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1.
Article in English | WPRIM | ID: wpr-785397

ABSTRACT

BACKGROUND: Pathogenic variants of USH1C, encoding a PDZ-domain-containing protein called harmonin, have been known to cause autosomal recessive syndromic or nonsyndromic hearing loss (NSHL). We identified a causative gene in a large Korean family with NSHL showing a typical pattern of autosomal dominant (AD) inheritance.METHODS: Exome sequencing was performed for five affected and three unaffected individuals in this family. Following identification of a candidate gene variant, segregation analysis and functional studies, including circular dichroism and biolayer interferometry experiments, were performed.RESULTS: A novel USH1C heterozygous missense variant (c.667G>T;p.Gly223Cys) was shown to segregate with the NSHL phenotype in this family. This variant affects an amino acid residue located in the highly conserved carboxylate-binding loop of the harmonin PDZ2 domain and is predicted to disturb the interaction with cadherin-related 23 (cdh23). The affinity of the variant PDZ2 domain for a biotinylated synthetic peptide containing the PDZ-binding motif of cdh23 was approximately 16-fold lower than that of the wild-type PDZ2 domain and that this inaccessibility of the binding site was caused by a conformational change in the variant PDZ2 domain.CONCLUSIONS: A heterozygous variant of USH1C that interferes with the interaction between cdh23 and harmonin causes novel AD-NSHL.


Subject(s)
Binding Sites , Circular Dichroism , Exome , Hearing Loss , Hearing , Humans , Interferometry , Phenotype , Wills
2.
Article in English | WPRIM | ID: wpr-761893

ABSTRACT

BACKGROUND: Nano-hydroxyapatite/polyamide 66 (nHA/PA66) is a composite used widely in the repair of bone defects. However, this material is insufficient bioactivity. In contrast, D-RADA16-RGD self-assembling peptide (D-RADA16-RGD sequence containing all D-amino acids is Ac-RADARADARADARADARGDS-CONH2) shows admirable bioactivity for both cell culture and bone regeneration. Here, we describe the fabrication of a favorable biomaterial material (nHA/PA66/D-RADA16-RGD). METHODS: Proteinase K and circular dichroism spectroscopy were employed to test the stability and secondary structural properties of peptide D-RADA16-RGD respectively. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to characterize the surface of these materials. Confocal laser scanning (CLS), cell counting kit-8 tests (CCK-8), alizarin red S staining, cell immunofluorescence analysis and Western blotting were involved in vitro. Also biosafety and bioactivity of them have been evaluated in vivo. RESULTS: Proteinase K and circular dichroism spectroscopy demonstrated that D-RADA16-RGD in nHA/PA66 was able to form stable-sheet secondary structure. SEM and TEM showed that the D-RADA16-RGD material was 7–33 nm in width and 130–600 nm in length, and the interwoven pore size ranged from 40 to 200 nm. CLS suggests that cells in nHA/PA66/D-RADA16-RGD group were linked to adjacent cells with more actin filaments. CCK-8 analysis showed that nHA/PA66/D-RADA16-RGD revealed good biocompatibility. The results of Alizarin-red S staining and Western blotting as well as vivo osteogenesis suggest nHA/PA66/D-RADA16-RGD exhibits better bioactivity. CONCLUSION: This study demonstrates that our nHA/PA66/D-RADA16-RGD composite exhibits reasonable mechanical properties, biocompatibility and bioactivity with promotion of bone formation.


Subject(s)
Actin Cytoskeleton , Blotting, Western , Bone Regeneration , Cell Count , Cell Culture Techniques , Circular Dichroism , Endopeptidase K , Fluorescent Antibody Technique , In Vitro Techniques , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Osteogenesis , Sincalide , Spectrum Analysis
3.
Biosci. j. (Online) ; 34(3): 778-789, mai/jun. 2018. graf
Article in English | LILACS | ID: biblio-967000

ABSTRACT

Usually, weak inorganic acids have been used to disperse collagen as green solvents for fabricating kinds of biomaterials all the time. However, it is an open question how much the dissolving process preserves or alters the native structure of collagen till now. Herein, we have examined the effect of three different solvents (HAc, HCl, H3PO4) on the secondary structures of collagen, based on circular dichroism (CD) spectra of collagen from 185 to 260 nm together with CDNN programs. We have found that collagen almost completely preserved its triple helical structure in the three inorganic acids at pH=3.0 or so, which demonstrated that it was the concentration of free H+ in the above three solutions whose pH was fixed at 3.0 that can maintain an proper amount of surface charge on the collagen colloidal particles and appropriately loose the three-helix structure, which can not only lead to a better dispersion behavior, but also maximize the preservation of the integrity of the collagen structure. Although the fractions of kinds of secondary structures in collagen were different from all the three solvents based on CDNN data, which gave very similar results for each other. These results was tested for the first time in this work to estimate the secondary structures for collagen in the different common inorganic acids, which provides a new avenue for green collagen solvents to prepare collagen-based composite with well triple-helical structure for tissue engineering.


