts was performed by tricine gel electrophoresis and Western blotting. The distinctive bands between genotypes in the electrophoresis profiles were subjected to mass spectrometry sequencing. Trypsin inhibition assays, reverse zymographic detection of protease inhibition and ß-1,3-glucanase activity assays were also performed and extracts were also tested for their ability to inhibit the growth of C. gloeosporioides fungi 'in vitro' There were several low molecular weight proteins in all treated samples, and some treatments in which antimicrobial peptides such as defensin, lipid transfer protein (LTP) and protease inhibitor have been identified. It was shown that the green fruits are more responsive to infection, showing the production of antimicrobial peptides in response to injury and inoculation of the fungus, what did not occur in ripe fruits under any treatment.

Leptodactylus latrans Amphibian Skin Secretions as a Novel Source for the Isolation of Antibacterial Peptides.

Amphibians´ skin produces a diverse array of antimicrobial peptides that play a crucial role as the first line of defense against microbial invasion. Despite the immense richness of wild amphibians in Argentina, current knowledge about the presence of peptides with antimicrobial properties is limited to a only few species. Here we used LC-MS-MS to identify antimicrobial peptides with masses ranging from 1000 to 4000 Da from samples of skin secretions of Leptodactylus latrans (Anura: Leptodactylidae). Three novel amino acid sequences were selected for chemical synthesis and further studies. The three synthetic peptides, named P1-Ll-1577, P2-Ll-1298, and P3-Ll-2085, inhibited the growth of two ATCC strains, namely Escherichia coli and Staphylococcus aureus. P3-Ll-2085 was the most active peptide. In the presence of trifluoroethanol (TFE) and anionic liposomes, it adopted an amphipathic α-helical structure. P2-Ll-1298 showed slightly lower activity than P3-Ll-2085. Comparison of the MIC values of these two peptides revealed that the addition of seven amino acid residues (GLLDFLK) on the N-terminal of P2-Ll-1298 significantly improved activity against both strains. P1-Ll-1577, which remarkably is an anionic peptide, showed interesting antimicrobial activity against E. coli and S. aureus strain, showing marked membrane selectivity and non-hemolysis. Due to this, P1-L1-1577 emerges as a potential candidate for the development of new antibacterial drugs.

Peptidomic analysis of the host-defense peptides in skin secretions of the Trinidadian leaf frog Phyllomedusa trinitatis (Phyllomedusidae).

Peptidomic analysis (reversed-phase HPLC combined with MALDI-TOF mass spectrometry and automated Edman degradation) of norepinephrine-stimulated skin secretions from the Trinidadian leaf frog Phyllomedusa trinitatis Mertens 1926 led to the identification and structural characterization of 26 host-defense peptides. On the basis of amino acid sequence similarity, the peptides may be divided into the followings groups: dermaseptins with the conserved N-terminal region GLWSKIK (6 peptides), dermaseptins with the N-terminal region ALWKXXLK (5 peptides), dermaseptins with the conserved N-terminal region GLFKTLIKGAGKMLGHVAK (4 peptides), C-terminally α-amidated and non-amidated forms of the phylloseptins (9 peptides), phyllocaerulein, a peptide (GLVSGLLNSVTGLLGNLAGGGL) with structural similarity to the plasticins, and a putative antioxidant peptide (LTWKIPTRFCGVT). The primary structures of the peptides support the claim based upon morphological criteria that P. trinitatis and Phyllomedusa tarsius are very closely related phylogenetically but are probably not conspecific. Among the phylloseptins, phylloseptin-1.1TR (FLSLIPKIAGGIASLVKNL.NH2) displayed the most potent antimicrobial activity.

An investigation of human beta-defensins and cathelicidin expression in patients with pterygium.

