RESUMO
AIMS: To evaluate the applicability of the Mimosa tenuiflora and Eucalyptus urograndis pyroligneous acids (PAs) as alternative antiseptics in dairy goats. METHODS AND RESULTS: Cytotoxicity was evaluated in vitro using bacteria, as well as in vivo using goats, and the influence of PAs on the physicochemical parameters of fresh milk were examined. The cytotoxicity of PAs was evaluated in terms of morphology, cell viability and metabolic activity of goat tegumentary cells. The PA of M. tenuiflora had results similar to those of 2% iodine. For the in vitro tests, strains of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were used with the well technique, demonstrating inhibition halos greater than 9 mm. In the in vivo test, 15 animals were used per phase of the experiment, and the plate counting technique showed that there was antiseptic action of both extracts, with emphasis on the M. tenuiflora PA. Physicochemical analysis of the milk showed that neither PAs interfered with its physical-chemical parameters. CONCLUSIONS: The PA of M. tenuiflora presented potential as an alternative antiseptic in dairy goats. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the use of PA as an antimicrobial agent in animals.
Assuntos
Antibacterianos/farmacologia , Eucalyptus , Leite/microbiologia , Mimosa , Terpenos/farmacologia , Animais , Antibacterianos/isolamento & purificação , Anti-Infecciosos Locais/isolamento & purificação , Anti-Infecciosos Locais/farmacologia , Eucalyptus/química , Cabras , Mimosa/química , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Terpenos/isolamento & purificaçãoRESUMO
BACKGROUND: This study evaluated the epidemiological and morphological features of the mandibular incisive canal (MIC) using cone beam computed tomography (CBCT) in a significant sample of subjects in Brazil. MATERIAL AND METHODS: This retrospective, multicenter study assessed 847 CBCT scans performed at four oral imaging centers. The sample comprised CBCT images acquired from dentate individuals who presented at least from tooth 35 to tooth 45 in the anterior mandible region. Data regarding patient sex and age, and MIC linear measurements (length and diameter in mm), anatomical distances (to the alveolar, buccal and lingual cortexes, inferior border of the mandible, and adjacent teeth apexes), and location were obtained. RESULTS: The MIC was more prevalent in women (76.3% [p<0.001]) between the fourth and sixth decades of life (p<0.001). It was present bilaterally (p<0.001) and exhibited a mean length of 7.7 mm (standard deviation [SD]=3.7 mm). Spearman correlation and logistic regression analysis revealed collinearity between age and linear measurements (p<0.05). The mean distances varied from the initial to the final portion of the MIC, respectively, in relation to the buccal cortex (mean=2.6 mm, SD=1.27; mean=3.96 mm; SD=1.43), to lingual cortex (mean=5.13 mm; SD=1.7; mean=4.61 mm, SD = 1.65), and to the inferior mandibular border (mean = 9.32 mm, SD=1.92; mean=8.76 mm, SD=2.07 mm). The difference in the proximity of the MIC to the apex of the inferior lateral incisor was statistically significant (p<0.05). CONCLUSIONS: Results of this study revealed a high prevalence of MIC with a bilateral pattern in women who were between the fourth and sixth decades of life. Both the distance between the MIC and the lingual cortex of the mandibular alveolar bone, and the diameter of the MIC, decreased as its trajectory assumed a more anterior position.