Habitualmente, os ácidos inorgânicos fracos têm sido usados para dispersar colágeno como solventes verdes para fabricar tipos de biomateriais o tempo todo. No entanto, é uma questão aberta quanto o processo de dissolução preserva ou altera a estrutura nativa do colágeno até agora. Aqui, examinamos o efeito de três solventes diferentes (HAc, HCl, H3PO4) nas estruturas secundárias de colágeno, com base em espectros de dicroísmo circular (CD) de colágeno de 185 a 260 nm em conjunto com programas CDNN. Descobrimos que o colágeno preservou quase completamente sua estrutura helicoidal tripla nos três ácidos inorgânicos a pH = 3,0 ou mais, o que demonstrou que foi a concentração de H+ livre nas três soluções acima cujo pH foi fixado em 3,0 que pode manter uma boa quantidade de carga superficial sobre as partículas coloidais de colágeno e destrói adequadamente a estrutura de três hélices, o que não só pode levar a um melhor comportamento de dispersão, mas também maximizar a preservação da integridade da estrutura de colágeno. Embora as frações de tipos de estruturas secundárias em colágeno fossem diferentes de todos os três solventes com base em dados CDNN, que deram resultados muito semelhantes entre si. Estes resultados foram testados pela primeira vez neste trabalho para estimar as estruturas secundárias para o colágeno nos diferentes ácidos inorgânicos comuns, o que fornece uma nova alternativa para solventes de colágeno verdes para preparar compósitos à base de colágeno com a estrutura helicoidal tripla para engenharia de tecidos.


Subject(s)
Circular Dichroism , Collagen Type I , Inorganic Acids , Solvents , Biocompatible Materials
4.
Article in English | WPRIM | ID: wpr-773605

ABSTRACT

One new sorbicillin derivative, 2-deoxy-sohirnone C (1), one new diketopiperazine alkaloid, 5S-hydroxynorvaline-S-Ile (2), and two naturally occurring diketopiperazines, 3S-hydroxylcyclo(S-Pro-S-Phe) (3) and cyclo(S-Phe-S-Gln) (4), together with three known compounds were isolated from the Chinese mangrove endophytic fungus Penicillium sp. GD6. Their structures were determined on the basis of extensive spectroscopic analyses and by comparison with literature data. The absolute configuration of 3-hydroxyl moiety in 3 was determined by Mosher's method, while the absolute stereochemistry of 2 and 4 was established by comparison with the CD spectra of natural and synthesized diketopiperazines. Compound 1 showed moderate antibacterial activity against Methicillin-resistant Staphylococcus aureus with a MIC value of 80 μg·mL.


Subject(s)
Alkaloids , Chemistry , Anti-Bacterial Agents , Chemistry , Pharmacology , China , Circular Dichroism , Diketopiperazines , Chemistry , Methicillin-Resistant Staphylococcus aureus , Microbial Sensitivity Tests , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Penicillium , Chemistry , Resorcinols , Chemistry , Pharmacology , Rhizophoraceae , Microbiology , Wetlands
5.
Article in English | WPRIM | ID: wpr-812394

ABSTRACT

One new sorbicillin derivative, 2-deoxy-sohirnone C (1), one new diketopiperazine alkaloid, 5S-hydroxynorvaline-S-Ile (2), and two naturally occurring diketopiperazines, 3S-hydroxylcyclo(S-Pro-S-Phe) (3) and cyclo(S-Phe-S-Gln) (4), together with three known compounds were isolated from the Chinese mangrove endophytic fungus Penicillium sp. GD6. Their structures were determined on the basis of extensive spectroscopic analyses and by comparison with literature data. The absolute configuration of 3-hydroxyl moiety in 3 was determined by Mosher's method, while the absolute stereochemistry of 2 and 4 was established by comparison with the CD spectra of natural and synthesized diketopiperazines. Compound 1 showed moderate antibacterial activity against Methicillin-resistant Staphylococcus aureus with a MIC value of 80 μg·mL.


Subject(s)
Alkaloids , Chemistry , Anti-Bacterial Agents , Chemistry , Pharmacology , China , Circular Dichroism , Diketopiperazines , Chemistry , Methicillin-Resistant Staphylococcus aureus , Microbial Sensitivity Tests , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Penicillium , Chemistry , Resorcinols , Chemistry , Pharmacology , Rhizophoraceae , Microbiology , Wetlands
6.
Electron. j. biotechnol ; 29: 7-12, sept. 2017. ilus, graf, tab
Article in English | LILACS | ID: biblio-1016095

ABSTRACT

Background: DegP is a serine protease that specifically cleaves and refolds unfolding proteins in the periplasmic space of the cells. To date, there is no information regarding DegP from halophilic bacteria. Chromohalobacter salexigens BKL5 is a moderately halophilic bacterium that has the ability to grow in a media containing more than 15% salt. Therefore, the objectives of this work were to clone and overexpress DegP-encoding gene from C. salexigens BKL5 and characterize its biochemical properties. Results: DegP-encoding gene was overexpressed in Escherichia coli BL21(DE3) CodonPlus in an active form. SDS-PAGE analysis showed that the molecular weight of the recombinant DegP was 45 kDa. Size-exclusion chromatography analysis suggested that recombinant DegP was present in two multimeric states, hexameric and dodecameric, with molecular weights of 297.9 and 579.12 kDa, respectively. Both conformations were enzymatically active when casein was used as substrate for enzymatic assay. Circular dichroism analysis showed that recombinant DegP was composed of 0.21­0.29 helical content, which was comparable to the helical content in the crystal structure of E. coli DegP. The basic/acidic residue ratio of recombinant DegP was 0.56, which was slightly higher than that of DegP from extreme halophiles (average, 0.45) but significantly lower than that of DegP from nonhalophiles (average, 0.94). Conclusions: Recombinant DegP from C. salexigens BKL5 showed proteolytic activity when ß-casein was used as a substrate. In silico analysis indicated that recombinant DegP had characteristics similar to those of halophilic proteins depending on its amino acid composition.