PURPOSE: To investigate human beta-defensins (HBDs) and cathelicidin LL-37 (LL-37) expressions in patients with pterygium. METHODS: In this retrospective consecutive case series, 26 pterygium specimens and 15 normal conjunctival specimens of 15 control subjects were in vestigated. Expressions of HBD-1, HBD-2, HBD-3, and LL-37 were assessed using immuno histochemical staining. A brown color in the cytoplasm and/or nuclei of epithelial cells indicated positive staining for HBDs and LL-37. For each antibody, the intensity of the reaction (negative [-], weak [1+], moderate [2+], or strong [3+]) was determined to describe the immunoreactions. RESULTS: The median age was 52 years in both groups. There were no significant differences in age and sex between the groups (p=0.583, p=0.355, respectively). Of the 26 pterygium specimens, 15 (57.7%) (14 weak, 1 moderate staining) showed HBD-2 expression, which was not observed in any of the control specimens. One (3.8%) pterygium and one (6.7%) control specimen demonstrated weak staining for HBD-3. HBD-2 expression was significantly higher in the pterygium specimens than in the controls (p=0.002). None of the tissue specimens had positive staining for HBD-1 or LL-37 in either group (both; p=1.00). CONCLUSIONS: HBD-2 expression was higher in pterygium specimens than in the controls. HBD-2 expression that might be stimulated by inflammatory cytokines may be related to inflammation and fibrovascular proliferation and may play a role in pterygium pathogenesis.

An investigation of human beta-defensins and cathelicidin expression in patients with pterygium / Uma investigação da expressão de beta-defensinas humanas e de catelidicina em pacientes com pterígio

ABSTRACT Purpose: To investigate human beta-defensins (HBDs) and cathelicidin LL-37 (LL-37) expressions in patients with pterygium. Methods: In this retrospective consecutive case series, 26 pterygium specimens and 15 normal conjunctival specimens of 15 control subjects were in vestigated. Expressions of HBD-1, HBD-2, HBD-3, and LL-37 were assessed using immuno histochemical staining. A brown color in the cytoplasm and/or nuclei of epithelial cells indicated positive staining for HBDs and LL-37. For each antibody, the intensity of the reaction (negative [-], weak [1+], moderate [2+], or strong [3+]) was determined to describe the immunoreactions. Results: The median age was 52 years in both groups. There were no significant differences in age and sex between the groups (p=0.583, p=0.355, respectively). Of the 26 pterygium specimens, 15 (57.7%) (14 weak, 1 moderate staining) showed HBD-2 expression, which was not observed in any of the control specimens. One (3.8%) pterygium and one (6.7%) control specimen demonstrated weak staining for HBD-3. HBD-2 expression was significantly higher in the pterygium specimens than in the controls (p=0.002). None of the tissue specimens had positive staining for HBD-1 or LL-37 in either group (both; p=1.00). Conclusions: HBD-2 expression was higher in pterygium specimens than in the controls. HBD-2 expression that might be stimulated by inflammatory cytokines may be related to inflammation and fibrovascular proliferation and may play a role in pterygium pathogenesis.

RESUMO Objetivo: Investigar as expressões beta defensinas humanas (HBD) e catelicidina em pacientes com pterígio. Métodos: Nesta série de casos retrospectivos consecutivos, 26 espécimes de pterígio e 15 espécimes conjuntivais normais de 15 indivíduos controle foram investigados. As expressões de HBD-1, HBD-2, HBD-3 e catelicidina (LL-37) foram avaliadas por coloração imuno-histoquímica. Uma cor castanha no citoplasma ou nos núcleos de células epiteliais foi definida como coloração positiva para HBDs e LL-37. Para cada anticorpo foi determinada a intensidade da reação (negativo [-], fraco [1+], moderado [2+] ou forte [3+]) para descrever as imunoreações. Resultados: A idade média foi de 52 anos em ambos os grupos. Não houve diferença significativa entre os grupos em termos de idade e sexo (p=0,583, p=0,355, respectivamente). Das 26 amostras de pterígio, 15 (57,7%) (14 fracas e 1 moderada) demonstraram a expressão de HBD-2 enquanto não foi encontrada em nenhum dos espécimes de controlo. Um dos pterígios (3,8%) e um dos espécimes de controlo (6,7%) demonstraram fraca coloração para HBD-3. A expressão de HBD-2 foi significati vamente maior nos espécimes de pterígio do que nos controles (p=0,002). Nenhum dos espécimes de tecido apresentou coloração positiva para HBD-1 ou LL-37 em ambos os grupos (ambos p=1,00). Conclusão: Encontramos aumento da expressão de HBD-2 em espécimes de pte rígio em relação aos controles. A expressão de HBD-2 que pode ser estimulada por citocinas inflamatórias pode estar relacionada com inflamação e proliferação fibrovascular e pode desempenhar um papel na patogênese do pterígio.