Assuntos
Tomografia Computadorizada de Feixe Cônico Espiral , Brasil , Tomografia Computadorizada de Feixe Cônico , Feminino , Humanos , Mandíbula , Prevalência , Estudos RetrospectivosRESUMO
BACKGROUND AND AIMS: Polyphenol-rich diets have been associated with reduced risk of cardiovascular disease (CVD). However, few prospective epidemiological studies have examined the relationship between classes of ingested polyphenols and risk of CVD. Our aim was to evaluate the association between polyphenol intake and risk of major cardiovascular events in a prospective Spanish cohort. METHODS AND RESULTS: We included 17,065 university graduates (60.7% women, mean age: 37.2 years, age range: 20-89) followed-up for a mean of 10.1 years. Polyphenol intake was assessed at baseline using a validated semi-quantitative 136-item food frequency questionnaire and matching food consumption data with the Phenol-Explorer database. Cox proportional hazards models were used to estimate the adjusted hazard ratios (HR) and 95% confidence intervals (95% CI) for incident cardiovascular events (myocardial infarction, stroke or cardiovascular death). Cherries, chocolate, coffee, apples, and olives were the major sources of variability in polyphenol intake. Participants with higher flavonoids intake (fifth quintile) had a 47% lower incidence of cardiovascular events compared to those in the lowest quintile (HR: 0.53, 95% CI: 0.29-0.98; P for trend = 0.09) after adjusting for potential confounders. The results were non-significant for other polyphenol types. CONCLUSION: The intake of flavonoids showed an inverse association with risk of cardiovascular events in a prospective cohort of Spanish middle-aged adult university graduates. REGISTRATION NUMBER FOR CLINICAL TRIALS: NCT02669602 in Clinical Trials.
Assuntos
Doenças Cardiovasculares/prevenção & controle , Dieta , Polifenóis/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/epidemiologia , Inquéritos sobre Dietas , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Polifenóis/classificação , Estudos Prospectivos , Fatores de Proteção , Fatores de Risco , Espanha/epidemiologia , Estudantes , Fatores de Tempo , Universidades , Adulto JovemRESUMO
AIMS: This work aimed to evaluate the antibacterial and antifungal activities of two types of pyroligneous acid (PA) obtained from slow pyrolysis of wood of Mimosa tenuiflora and of a hybrid of Eucalyptus urophylla × Eucalyptus grandis. METHODS AND RESULTS: Wood wedges were carbonized on a heating rate of 1·25°C min-1 until 450°C. Pyrolysis smoke was trapped and condensed to yield liquid products. Crude pyrolysis liquids were bidistilled under 5 mmHg vacuum yielding purified PA. Multi-antibiotic-resistant strains of Escherichia coli, Pseudomonas aeruginosa (ATCC 27853) and Staphylococcus aureus (ATCC 25923) had their sensitivity to PA evaluated using agar diffusion test. Two yeasts were evaluated as well, Candida albicans (ATCC 10231) and Cryptococcus neoformans. GC-MS analysis of both PAs was carried out to obtain their chemical composition. Regression analysis was performed, and models were adjusted, with diameter of inhibition halos and PA concentration (100, 50 and 20%) as parameters. Identity of regression models and equality of parameters in polynomial orthogonal equations were verified. Inhibition halos were observed in the range 15-25 mm of diameter. CONCLUSIONS: All micro-organisms were inhibited by both types of PA even in the lowest concentration of 20%. SIGNIFICANCE AND IMPACT OF THE STUDY: The feasibility of the usage of PAs produced with wood species planted in large scale in Brazil was evident and the real potential as a basis to produce natural antibacterial and antifungal agents, with real possibility to be used in veterinary and zootechnical applications.
Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Eucalyptus/química , Mimosa/química , Terpenos/farmacologia , Antibacterianos/isolamento & purificação , Antifúngicos/isolamento & purificação , Brasil , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Terpenos/isolamento & purificação , Madeira/química , Leveduras/efeitos dos fármacosRESUMO
BACKGROUND: and purpose: The peptide PnPP-19, derived from the spider toxin PnTx2-6 (renamed as δ-CNTX-Pn1c), potentiates erectile function by activating the nitrergic system. Since NO has been studied as an antinociceptive molecule and PnPP-19 is known to induce peripheral antinociception, we intended to evaluate whether PnPP-19 could induce peripheral antinociception through activation of this pathway. EXPERIMENTAL APPROACH: Nociceptive thresholds were measured by paw pressure test. PGE2 (2 µg/paw) was administered intraplantarly together with PnPP-19 and inhibitors/blockers of NOS, guanylyl cyclase and KATP channels. The nitrite concentration was accessed by Griess test. The expression and phosphorylation of eNOS and nNOS were determined by western blot. KEY RESULTS: PnPP-19 (5, 10 and 20 µg/paw) induced peripheral antinociception in rats. Administration of NOS inhibitor (L-NOarg), selective nNOS inhibitor (L-NPA), guanylyl cyclase inhibitor (ODQ) and the blocker of KATP (glibenclamide) partially inhibited the antinociceptive effect of PnPP-19 (10 µg/paw). Tissue nitrite concentration increased after PnPP-19 (10 µg/paw) administration. Expression of eNOS and nNOS remained the same in all tested groups, however the phosphorylation of nNOS Ser852 (inactivation site) increased and phosphorylation of eNOS Ser1177 (activation site) decreased after PGE2 injection. Administration of PnPP-19 reverted this PGE2-induced effect. CONCLUSIONS AND IMPLICATIONS: The peripheral antinociceptive effect induced by PnPP-19 is resulting from activation of NO-cGMP-KATP pathway. Activation of eNOS and nNOS might be required for such effect. Our results suggest PnPP-19 as a new drug candidate to treat pain and reinforce the importance of nNOS and eNOS activation, as well as endogenous NO release, for induction of peripheral antinociception.
Assuntos
Analgésicos/farmacologia , GMP Cíclico/metabolismo , Canais KATP/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico/metabolismo , Peptídeos/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Pé/fisiopatologia , Masculino , Óxido Nítrico Sintase Tipo I/análise , Óxido Nítrico Sintase Tipo III/análise , Manejo da Dor , Sistema Nervoso Periférico/efeitos dos fármacos , Fosforilação , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Venenos de AranhaRESUMO
The aim of this work was to analyze the activity of novel synthetic lactams on preventing biofilm formation on titanium surfaces. Titanium (Ti6Al4V) samples were exposed to Streptococcus mutans cultures in the presence or absence of a synthetic lactam. After 48h incubation, planktonic growth was determined by spectrophotometry. Biofilm was evaluated by crystal violet staining and colony forming units (CFU·ml(-)(1)), followed by scanning electron microscopy (SEM). Results showed that the average of adhered viable cells was approximately 1.5×10(2)CFU/ml in the presence of lactam and 4×10(2)CFU/ml in its absence. This novel compound was considerable active in reducing biofilm formation over titanium surfaces, indicating its potential for the development of antimicrobial drugs targeting the inhibition of the initial stages of bacterial biofilms on dental implants abutments.
Assuntos
Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Lactamas , Streptococcus mutans/fisiologia , Titânio/química , Ligas , Lactamas/química , Lactamas/farmacologia , Microscopia Eletroquímica de Varredura , Streptococcus mutans/ultraestruturaRESUMO
A new fibrinogenolytic metalloproteinase (Bmoo FIBMP-I) was purified from Bothrops moojeni snake venom. This enzyme was isolated through a combination of three chromatographic steps (ion-exchange, molecular exclusion, and affinity chromatography). Analyses by reverse phase chromatography, followed by mass spectrometry, showed the presence of enzyme isoforms with average molecular mass of 22.8 kDa. The SDS-PAGE analyses showed a single chain of 27.6 kDa, in the presence and absence of reducing agent. The protein has a blocked N-terminal. One of the peptides obtained by enzymatic digestion of a reduced and S-alkylated isoform was completely sequenced by mass spectrometry (MS/MS). Bmoo FIBMP-I showed similarity with hemorrhagic factor and several metalloproteinases (MP). This enzyme degraded Aα-chain faster than the Bß-chain and did not affect the γ-chain of bovine fibrinogen. The absence of proteolytic activity after treatment with EDTA, together with the observed molecular mass, led us to suggest that Bmoo FIBMP-I is a member of the P-I class of the snake venom MP family. Bmoo FIBMP-I showed pH-dependent proteolytic activity on azocasein, but was devoid of coagulant, defibrinating, or hemorrhagic activities. The kinetic parameters of proteolytic activity in azocasein were determined (V max = 0.4596 Uh(-1)nmol(-1) ± 0.1031 and K m = 14.59 mg/mL ± 4.610).