Subject(s)
Serine Endopeptidases/genetics , Periplasmic Proteins/genetics , Chromohalobacter/enzymology , Proteolysis , Heat-Shock Proteins/genetics , Recombinant Proteins , Serine Endopeptidases/metabolism , Caseins , Chromatography, Gel , Circular Dichroism , Cloning, Molecular , Periplasmic Proteins/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Salinity , Chromohalobacter/genetics , Heat-Shock Proteins/metabolism , Molecular Weight
7.
Article in English | ID: biblio-954808

ABSTRACT

Background The availability of antimicrobial peptides from several different natural sources has opened an avenue for the discovery of new biologically active molecules. To the best of our knowledge, only two peptides isolated from the frog Leptodactylus labyrinthicus, namely pentadactylin and ocellatin-F1, have shown antimicrobial activities. Therefore, in order to explore the antimicrobial potential of this species, we have investigated the biological activities and membrane interactions of three peptides isolated from the anuran skin secretion. Methods Three peptide primary structures were determined by automated Edman degradation. These sequences were prepared by solid-phase synthesis and submitted to activity assays against gram-positive and gram-negative bacteria and against two fungal strains. The hemolytic properties of the peptides were also investigated in assays with rabbit blood erythrocytes. The conformational preferences of the peptides and their membrane interactions have been investigated by circular dichroism spectroscopy and liposome dye release assays. Results The amino acid compositions of three ocellatins were determined and the sequences exhibit 100% homology for the first 22 residues (ocellatin-LB1 sequence). Ocellatin-LB2 carries an extra Asn residue and ocellatin-F1 extra Asn-Lys-Leu residues at C-terminus. Ocellatin-F1 presents a stronger antibiotic potential and a broader spectrum of activities compared to the other peptides. The membrane interactions and pore formation capacities of the peptides correlate directly with their antimicrobial activities, i.e., ocellatin-F1 > ocellatin-LB1 > ocellatin-LB2. All peptides acquire high helical contents in membrane environments. However, ocellatin-F1 shows in average stronger helical propensities. Conclusions The obtained results indicate that the three extra amino acid residues at the ocellatin-F1 C-terminus play an important role in promoting stronger peptide-membrane interactions and antimicrobial properties. The extra Asn-23 residue present in ocellatin-LB2 sequence seems to decrease its antimicrobial potential and the strength of the peptide-membrane interactions.(AU)


Subject(s)
Peptides , Biological Products , Circular Dichroism , Anti-Bacterial Agents , Anura/physiology , Anti-Infective Agents
8.
Article in English | WPRIM | ID: wpr-219681

ABSTRACT

PURPOSE: The sequence variations of the Der p 2 allergen of Dermatophagoides pteronyssinus diverge along 2 pathways with particular amino acid substitutions at positions 40,47,111, and 114. The environmental prevalence and IgE binding to Der p 2 variants differ among regions. To compare IgE binding to Der p 2 variants between sera from Bangkok, Thailand and Perth, Western Australia with different variants and to determine the variant-specificity of antibodies induced by vaccination with recombinant variants. METHODS: The structures of recombinant variants produced in yeast were compared by circular dichroism and 1-anilinonaphthalene 8-sulfonic acid staining of their lipid-binding cavity. Sera from subjects in Bangkok and Perth where different variants are found were compared by the affinity (IC50) of IgE cross-reactivity to different variants and by direct IgE binding. Mice were immunized with the variants Der p 2.0101 and Der p 2.0110, and their IgG binding to Der p 2.0103, 2.0104, and 2.0109 was measured. RESULTS: The secondary structures of the recombinant variants resembled the natural allergen but with differences in ANS binding. The IC50 of Der p 2.0101 required 7-fold higher concentrations to inhibit IgE binding to the high-IgE-binding Der p 2.0104 than for homologous inhibition in sera from Bangkok where it is absent, while in sera from Perth that have both variants the IC50 was the same and low. Reciprocal results were obtained for Der p 2.0110 not found in Perth. Direct binding revealed that Der p 2.0104 was best for detecting IgE in both regions, followed by Der p 2.0101 with binding to other variants showing larger differences. Mouse anti-Der p 2.0101 antibodies had a high affinity of cross-reactivity but bound poorly to other variants. CONCLUSIONS: The affinity of IgE antibody cross-reactivity, the direct IgE binding, and the specificities of antibodies induced by vaccination show that measures of allergic sensitization and therapeutic strategies could be optimized with knowledge of Der p 2 variants.


Subject(s)
Amino Acid Substitution , Animals , Antibodies , Circular Dichroism , Dermatophagoides pteronyssinus , Dust , Hypersensitivity , Immunoglobulin E , Immunoglobulin G , Inhibitory Concentration 50 , Mice , Prevalence , Pyroglyphidae , Thailand , Vaccination , Western Australia , Yeasts
9.
São Paulo; s.n; s.n; 2016. 190 p. graf, tab.
Thesis in Portuguese | LILACS | ID: biblio-881507