Activity of the antimicrobial peptide P34 against bovine alphaherpesvirus type 1 / Atividade do peptídeo antimicrobiano P34 contra o alfaherpesvírus bovino tipo 1

Previous studies have demonstrated the antimicrobial activity of the peptide P34. In this study, the antiviral potential of P34 and the in vitro mechanism of action were investigated against bovine alphaherpesvirus type 1 (BoHV1). P34 exhibited low toxicity, a high selectivity index (22.9) and a percentage of inhibition of up to 100% in MDBK cells. Results from antiviral assays indicated that P34 did not interact with cell receptors, but it was able to inhibit the viral penetration immediately after pre-adsorption. In addition, BoHV1 growth curve in MDBK cells in the presence of P34 revealed a significant reduction in virus titer only 8h post-infection, also suggesting an important role at late stages of the replicative cycle. Virucidal effect was observed only in cytotoxic concentrations of the peptide. These findings showed that the antimicrobial peptide P34 may be considered as a potential novel inhibitor of in vitro herpesviruses and must encourage further investigation of its antiherpetic activity in animal models as well as against a wide spectrum of viruses.(AU)

A atividade antimicrobiana do peptídeo P34 já foi previamente demonstrada. Neste estudo, o potencial antiviral do P34 e o mecanismo de ação in vitro contra o alfaherpesvírus bovino tipo 1 (BoHV1) foram investigados. O P34 exibiu baixa toxicidade, alto índice de seletividade (22.9) e percentagem de inibição viral de até 100% em células MDBK. Os resultados dos ensaios antivirais indicaram que não interage com receptores celulares, mas é capaz de inibir a penetração viral, imediatamente após a pré-adsorção. Além disso, a curva de crescimento do BoHV1 em células MDBK na presença do P34 revelou uma significativa redução no título somente após 8h de infecção, sugerindo também uma importante atividade do peptídeo nas fases finais do ciclo replicativo. Efeito virucida frente / BoHV1 foi observado apenas em concentrações citotóxicas do peptídeo. Os dados obtidos indicam que o peptídeo antimicrobiano P34 pode ser considerado um potencial composto inibidor de herpesvírus, in vitro, e estimulam posteriores investigações sobre sua atividade anti-herpética em modelos animais, bem como contra outros vírus.(AU)

Activity of the antimicrobial peptide P34 against bovine alphaherpesvirus type 1 / Atividade do peptídeo antimicrobiano P34 contra o alfaherpesvírus bovino tipo 1

Previous studies have demonstrated the antimicrobial activity of the peptide P34. In this study, the antiviral potential of P34 and the in vitro mechanism of action were investigated against bovine alphaherpesvirus type 1 (BoHV1). P34 exhibited low toxicity, a high selectivity index (22.9) and a percentage of inhibition of up to 100% in MDBK cells. Results from antiviral assays indicated that P34 did not interact with cell receptors, but it was able to inhibit the viral penetration immediately after pre-adsorption. In addition, BoHV1 growth curve in MDBK cells in the presence of P34 revealed a significant reduction in virus titer only 8h post-infection, also suggesting an important role at late stages of the replicative cycle. Virucidal effect was observed only in cytotoxic concentrations of the peptide. These findings showed that the antimicrobial peptide P34 may be considered as a potential novel inhibitor of in vitro herpesviruses and must encourage further investigation of its antiherpetic activity in animal models as well as against a wide spectrum of viruses.

A atividade antimicrobiana do peptídeo P34 já foi previamente demonstrada. Neste estudo, o potencial antiviral do P34 e o mecanismo de ação in vitro contra o alfaherpesvírus bovino tipo 1 (BoHV1) foram investigados. O P34 exibiu baixa toxicidade, alto índice de seletividade (22.9) e percentagem de inibição viral de até 100% em células MDBK. Os resultados dos ensaios antivirais indicaram que não interage com receptores celulares, mas é capaz de inibir a penetração viral, imediatamente após a pré-adsorção. Além disso, a curva de crescimento do BoHV1 em células MDBK na presença do P34 revelou uma significativa redução no título somente após 8h de infecção, sugerindo também uma importante atividade do peptídeo nas fases finais do ciclo replicativo. Efeito virucida frente / BoHV1 foi observado apenas em concentrações citotóxicas do peptídeo. Os dados obtidos indicam que o peptídeo antimicrobiano P34 pode ser considerado um potencial composto inibidor de herpesvírus, in vitro, e estimulam posteriores investigações sobre sua atividade anti-herpética em modelos animais, bem como contra outros vírus.