RESUMO
Cognitive dysfunction following surgery is a common complication, which increases the incidence of other co-morbid conditions, hospital and health-care costs. The reported rate of the occurrence of post-operative cognitive decline varies with different studies, depending on population profile, type of surgery, definition of cognitive disorder and detection methods, design of study, etc. It remains unclear whether these psychiatric signs and symptoms are direct results of the effects of surgery or general anesthesia. Nonetheless they are more frequent after cardiac surgery and are likely to be multi-factorial, but the patho-mechanisms are not yet fully characterized. This communication provides a synopsis of proteomics tools and delineates novel SELDI-TOF results to evaluate biomarkers in this regard. Presented for the first time is a classification of the clinically relevant forms of post-operative cognitive decline with the advent of a novel subclass.
Assuntos
Líquido Cefalorraquidiano/química , Cognição/fisiologia , Ponte de Artéria Coronária , Análise Serial de Proteínas , Proteoma/análise , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Animais , Humanos , SíndromeRESUMO
A new vasoactive cytolytic toxin, referred to as Sp-CTx, has been purified from the venom of the scorpionfish Scorpaena plumieri by a combination of gel filtration and anion exchange chromatographies. An estimation of Sp-CTx native molecular mass, performed by size exclusion chromatography, demonstrated that it is a 121 kDa protein. Further physicochemical studies revealed its glycoproteic nature and dimeric constitution, comprising subunits of approximately 65 kDa (MALDI-TOF-MS). Such protein has proved to possess a potent hemolytic activity on washed rabbit erythrocytes (EC(50) 0.46 nM), whose effect was strongly reduced after treatment with antivenom raised against stonefish venom -Synanceja trachynis (SFAV). This cross-reactivity has been confirmed by western blotting. Like S. plumieri whole venom (100 microg/mL), Sp-CTx (1-50 nM) caused a biphasic response on phenylephrine pre-contracted rat aortic rings, characterized by an endothelium- and dose-dependent relaxation phase followed by a contractile phase. The vasorelaxant activity has been abolished by l-NAME, demonstrating the involvement of nitric oxide on the response. We report here the first isolation of a cytolytic/vasoactive protein from scorpionfish venom and the data provided suggest structural and functional similarities between Sp-CTx and previously published stonefish hemolytic toxins.
Assuntos
Citotoxinas/química , Venenos de Peixe/química , Peixes Venenosos , Hemolíticos/química , Vasodilatadores/química , Animais , Aorta/efeitos dos fármacos , Citotoxinas/isolamento & purificação , Citotoxinas/toxicidade , Eritrócitos/efeitos dos fármacos , Venenos de Peixe/isolamento & purificação , Venenos de Peixe/toxicidade , Hemolíticos/isolamento & purificação , Hemolíticos/toxicidade , Técnicas In Vitro , Contração Miocárdica/efeitos dos fármacos , Coelhos , Ratos , Vasodilatadores/isolamento & purificação , Vasodilatadores/toxicidadeRESUMO
In the current study, the putative cDNA for PnTx2-6 toxin of the Phoneutria nigriventer spider venom was cloned and expressed as tioredoxin fusion protein in the cytoplasm of Escherichia coli. The fusion protein was purified from the bacterial extracts by combination of immobilized Ni-ion affinity and gel filtration chromatographies. Then, it was cleaved by enterokinase and the generated recombinant PnTx2-6 (rPnTx2-6) was further purified by reverse-phase HPLC. Likewise the native toxin purified from the spider venom, rPnTx2-6 potentiates the erectile function when injected in rats. This result indicates that the production of functional recombinant PnTx2-6 might be an alternative to provide this basic and valuable tool for study, as well as for further understanding such complex physiological system, including its correlation with the central nervous system and local tissue factors.