ABSTRACT

O nadolol é um agente bloqueador de receptores ß-adrenérgicos empregado principalmente, na "angina pectoris", hipertensão, certas arritmias cardíacas e no tratamento do glaucoma (SING, 2006). A ivermectina e a abamectina são fármacos que apresentam ação antiparasitária (SHOOP, 1995). Na presente pesquisa, a cromatografia em fase líquida de alta eficiência foi uma das técnicas estudadas para a quantificação dos enantiômeros do nadolol e dos homólogos presentes na abamectina e ivermectina. A versatilidade desta técnica reside no grande número de fases estacionárias existentes, as quais possibilitam análises, separações e determinações quantitativas de uma ampla gama de compostos com alta eficiência (Aquino Neto e Nunes, 2003). Para identificação dos enantiômeros do nadolol foi utilizado o dicroísmo circular que permite a determinação da configuração absoluta de enantiômeros (LIMA, 1997). Para os enantiômeros do nadolol e dos homólogos presentes na abamectina e na ivermectina também foram realizados testes para desenvolvimento de uma metodologia de quantificação por meio de uma técnica relativamente recente chamada de eletroforese capilar (EC), a qual tem alcançado desde sua introdução um rápido desenvolvimento e ampla aplicação na análise de fármacos em medicamentos (SANTORO, 2000). Para a comprovação da qualidade e segurança dos sistemas computadorizados dos equipamentos de cromatografia em fase líquida de alta eficiência (CLAE) e de eletroforese capilar (EC) foram efetuadas, neste trabalho, as respectivas validações. Após esta validação, pode-se confirmar o correto funcionamento de um software, e suas interações com o hardware, onde devem ser levados em consideração, dentre outros, os aspectos relacionados à infra-estrutura, segurança e manutenção de dados (AGÊNCIA NACIONAL DE VIGILÂNIA SANITÁRIA, 2010). As metodologias analíticas desenvolvidas a para quantificação do nadolol, abamectina e ivermectina por cromatografia em fase líquida de alta eficiência foram validadas. A validação analítica deve garantir, por meio de estudos experimentais, que o método atenda às exigências das aplicações analíticas, assegurando a confiabilidade dos resultados. Para tanto, o método deve apresentar especificidade, linearidade, intervalo, precisão, sensibilidade, limite de quantificação e detecção, exatidão, adequados à análise (AGÊNCIA NACIONAL DE VIGILÂNCIA SANITÁRIA, 2003). Portanto, o objetivo proposto nesta pesquisa é primeiramente a validação dos sistemas computadorizados dos equipamentos de cromatografia em fase líquida de alta eficiência (CLAE) e de eletroforese capilar (EC). Para isto, serão desenvolvidos e validados os métodos analíticos de separação, identificação e quantificação dos enantiômeros do nadolol e dos homólogos presentes na abamectina e na ivermectina, em medicamentos, empregando as técnicas analíticas selecionadas


Nadolol is a blocking agent with activity in the ß -adrenergic receptors. It is mainly used in angina, hypertension, certain heart arrhythmias and in the treatment of glaucoma (SING, 2006). Ivermectin and abamectin are drugs with antiparasitic activity (SHOOP, 1995). In the present research, high performance liquid chromatography is one of the techniques used in the quantification of the enantiomers of nadolol and homologues present in abamectin and ivermectin. The versatility of this technique and the large number of existing stationary phases, enables the separation and quantitative determination of a wide range of compounds with high efficiency (Aquino Neto e Nunes, 2003). For identification of the nadolol enantiomers, circular dichroism was used which allows the determination of the absolute configuration of the enantiomers (LIMA, 1997). Nadolol enantiomers and the homologues present in abamectin and ivermectin will be also quantified by capillary zone electrophoresis (CE), a separation technique relatively recent, which has achieved, since its introduction, a wide application in the analysis of drugs in pharmaceutical preparations (SANTORO, 2000). In order to assure the quality of the analytical results, the computer systems of the liquid chromatograph and capillary electrophoresis equipments, must be validated prior to the analytical methods validation. Computer systems validation is used to verify and confirm the proper operation of softwares, and their interactions with the hardwares, besides the infrastructure, safety and storage of data (AGÊNCIA NACIONAL DE VIGILÂNIA SANITÁRIA, 2010). The analytical methodologies developed for quantification of nadolol, abamectin, ivermectin by using high efficiency liquid chromatography and capillary electrophoresis were validated. The analytical methods validation should ensure, through experimental studies, that the method meets the requirements for analytical applications, ensuring the reliability of the results. Parameters like, specificity, linearity, range, accuracy, sensitivity, limits of detection and quantification and accuracy, must be determined (AGÊNCIA NACIONAL DE VIGILÂNCIA SANITÁRIA, 2003). The objective of this study is to validate the computer systems of the high performance liquid chromatograph and capillary electrophoresis equipments and then to develop and validate analytical methods for separation, identification and quantification of nadolol enantiomers and the homologues of abamectin and ivermectin


Subject(s)
Device Approval , Ivermectin , Nadolol/analysis , Validation Study , Analytical Methods/methods , Chromatography, High Pressure Liquid/instrumentation , Circular Dichroism , Electrophoresis, Capillary/instrumentation , Software Validation
10.
São Paulo; s.n; s.n; 2016. 201 p. tab, graf, ilus.
Thesis in Portuguese | LILACS | ID: biblio-847477