Expression of Antimicrobial Peptides in Human Monocytic Cells and Neutrophils in Response to Dengue Virus Type 2.

BACKGROUND/AIMS: The innate immune response is remarkably important for controlling infections. Information about the participation of antimicrobial peptides (AMPs) in response to dengue virus (DENV) is scarce. The aim of this study was to examine the AMP response to DENV-2 in human THP-1 cells and neutrophils. METHODS: Protein and mRNA levels of two AMPs - hBD-1 and cathelicidin LL-37 - were assessed in DENV-infected macrophage-like THP-1 cells using qRT-PCR and indirect immunofluorescence. Also, mRNA levels of α-defensins (hDEFAs) and LL-37 were examined by qRT-PCR in human neutrophils taken from peripheral blood and treated with DENV-2. RESULTS: mRNA expression of hBD-1 rose in THP-1 cells at 24-72 h, while protein expression increased later, from 48 to 72 h after infection. Cathelicidin LL-37 mRNA expression of DENV-infected THP-1 cells was observed at 6-48 h after infection, while protein levels increased importantly up to 72 h after infection. Regarding neutrophils, the mRNA expression of hDEFAs and LL-37 increased slightly at 2 and 5 h after the contact with DENV-2. CONCLUSION: THP-1 cells and human neutrophils strongly respond to DENV by producing AMPs: hBD-1 and LL-37 for the THP-1 cells and hDEFAs and LL-37 for neutrophils. However, the direct effect of these molecules on DENV particles remains unclear.

Proteomic profile of hemolymph and detection of induced antimicrobial peptides in response to microbial challenge in Diatraea saccharalis (Lepidoptera: Crambidae).

Insects are organisms extremely well adapted to diverse habitats, primarily due to their innate immune system, which provides them with a range of cellular and humoral responses against microorganisms. Lepidoptera hemolymph proteins involved in humoral responses are well known; however, there is a lack of knowledge about the sugarcane borer Diatraea saccharalis. In this present work, the hemolymph proteins of this pest insect were studied by applying proteomic methodologies. Two-dimensional electrophoresis (2-DE) gels of proteins extracted from naive larvae and larvae challenged with Escherichia coli (ATCC 11224) and Bacillus subtilis (ATCC 6623) showed an average of 300 spots, and 92 of these spots corresponded in all three 2-DE gels. Forty-one spots were excised and digested with trypsin and analyzed using mass spectrometry. After analysis, 10 proteins were identified, including some proteins of the immune system: ß-defensin-like protein, Turandot A-like protein, attacin-like protein, peptidoglycan recognition protein and cyclophilin-like protein. Nine proteins were present in both experimental conditions; however, ß-defensin-like protein was present only in hemolymph challenged by B. subtilis. Notably, attacin-like protein was strongly induced by challenge with E. coli, suggesting an immune response against the infection. However, antimicrobial activity was observed in the test zone of microbial growth inhibition of B. subtilis solely with the hemolymph extract of the larvae challenged with B. subtilis. We made for the first time a proteomic profile of the hemolymph of D. saccharalis in which it was possible to identify the presence of important proteins involved in the immune response.