ABSTRACT

Esticolisinas I e II, citolisinas purificadas da anêmona marinha Stichodactyla helianthus, agem lisando membranas biológicas e modelo. O mecanismo de ação proposto consiste na formação de um poro toroidal com o envolvimento do domínio N-terminal. Diferentes aspectos da interação entre peptídeos derivados do N-terminal das toxinas (StI1-31 and StI12-31 SELAGTIIDGASLTFEVLDKVLGELGKVSRK, e StII1-30 and StII11-30 ALAGTIIAGASLTFQVLDKVLEELGKVSRK) com membranas modelo - micelas e bicamadas - foram estudados com o objetivo de contribuir para a elucidação do mecanismo de ação das toxinas em nível molecular. O emprego dos peptídeos teve como base a hipótese de que fragmentos proteicos podem ser capazes de mimetizar a estrutura e atividade das proteínas inteiras. O análogo contendo o aminoácido paramagnético TOAC (N-TOAC-StII11-30) também foi estudado. Estudos conformacionais foram realizados empregando-se as técnicas espectroscópicas de dicroísmo circular (CD), ressonância paramagnética eletrônica (EPR) e fluorescência. Foram ainda realizados estudos de predição de estrutura e modelagem molecular. Espectros de CD mostraram que os peptídeos adquirem conformação em α-hélice ao interagir com membranas modelo, de acordo com a conformação observada nessa região para as toxinas. Variando a composição lipídica das membranas modelo estudadas, foi possível investigar a contribuição de forças eletrostáticas de de interações hidrofóbicas para a ligação do peptídeo. Ensaios de supressão de fluorescência de lípidos contendo grupamentos fluorescentes em diferentes posições pelo resíduo paramagnético TOAC e espectros de ressonância paramagnética eletrônica (EPR) permitiram localizar o resíduo TOAC na interface membrana-água, corroborando o modelo proposto do poro toroidal. A análise dos espectros de CD e EPR também permitiu obter as constantes de ligação dos peptídeos com micelas e bicamadas. Os peptídeos também foram capazes de mimetizar as toxinas do ponto de vista funcional, como mostrado por testes de vazamento de carboxifluoresceína e atividade hemolítica. Peptídeos curtos, contendo partes da sequência de StII1-30, sintetizados com o objetivo de examinar uma eventual atividade antimicrobiana, demonstraram baixa atividade, bem como ausência de atividade hemolítica e de toxicidade para células humanas


Sticholysins I and II, cytolysins purified from the sea anemone Stichodactyla helianthus, act by lysing biological and model membranes. The proposed mechanism of action consists in the formation of a toroidal pore with the involvement of the N-terminal domain [1]. Different aspects of the interaction between peptides from the toxins' N-termini (StI1-31 and StI12-31 SELAGTIIDGASLTFEVLDKVLGELGKVSRK, and StII1-30 and StII11-30 ALAGTIIAGASLTFQVLDKVLEELGKVSRK) and model membranes - micelles and bilayers - have been studied to contribute to the elucidation of the toxins mechanism of action at the molecular level. The use of peptides was based on the hypothesis that potein fragments can eventually mimic the structure and activity of the whole protein. An analogue containing the paramagnetic amino acid TOAC (N-TOAC-StII11-30) was also studied. Conformational studies were performed making use of the spectroscopic techniques circular dichroism (CD), electron paramagnetic resonance (EPR), and fluorescence. Studies of structure prediction and molecular modeling were also performed. CD spectra showed that the peptides acquired α-helical conformation upon interaction with model lipid membranes, in agreement with the conformation found for these segments in the whole proteins. Making use of membranes of variable lipid composition, it was possible to assess the contribution of electrostatic and hydrophobic interactions for peptide binding. Fluorescence quenching of labeled lipids by paramagnetic TOAC and EPR spectra allowed us to locate the TOAC residue at the membrane-water interface, corroborating the proposed model of the toroidal pore. The CD and EPR studies also allowed us to obtain the binding constants for the peptide-micelle and peptide-bilayer interaction. The peptides were also capable of mimicking the toxins function, as shown by assays of carboxyfluorescein leakage and hemolytic activity. Short peptides containing parts of StII1-30's sequence were synthesized with the aim of testing their antimicrobial activity. The peptides displayed low antimicrobial activity, as well as lack of hemolytic activity and toxicity against human cells


Subject(s)
Electron Spin Resonance Spectroscopy/methods , Peptides/analysis , Spectrum Analysis/methods , Bacterial Infections/prevention & control , Circular Dichroism/instrumentation , Models, Structural , Structure-Activity Relationship , Tomography, Spiral Computed
11.
Natural Product Sciences ; : 273-277, 2015.
Article in English | WPRIM | ID: wpr-7747

ABSTRACT

Comprehensive chemical analysis of extracts and fractions of marine actinomycete strains led to the discovery of a new minor secondary metabolite, salternamide E (1), from a saltern-derived halophilic Streptomyces strain. The planar structure of salternamide E (1) was elucidated by a combinational analysis of spectroscopic data including NMR, MS, UV, and IR. The absolute configuration of salternamide E (1) was determined by circular dichroism spectroscopic analysis. Salternamide E displayed weak cytotoxicity against various human carcinoma cell lines.


Subject(s)
Cell Line , Circular Dichroism , Humans , Streptomyces
12.
Chinese Journal of Biotechnology ; (12): 1651-1659, 2015.
Article in Chinese | WPRIM | ID: wpr-240546

ABSTRACT

The relationship between the conformation of interferon-α (IFN-α) and its anti-viral activity were analyzed by circular dichroism (CD) and flow cytometry (FCM) techniques. The recombinant human IFN-α (rIFN-α2b and rIFN-α2a) were used. CD spectra from 190 nm to 240 nm indicated that two the IFN-α showed stable secondary structure at 65 degrees C, but unstable when the temperature was above 65 degrees C, and the change was irreversible. FCM data of the anti-viral activity of IFN-α indicated that the change of its secondary structures partly weakened its anti-viral activity. The rIFN-α2b and rIFN-α2a showed the same phenomenon. These data indicated that the conformation of IFN-α is one of the factors to influence its anti-viral activity and the combination of CD and FCM is a good method to analyze the relationship between the conformation of protein drugs and their biological activities in single cell level.


Subject(s)
Antiviral Agents , Chemistry , Circular Dichroism , Flow Cytometry , Humans , Interferon-alpha , Chemistry , Protein Structure, Secondary , Recombinant Proteins , Chemistry
13.
Article in English | WPRIM | ID: wpr-812480

ABSTRACT

The stereochemistry of two 6, 9-oxygen bridge dibenzocyclooctadiene lignans from Kadsura coccinea, are difficult to separate and very unstable. The present study was designed to develop a high-performance liquid chromatography using circular dichroism detection for the analysis of the stereochemistry. A new 6, 9-oxygen bridge dibenzocyclooctadiene lignans named Kadsulignan Q was firstly found with an S-biphenyl configuration. The other compound was identified as Kadsulignan L with an R- biphenyl configuration. In order to obtain kinetic data on their reversible interconversion, the stability was measured at different deuterated solvents such as deuterated methanol, deuterated chloroform and deuterated dimethylsulfoxide. The lignans were more unstable and converted more easily in deuterated methanol than in deuterated chloroform and deuterated dimethylsulfoxide.


Subject(s)
Chromatography, High Pressure Liquid , Circular Dichroism , Cyclooctanes , Chemistry , Kadsura , Chemistry , Lignans , Chemistry , Molecular Structure , Oxygen , Plant Extracts , Chemistry , Stereoisomerism
14.
Protein & Cell ; (12): 631-642, 2014.
Article in English | WPRIM | ID: wpr-757476

ABSTRACT

A major barrier to the use of antimicrobial peptides as antibiotics is the toxicity or ability to lyse eukaryotic cells. In this study, a 26-residue amphipathic α-helical antimicrobial peptide A12L/A20L (Ac-KWKSFLKTFKSLKKTVLHTLLKAISS-amide) was used as the framework to design a series of D- and L-diastereomeric peptides and study the relationships of helicity and biological activities of α-helical antimicrobial peptides. Peptide helicity was measured by circular dichroism spectroscopy and demonstrated to correlate with the hydrophobicity of peptides and the numbers of D-amino acid substitutions. Therapeutic index was used to evaluate the selectivity of peptides against prokaryotic cells. By introducing D-amino acids to replace the original L-amino acids on the non-polar face or the polar face of the helix, the hemolytic activity of peptide analogs have been significantly reduced. Compared to the parent peptide, the therapeutic indices were improved of 44-fold and 22-fold against Gram-negative and Gram-positive bacteria, respectively. In addition, D- and L-diastereomeric peptides exhibited lower interaction with zwitterionic eukaryotic membrane and showed the significant membrane damaging effect to bacterial cells. Helicity was proved to play a crucial role on peptide specificity and biological activities. By simply replacing the hydrophobic or the hydrophilic amino acid residues on the non-polar or the polar face of these amphipathic derivatives of the parent peptide with D-amino acids, we demonstrated that this method could have excellent potential for the rational design of antimicrobial peptides with enhanced specificity.


Subject(s)
Anti-Infective Agents , Chemistry , Pharmacology , Circular Dichroism , Drug Design , Erythrocytes , Gram-Negative Bacteria , Gram-Positive Bacteria , Hemolysis , Humans , Peptide Fragments , Chemistry , Pharmacology , Protein Structure, Secondary , Structure-Activity Relationship , Substrate Specificity
15.
Pakistan Journal of Pharmaceutical Sciences. 2013; 26 (4): 733-740
in English | IMEMR | ID: emr-126966

ABSTRACT

The enzyme isopentenyl diphosphate isomerase [IDI, EC 5.3.3.2] interconverts isopentenyl diphosphate and dimethylallyl diphosphate. We had previously cloned Tk-idi gene encoding the thermostable Tk-IDI enzyme from Thermococcus kodakaraensis KOD1. Four putative start codons were found on Tk-idi gene at 123, 213, 297 and 321 positions downstream of the first start codon. In the present work four mutants were obtained by deleting 123, 213, 297 and 321 nucleotides from the 5'-end of Tk-idi gene to obtain Tk-idim, Tk-idim1, Tk-idim2, and Tk-idim3, respectively. When we tried to express these truncated genes in Escherichia coli only Tk-idim was expressed in the active form. The product, Tk-IDIM, was purified and characterized. The molecular mass of the enzyme, estimated by gel filtration chromatography, was 300 kDa which indicated that the truncated enzyme retained the octameric form. The removal of 41 N-terminal amino acids did not exhibit a significant effect on the enzyme activity however, the thermostability of the enzyme decreased. The decrease in thermostability of Tk-IDIM correlated well with the results of circular dichroism [CD] analysis and structural modeling


Subject(s)
Thermococcus , Organophosphorus Compounds , Hemiterpenes , Codon , Genes , Escherichia coli , Chromatography, Gel , Circular Dichroism , Models, Structural
16.
Acta Pharmaceutica Sinica ; (12): 366-371, 2013.
Article in Chinese | WPRIM | ID: wpr-235658