Efeito antimicrobiano do peptídeo Lye Tx-I associado à ß-Ciclodextrina em biofilmes de bacterias patógenas periodontais: estudo in vitro / Effect of antimicrobial Peptide Lye Tx-I associated with ß-Cyclodextrin in biofilms of bacterial periodontal patógenas: in vitro study

Biofilmes bacterianos estão associados como fator etiológico da doença periodontal, uma vez que são difíceis de controlar por meio da terapia mecânica e antibiótica convencional. As células bacterianas dos biofilmes possuem mecanismos de resistência bacteriana tornando-as difíceis de neutralizar tanto pela resposta imune do hospedeiro quanto à ação dos antibacterianos. Uma alternativa para o controle dos biofilmes bacterianos, é o uso de peptídeos antimicrobianos, uma vez que tem amplo espectro e baixa incidência de resistência bacteriana. O presente estudo investigou a atividade antibacteriana do peptídeo sintético denominado LyeTx I, originário do veneno de uma aranha (Lycosaerithognata), em sua forma livre ou associado em ßciclodextrina (ßCD) contra bactérias periodontopatógenas colonizadoras iniciais e tardias tanto em estado planctônico quanto em biofilme. A mistura entre LyeTx I e a ßCD foi caracterizada por espectroscopia de infravermelho. Para conhecer a cinética das bactérias testadas, foram feitas curvas de crescimento bacteriano tanto em estado planctônico quanto em biofilmes formados sobre pinos de polietileno no dispositivo de biofilme da Universidade de Calgary. Para confirmar a formação e estrutura dos biofilmes, os pinos foram examinados por microscopia eletrônica de varredura em diferentes tempos (2, 5, 10 dias). Em seguida, foram determinadas as concentrações inibitória mínima (CIM) e bactericida mínima (CBM) dos S. mutans, S. oralis, S. sanguinis, E. corrodens, L. acidophilus, L. casei, F. nucleatum e P. intermedia. Inicialmente, o efeito da cinética do LyeTxI nas bactérias foi determinado por meio do ensaio de curva de morte usando as CIMs. Adicionalmente, foram determinadas a concentração mínima de erradicação do biofilme (CMEB) e a porcentagem de redução da atividade metabólica tanto em biofilme jovem (2 dias) quanto para biofilme maturo (4 dias). As CIMs tanto do LyeTxI quanto do LyeTx I/ßCD foram entre 7,81- 62,5 µg/mL em estado planctônico sendo que o S. mutans, S. oralis, S. sanguinis e E. corrodens foram as mais sensíveis ao LyeTx I e ao LyeTx I/ßCD. O efeito antibacteriano do LyeTx I começou dentro dos primeiros 15 minutos e se manteve durante 10 horas de acordo com os resultados obtidos nas curvas de morte das bactérias testadas. A porcentagem de redução da atividade metabólica das células do biofilme foi determinada usando resazurina e leitura da fluorescência (λex570 nm/ λem 590 nm). Os resultados mostraram que tanto o LyeTx I quanto o LyeTx I/ßCD quando comparados à clorexidina (controle), apresentaram uma redução da atividade metabólica das células dos biofilmes de 2 dias na maior concentração (250 µg/mL), porém não houve diferença estatisticamente significativa após análise da variância e o teste Tukey de multiplex comparações entre os tratamentos testados (p>0,05). Contudo, dentre os agentes testados nenhum foi capaz de reduzir a atividade metabólica das células do biofilme de 4 dias, sendo que a clorexidina diminuiu levemente a atividade metabólica do biofilme maturo (p< 0,05). Os resultados do presente trabalho sugerem que, o peptídeo LyeTx I e o composto de inclusão LyeTx I/ßCD possuem efeito antibacteriano contra bactérias patógenas periodontais, agindo de forma bacteriostática por um período de até 10 horas. LyeTx I e LyeTx I/ßCD possuem efeito antibacteriano em biofilmes jovens (2 dias), indicando o potencial uso destes como coadjuvantes na terapia mecânica periodontal