ABSTRACT

A novel peptide, named BF2-X, was designed based on the structure-activity analysis of an analogue of Buforin II, named BF2-A. The BF2-X was a hybrid peptide containing the N-terminal residues 5 to 13 of BF2-A and three repeats of the C-terminal regular alpha-helical motif RLLR, and the residues 8 valine were replaced by leucine. The results of bioinformatics analysis had showed that compared with BF2-A, the helicity, positive charge, hydrophobicity rate and C-terminal amphipathy of BF2-X had remarkably enhanced. Both peptides showed a random coil structure in an aqueous solution, while displaying a typical alpha-helical structure in 50% trifluoroethanol solution (a membrane mimic condition). BF2-X exhibited higher alpha-helical contents than BF2-A in hydrophobic environment. BF2-X displayed potent antimicrobial activities against a broad spectrum of microorganisms. And BF2-X showed stronger antimicrobial activities against bacteria tested than parent peptide BF2-A. These results suggest that the alpha-helical content was directly correlated with the enhanced antibacterial activity. Both peptides had no hemolytic action on mouse erythrocyte.


Subject(s)
Amino Acid Sequence , Animals , Anti-Bacterial Agents , Chemistry , Pharmacology , Antimicrobial Cationic Peptides , Chemistry , Pharmacology , Bacteria , Circular Dichroism , Hemolysis , Hydrophobic and Hydrophilic Interactions , Mice , Protein Structure, Secondary , Proteins , Chemistry , Pharmacology , Structure-Activity Relationship
17.
São Paulo; s.n; s.n; 2013. 129 p. tab, graf, ilus.
Thesis in Portuguese | LILACS | ID: biblio-846885

ABSTRACT

A Esclerose Lateral Amiotrófica (ELA) é uma doença progressiva e fatal causada pela degeneração seletiva dos neurônios motores do cérebro e medula. Dos casos familiares de ELA (fELA), 20% são causados por mutações pontuais no gene da sod1. O ácido docosahexaenoico (C22:6, n-3, DHA) é um ácido graxo altamente insaturado, sendo um dos principais ácidos graxos da massa cinzenta do cérebro. Estudos têm correlacionado mutações de SOD1 com a formação de agregados que poderiam ser induzidos por ácidos graxos insaturados. O objetivo deste estudo foi avaliar os efeitos e mecanismos do DHA e de seus hidroperóxidos (DHAOOH) na agregação de SOD1 in vitro. As análises de dicroísmo circular (CD) mostraram mudanças na estrutura secundária de ambas as proteínas apo-SOD1WT e G93A promovidas pelo DHA, resultando em aumento de superfície hidrofóbica e formação de estruturas do tipo beta-amilóide, como mostrado pelos ensaios do bis- ANS e Tioflavina, respectivamente. Estas mudanças resultam na formação de agregados amorfos como observado por microscopia eletrônica de varredura (MEV). Espécies de alto peso molecular foram observadas nas incubações do DHA com as formas apo da SOD1 por SDS-PAGE sob condições não redutoras e também por cromatografia de exclusão por tamanho. A formação dos agregados mostrou-se dependente de resíduos de Cys na sua forma desprotonada, visto que agregados não foram observados na presença de beta-mercaptoetanol e sua formação foi inibida na presença de bloqueador de tióis e em pH ácido. Além disso, análises por cromatografia de exclusão mostraram que a agregação é dependente da insaturação e conformação cis dos ácidos graxos. Comparativamente ao DHA, os hidroperóxidos do DHA tiveram um efeito menor na agregação de SOD1, porém revelaram a propriedade de induzir a dimerização covalente de SOD1. No geral, os dados mostram que o DHA induz a agregação de SOD1, através de um processo envolvendo a exposição de superfícies hidrofóbicas, formação de pontes dissulfeto e também de possíveis cross-links envolvendo reações do tipo "ene-tiol"


ALS is a progressive and fatal disease caused by selective degeneration of motor neurons in the brain and spinal cord. Twenty percent of familial ALS (fALS) cases are caused mainly by point mutations in the sod1 gene. Docosahexaenoic acid (C22:6, n-3, DHA) is a highly unsaturated fatty acid, wich is one of the main fatty acids in the cerebral gray matter. Studies have linked SOD1 mutations to the formation of aggregates that could be induced by unsaturated fatty acids. The aim of this study was to evaluate the effect of DHA on aggregation of SOD1 fALS mutants in vitro and its mechanisms. CD analysis shows changes in the secondary structure of both apo-SOD1WT and G93A promoted by DHA resulting in an increase in the surface hydrophobicity and formation of structures such as beta amyloid, which was also confirmed by bis-ANS assay and Thioflavin, respectively. These changes enhance the interaction of SOD1 and DHA, leading to amorphous aggregates as revealed by FESEM. Incubation of DHA with apo-SOD1 forms results in high-molecular weight species as detected by SDS-PAGE analyses under non-reducing conditions and also by size exclusion chromatography. This appears to require Cys residues in their thiolate forms because high aggregates are not observed under reducing conditions and also by size exclusion chromatography or at acidic pH. Also, size-exclusion chromatography indicates that the mutant apo-SOD1 aggregation is dependent on the unsaturation and cis-conformation of fatty acids. Compared to the DHA, DHAOOH had a minor effect on SOD1 aggregation, however revealed the ability to induce covalent dimerization of SOD1. Overall, the data suggest a mechanism of DHA aggregation, by a process involving exposure to hydrophobic surfaces, formation of disulfide bonds and also for possible cross-links involving reactions such "thiol-ene"