Biofilms are involved as etiology of bacterial infections such as periodontal disease, once they difficult to control with conventional mechanic and antibiotic therapy. Biofilms cells display mechanism of resistance making them tolerant against immune response and antibiotics. Antimicrobial peptides (AMPs) are an alternative to control bacterial biofilms because their wide spectrum and low rate of bacterial resistance. Therefore, the aim of this study was to evaluate the antibacterial activity of the synthetic peptide LyeTx I (originary of the poison of a spider Lycosaerithognata), free and associated with beta-cyclodextrin (ßCD) against both, first and late colonizer bacteria in planktonic state and in biofilms. The mixture of LyeTx I and ßCDwas characterized by infrared absorption spectroscopy. Planktonic and biofilm growth curves were done to identify the kinetics of tested bacteria. Biofilms were formed on polyethylene pegs using the calgary biofilm device (CBD) of the University of Calgary. To determine whether differences in biofilm architecture and composition exist between immature biofilms to mature biofilms formed on pegs, we examined biofilms by scanning electron microscopy at 2, 5 and 10 days. Then, the minimal inhibitory concentrations (MIC) and minimal bactericidal concentration (MBC) were determined for S. mutans, S. oralis, S. sanguinis, E. corrodens, L. acidophilus, L. casei, F. nucleatum and P. intermedia. Killing kinetics assays were performed for each species at MICs to determine the kinetic of LyeTx I. Additionally, the minimal biofilm eradication concentration (MBEC) and the percent of reduction in metabolic activity were determined for young (2-day) and mature (4-day) multispecies biofilms. The band detected between 1200-1700 cm1 regions shows a decrease of functional group C-H aliphatic stretching vibrations that may be attributed to the formation of van der Waals interactions between ßCD and LyeTx I, thus confirming the presence of the association compound LyeTx I/ßCD. The MICs of the LyeTx I and the LyeTx I /ßCD were found between 62.5 - 7.81 µg /mL in planktonic state. S. mutans, S. oralis, S. sanguinis and E. corrodens were the most sensitive to LyeTx I and LyeTx I /ßCD. The antibacterial effect of LyeTx I started within the first 15 minutes and continued for 10 hours according to results of death curves on tested bacteria. The percentage reduction of metabolic activity of biofilm cells was determined using resazurin and fluorometry (λex 570 nm / λof 590 nm). LyeTx I, LyeTx I/ßCD and chlorhexidine (control) decreased the metabolic activity of 2-day biofilm cells at 250 µg/mL and there were no statistically significant difference between the treatments (p> 0.05). In contrast, any of the tested agents decreased metabolic activity of 4-day biofilm cells. Chlorhexidine was able to slightly reduce metabolic activity of mature biofilm (p<0.05). In conclusion, the LyeTx I peptide free or associated with ßCD possesses antibacterial effect against planktonic periodontal pathogenic bacteria and reduce the metabolic activity in young biofilm cells acting as a bacteriostatic agent for a period of 10 hours. Therefore, this study suggests that LyeTx I and LyeTx I/ßCD are promising antibacterial agents for the control of 2-day biofilms, which suggest their use as coadjuvant in periodontal mechanical therapy

Dynamics and conformational studies of TOAC spin labeled analogues of Ctx(Ile(21))-Ha peptide from Hypsiboas albopunctatus.

Antimicrobial peptides (AMPs) isolated from several organisms have been receiving much attention due to some specific features that allow them to interact with, bind to, and disrupt cell membranes. The aim of this paper was to study the interactions between a membrane mimetic and the cationic AMP Ctx(Ile(21))-Ha as well as analogues containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) incorporated at residue positions n = 0, 2, and 13. Circular dichroism studies showed that the peptides, except for [TOAC(13)]Ctx(Ile(21))-Ha, are unstructured in aqueous solution but acquire different amounts of α-helical secondary structure in the presence of trifluorethanol and lysophosphocholine micelles. Fluorescence experiments indicated that all peptides were able to interact with LPC micelles. In addition, Ctx(Ile(21))-Ha and [TOAC(13)]Ctx(Ile(21))-Ha peptides presented similar water accessibility for the Trp residue located near the N-terminal sequence. Electron spin resonance experiments showed two spectral components for [TOAC(0)]Ctx(Ile(21))-Ha, which are most likely due to two membrane-bound peptide conformations. In contrast, TOAC(2) and TOAC(13) derivatives presented a single spectral component corresponding to a strong immobilization of the probe. Thus, our findings allowed the description of the peptide topology in the membrane mimetic, where the N-terminal region is in dynamic equilibrium between an ordered, membrane-bound conformation and a disordered, mobile conformation; position 2 is most likely situated in the lipid polar head group region, and residue 13 is fully inserted into the hydrophobic core of the membrane.

Salivary peptide profile and its association with early childhood caries.

BACKGROUND: As dietary management during early childhood is a great barrier in caries control, there is a need for the identification of intrinsic risk factors, capable of allowing the use of a more cost-effective approach to early childhood caries (ECC). OBJECTIVE: To evaluate the salivary peptide profile of children with and without ECC and its association with caries experience. METHODS: One hundred and six 10- to 71-month-old children participated in the study. Caries experience was determined through the visual/tactile method, based on the number of decayed, missing, and filled teeth, and surface scores (dmft/dmfs). Whole saliva was collected for mutans streptococci (MS) detection and peptide analysis. RESULTS: Chromatograms from CF (children without caries experience, n = 58) and CE (children with caries experience, n = 48) saliva pools expressed different patterns. Identification of molecular masses suggested the presence of nine peptides. Three of them were significantly related with caries experience. HNP-3 (α-defensin 3) (P = 0.019) and HBD-3 (ß-defensin 3) (P = 0.034) reduced the chances of experiencing ECC. Proline-rich peptides IB-4 significantly increased caries experience (P = 0.035). Age (P = 0.020) and MS counts (P = 0.036) increased caries experience; however, gender was not associated with dental caries (P = 0.877). CONCLUSION: Specific salivary peptides of CF or CE children in early childhood predispose to a higher or lower risk of caries experience.

Potamotrygon cf. henlei stingray mucus: biochemical features of a novel antimicrobial protein.

Antimicrobial molecules are important components of the innate immune system in vertebrates. They have been studied widely in several fishes, but little is known about these defence factors in stingrays, which are thought to have less sophisticated adaptive immune systems when compared to other teleosts. Stingrays from the specie Potamotrygon cf. henlei are distributed throughout the rivers of central-west Brazil, being the cause of numerous envenomations occurring in the dry seasons. In a previous study, we reported that the mucus of the stingray P. cf. henlei shows antimicrobial effects. Here, to analyze the antimicrobial compounds from the mucus of P. cf. henlei, we employed solid-phase extraction, chromatographic separation followed by ESI-MS, and Edman degradation. A protein similar to the ß-chain of hemoglobin was identified, isolated and partially sequenced by Edman degradation. This protein has a molecular weight of 16072.8 Da, and was shown to be active against bacteria (Micrococcus luteus and Escherichiacoli) and yeast (Candida tropicalis) without hemolytic activity. Effects of this new protein in the microcirculation environment were also evaluated. The results obtained provide fundamental information for future basic research, clinical diagnosis and development of new therapies to accident treatment. To the best of our knowledge, this is the first description of a bioactive polypeptide from the mucus of a stingray.

Evaluation of the in vitro Activity of Dermaseptin 01, a CationicAntimicrobial Peptide, against Schistosoma mansoni

Schistosomiasis is a neglected tropical disease that remains a considerable public health problem worldwide. Since the mainstay of schistosomiasis control is chemotherapy with a single drug,praziquantel, drug resistance is a concern. Here, we examined the in vitro effects of dermaseptin 01(DS 01), an antimicrobial peptide found in the skin secretion of frogs of the genus Phyllomedusa, onSchistosoma mansoni adult worms. DS 01 at a concentration of 100 mg/ml reduced the worm motor activity and caused the death of all worms within 48 h in RPMI 1640 medium. At the highest sublethal concentration of antimicrobial peptide (75 mg/ml), a 100% reduction in egg output of paired female worms was observed. Additionally, DS 01 induced morphological alterations on the tegument of S. mansoni, and a quantitative analysis carried out by confocal microscopy revealed extensive destruction of the tubercles in a dose-dependent manner over the concentration range of 50 –200 mg/ml. It was the first time that an anthelmintic activity towards schistosomes has been reported for a dermaseptin.

Expression of mouse beta defensin 2 in Escherichia coli and its broad-spectrum antimicrobial activity