Subject(s)
Amyotrophic Lateral Sclerosis/pathology , Docosahexaenoic Acids/analysis , Peracetic Acid , Superoxide Dismutase-1 , Chromatography, High Pressure Liquid/methods , Circular Dichroism/instrumentation , Microscopy, Electron, Scanning/methods
18.
Article in English | WPRIM | ID: wpr-727603

ABSTRACT

We recently reported a Philyra pisum lectin (PPL) that exerts mitogenic effects on human lymphocytes, and its molecular characterization. The present study provides a more detailed characterization of PPL based on the results from a monosaccharide analysis indicating that PPL is a glycoprotein, and circular dichroism spectra revealing its estimated alpha-helix, beta-sheet, beta-turn, and random coil contents to be 14.0%, 39.6%, 15.8%, and 30.6%, respectively. These contents are quite similar to those of deglycosylated PPL, indicating that glycans do not affect its intact structure. The binding properties to different pathogen-associated molecular patterns were investigated with hemagglutination inhibition assays using lipoteichoic acid from Gram-positive bacteria, lipopolysaccharide from Gram-negative bacteria, and both mannan and beta-1,3-glucan from fungi. PPL binds to lipoteichoic acids and mannan, but not to lipopolysaccharides or beta-1,3-glucan. PPL exerted no significant antiproliferative effects against human breast or bladder cancer cells. These results indicate that PPL is a glycoprotein with a lipoteichoic acid or mannan-binding specificity and which contains low and high proportions of alpha-helix and beta-structures, respectively. These properties are inherent to the innate immune system of P. pisum and indicate that PPL could be involved in signal transmission into Gram-positive bacteria or fungi.


Subject(s)
beta-Glucans , Breast , Circular Dichroism , Fungi , Glycoproteins , Gram-Negative Bacteria , Gram-Positive Bacteria , Hemagglutination , Humans , Immune System , Lipopolysaccharides , Lymphocytes , Mannans , Polysaccharides , Sensitivity and Specificity , Teichoic Acids , Urinary Bladder Neoplasms
19.
Article in English | WPRIM | ID: wpr-812705

ABSTRACT

Structural elucidation of natural products is always one of the most important tasks for natural product researchers in related fields. Particularly, the absolute configuration (AC), being a great challenge for natural product chemists, has attracted much attention. During the past few decades, many techniques and approaches have been developed to determine the AC of natural products, including direct (or absolute) methods, e.g. X-ray diffraction (XRD), electronic and vibrational circular dichroism (ECD and VCD), and Raman optical activity (ROA), as well as indirect (or relative) methods using a reference or a derivatizing agent with known AC, e.g. CD with empirical rules and nuclear magnetic resonance (NMR) utilizing anisotropic effects of chiral derivatizing agents. However, none of the currently applied techniques is capable of dominating AC determination, since they each have their respective limitations corresponding to the different structural features. This mini review summarizes most of the techniques and methods which are commonly used in AC assignment of natural products, or have potential application prospects, and briefly describes their principles, advantages and limitations.


Subject(s)
Biological Products , Chemistry , Circular Dichroism , Molecular Structure , Optical Rotation , X-Ray Diffraction
20.
Journal of Integrative Medicine ; (12): 405-415, 2013.
Article in English | WPRIM | ID: wpr-308227

ABSTRACT

<p><b>OBJECTIVE</b>To test if myricanone (C21H24O5), a cyclic diarylheptanoid, has anticancer effects on two different cancer cell lines HeLa and PC3. The present study was conducted with a note on the drug-DNA interaction and apoptotic signalling pathway.</p><p><b>METHODS</b>Several studies like cytotoxicity, nuclear damage, annexin-V-fluorescein isothiocyanate (FITC)/propidium iodide (PI)-labelled apoptotic assay and cell cycle arrest, immunoblot and reverse transcriptase-polymerase chain reaction (RT-PCR) were used following standard protocols. Circular dichroism (CD) spectroscopy was also done to evaluate whether myricanone effectively interacted with DNA to bring about conformational changes that could strongly inhibit the cancer cell proliferation.</p><p><b>RESULTS</b>Myricanone showed a greater cytotoxic effect on PC3 cells than on HeLa cells. Myricanone promoted G0/G1 arrest in HeLa cells and S phase arrest in PC3 cells. Nuclear condensation and annexin V-FITC/PI studies revealed that myricanone promoted apoptotic cell death. CD spectroscopic data indicated that myricanone had an interaction with calf thymus DNA that changed DNA structural conformation. RT-PCR and immunoblot studies revealed that myricanone activated the apoptotic signalling cascades through down-regulation of transcription factors like nuclear factor-κB (NF-κB) (p65), and signal transducers and activators of transcription 3 (STAT3); cell cycle regulators like cyclin D1, and survivin and other signal proteins like Bcl-2 and up-regulation of Bax, caspase-9 and caspase-3.</p><p><b>CONCLUSION</b>Myricanone induced apoptosis in both types of cancer cells by triggering caspase activation, and suppression of cell proliferation by down-regulation of NF-κB and STAT3 signalling cascades, which makes it a suitable candidate for possible use in the formulation of therapeutic agent for combating cancer.</p>


Subject(s)
Antineoplastic Agents, Phytogenic , Pharmacology , Apoptosis , Cell Cycle , Cell Line, Tumor , Cell Survival , Circular Dichroism , DNA , Metabolism , Diarylheptanoids , Metabolism , Pharmacology , Female , Humans , Male , Myrica , Chemistry , Plant Extracts , Signal Transduction , Spectroscopy, Fourier Transform Infrared
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