Mature mouse beta defensin 2 (mBD2) is a small cationic peptide with antimicrobial activity. Here we established a prokaryotic expression vector containing the cDNA of mature mBD2 fused with thioredoxin (TrxA), pET32a-mBD2. The vector was transformed into Escherichia Coli (E. coli) Rosseta-gami (2) for expression fusion protein. Under the optimization of fermentation parameters: induce with 0.6 mM isopropylthiogalactoside (IPTG) at 34ºC in 2×YT medium and harvest at 6 h postinduction, fusion protein TrxA-mBD2 was high expressed in the soluble fraction (>95 percent). After cleaved fusion protein by enterokinase, soluble mature mBD2 was achieved 6 mg/L with a volumetric productivity. Purified recombinant mBD2 demonstrated clear broad-spectrum antimicrobial activity for fungi, bacteria and virus. The MIC of antibacterial activity of against Staphylococcus aureus was 50 µg/ml. The MIC of against Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans) was 12.5µg/ml and 25µg/ml, respectively. Also, the antimicrobial activity of mBD2 was effected by NaCl concentration. Additionally, mBD2 showed antiviral activity against influenza A virus (IAV), the protective rate for Madin-Darby canine kidney cells (MDCK) was 93.86 percent at the mBD2 concentration of 100 µg/ml. These works might provide a foundation for the following research on the mBD2 as therapeutic agent for medical microbes.

Novel dermaseptin, adenoregulin and caerin homologs from the Central American red-eyed leaf frog, Agalychnis callidryas, revealed by functional peptidomics of defensive skin secretion.

By integrating systematic peptidome and transcriptome studies of the defensive skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas, we have identified novel members of three previously described antimicrobial peptide families, a 27-mer dermaseptin-related peptide (designated DRP-AC4), a 33-mer adenoregulin-related peptide (designated ARP-AC1) and most unusually, a 27-mer caerin-related peptide (designated CRP-AC1). While dermaseptin and adenoregulin were originally isolated from phyllomedusine leaf frogs, the caerins, until now, had only been described in Australian frogs of the genus, Litoria. Both the dermaseptin and adenoregulin were C-terminally amidated and lacked the C-terminal tripeptide of the biosynthetic precursor sequence. In contrast, the caerin-related peptide, unlike the majority of Litoria analogs, was not C-terminally amidated. The present data emphasize the need for structural characterization of mature peptides to ensure that unexpected precursor cleavages and/or post-translational modifications do not produce mature peptides that differ in structure to those predicted from cloned biosynthetic precursor cDNA. Additionally, systematic study of the secretory peptidome can produce unexpected results such as the CRP described here that may have phylogenetic implications. It is thus of the utmost importance in the functional evaluation of novel peptides that the primary structure of the mature peptide is unequivocally established -- something that is often facilitated by cloning biosynthetic precursor cDNAs but obviously not reliable using such data alone.

Under-expression of VHL and over-expression of HDAC-1, HIF-1alpha, LL-37, and IAP-2 in affected skin biopsies of patients with psoriasis.

BACKGROUND: A feature of psoriasis is the rapid proliferation of keratinocytes, during which apoptosis is blocked and angiogenesis starts. It is known that tumor hypoxic cells produce histone deacetylase-1 (HDAC-1), which up-regulates hypoxia-inducible factor-1alpha (HIF-1alpha) and down-regulates von Hippel-Lindau (VHL) protein by up-regulating vascular endothelial growth factor (VEGF) expression. It has been reported recently that the porcine peptide PR39 (homologous to human LL-37) has angiogenic and antiapoptotic activity. Thus, LL-37, induced by insulin-like growth factor-1 (IGF-1), could help in the production of VEGF. PR39 also induces the expression of inhibitor of apoptosis protein-2 (IAP-2), which blocks apoptosis. The purpose of this work was to analyze whether these genes and their proteins are expressed in psoriatic biopsies. METHODS: Using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) messenger RNA (mRNA) expression and immunohistochemical staining, we studied VHL, IAP-2, and related genes in skin biopsies from psoriatic patients and healthy subjects. RESULTS: An over-expression of the mRNA for HDAC-1, HIF-1alpha, LL-37, and IGF-1 in psoriatic skin, in comparison with skin from healthy subjects, was found. The antiangiogenic VHL mRNA and protein were under-expressed in psoriatic skin and highly expressed in healthy skin. The antiapoptotic IAP-2 was over-expressed in dermal endothelial cells from psoriatic skin. The pro-apoptotic Bax, Fas, and FasL mRNAs were expressed. CONCLUSIONS: These findings suggest that there could be an association of HDAC-1, HIF-1alpha, LL-37, VHL, and IAP-2 with angiogenic and apoptotic mechanisms in psoriasis.