Assuntos
Ereção Peniana/efeitos dos fármacos , Peptídeos/farmacologia , Venenos de Aranha/farmacologia , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Escherichia coli/genética , Injeções Subcutâneas , Masculino , Dados de Sequência Molecular , Peptídeos/administração & dosagem , Peptídeos/isolamento & purificação , Priapismo/induzido quimicamente , Ratos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Venenos de Aranha/administração & dosagem , Venenos de Aranha/isolamento & purificaçãoRESUMO
BACKGROUND AIMS: Stem cells derived from human adipose tissue (ASC) have the capacity for renewal, are easily obtained and have plasticity properties that allow them to differentiate into several cell types, including osteoblast cells. With the aim of understanding the issue of the osteogenic process and finding reliable biomarkers in cells undergoing the osteogeneic differentiation process, this work took advantage of a proteomic approach to identify proteins involved in osteogenesis. METHODS: For this purpose, ASC were analyzed under three conditions: S0, in the absence of stimulation; S1, with 2 weeks of osteogenic medium stimulation; and S2, with 4 weeks of osteogenic medium stimulation. The identification of ASC was carried out by flow cytometry using antibodies specific to known undifferentiated stem cell-surface markers. Cell viability, enzymatic activity, mineral deposition, collagen structure and production and gene analyzes were evaluated for each condition. RESULTS: Phenotypic modifications were observed during the in vitro osteogenic differentiation process by two-dimensional (2-D) differential image gel electrophoresis (DIGE). The proteins were identified by mass espectrometry in tandem (MS/MS) analyzes using Matrix-assisted laser desorption/ionization with TOF/TOF is a tandem mass spectrometry method where two time-of-flight mass spectrometers are used consecutively (MALDI-TOF/TOF). A total of 51 differentially expressed proteins was identified when comparing the three observed conditions. Sixteen different spots were identified in the S0 stage compared with S2, while 28 different spots were found in S2 compared with S0. S1 expressed seven different spots compared with S0 and S2. CONCLUSIONS: These findings suggest the involvement of several proteins directly related to the osteogenic pathway, which can be used to improve understanding of the osteogenic process.
Assuntos
Tecido Adiposo/citologia , Biomarcadores , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Proteômica , Células Estromais/metabolismo , Tecido Adiposo/cirurgia , Adulto , Biomarcadores/metabolismo , Diferenciação Celular , Separação Celular , Células Cultivadas , Eletroforese em Gel Bidimensional , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/citologia , Pessoa de Meia-Idade , Osteoblastos/citologia , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Células Estromais/citologiaRESUMO
LyeTx I, an antimicrobial peptide isolated from the venom of Lycosa erythrognatha, known as wolf spider, has been synthesised and its structural profile studied by using the CD and NMR techniques. LyeTx I has shown to be active against bacteria (Escherichia coli and Staphylococcus aureus) and fungi (Candida krusei and Cryptococcus neoformans) and able to alter the permeabilisation of L: -alpha-phosphatidylcholine-liposomes (POPC) in a dose-dependent manner. In POPC containing cholesterol or ergosterol, permeabilisation has either decreased about five times or remained unchanged, respectively. These results, along with the observed low haemolytic activity, indicated that antimicrobial membranes, rather than vertebrate membranes seem to be the preferential targets. However, the complexity of biological membranes compared to liposomes must be taken in account. Besides, other membrane components, such as proteins and even specific lipids, cannot be discarded to be important to the preferential action of the LyeTx I to the tested microorganisms. The secondary structure of LyeTx I shows a small random-coil region at the N-terminus followed by an alpha-helix that reached the amidated C-terminus, which might favour the peptide-membrane interaction. The high activity against bacteria together with the moderate activity against fungi and the low haemolytic activity have indicated LyeTx I as a good prototype for developing new antibiotic peptides.
Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Venenos de Aranha/química , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antifúngicos/química , Antifúngicos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Candida/efeitos dos fármacos , Cryptococcus neoformans/efeitos dos fármacos , Relação Dose-Resposta a Droga , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Fosfatidilcolinas/antagonistas & inibidores , Estrutura Secundária de Proteína , Aranhas , Staphylococcus aureus/efeitos dos fármacosRESUMO
Using a proteomic approach, a new structural family of peptides was put in evidence in the venom of the yellow scorpion Tityus serrulatus. Tityus serrulatus Hypotensins (TsHpt) are random-coiled linear peptides and have a similar bradykinin-potentiating peptide (BPP) amino acid signature. TsHpt-I (2.7kDa), the first member of this family, was able to potentiate the hypotensive effects of bradykinin (BK) in normotensive rats. Using the C-terminal of this peptide as a template, a synthetic analog peptide (TsHpt-I([17-25])) was designed to held the BK-potentiating effect. A relevant hypotensive effect, independent on BK, was also observed on both TsHpt (native and synthetic). To better evaluate this hypotensive effect, we examined the vasorelaxation of aortic rings from male Wistar rats and the peptides were able to induce endothelium-dependent vasorelaxation dependent on NO release. Both TsHpt could not inhibit ACE activity. These peptides appear to exert their anti-hypertensive effect through NO-dependent and ACE-independent mechanisms.
Assuntos
Anti-Hipertensivos/química , Anti-Hipertensivos/farmacologia , Venenos de Escorpião/química , Venenos de Escorpião/farmacologia , Vasodilatadores/química , Vasodilatadores/farmacologia , Sequência de Aminoácidos , Animais , Anti-Hipertensivos/isolamento & purificação , Bradicinina/farmacologia , Sinergismo Farmacológico , Masculino , Dados de Sequência Molecular , Óxido Nítrico/metabolismo , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Peptidil Dipeptidase A/efeitos dos fármacos , Ratos , Ratos Wistar , Venenos de Escorpião/isolamento & purificação , Vasodilatação , Vasodilatadores/isolamento & purificaçãoRESUMO
The venom of the spider Phoneutria nigriventer contains several toxins that have bioactivity in mammals and insects. Accidents involving humans are characterized by various symptoms including penile erection. Here we investigated the action of Tx2-6, a toxin purified from the P. nigriventer spider venom that causes priapism in rats and mice. Erectile function was evaluated through changes in intracavernosal pressure/mean arterial pressure ratio (ICP/MAP) during electrical stimulation of the major pelvic ganglion (MPG) of normotensive and deoxycorticosterone-acetate (DOCA)-salt hypertensive rats. Nitric oxide (NO) release was detected in cavernosum slices with fluorescent dye (DAF-FM) and confocal microscopy. The effect of Tx2-6 was also characterized after intracavernosal injection of a non-selective nitric oxide synthase (NOS) inhibitor, L-NAME. Subcutaneous or intravenous injection of Tx2-6 potentiated the elevation of ICP/MAP induced by ganglionic stimulation. L-NAME inhibited penile erection and treatment with Tx2-6 was unable to reverse this inhibition. Tx2-6 treatment induced a significant increase of NO release in cavernosum tissue. Attenuated erectile function of DOCA-salt hypertensive rats was fully restored after toxin injection. Tx2-6 enhanced erectile function in normotensive and DOCA-salt hypertensive rats, via the NO pathway. Our studies suggest that Tx2-6 could be important for development of new pharmacological agents for treatment of erectile dysfunction.
Assuntos
Neuropeptídeos/farmacologia , Neurotoxinas/farmacologia , Ereção Peniana/efeitos dos fármacos , Pênis/efeitos dos fármacos , Venenos de Aranha/farmacologia , Aranhas , Animais , Modelos Animais de Doenças , Sinergismo Farmacológico , Estimulação Elétrica , Disfunção Erétil/complicações , Disfunção Erétil/tratamento farmacológico , Disfunção Erétil/fisiopatologia , Hipertensão/induzido quimicamente , Hipertensão/complicações , Hipertensão/fisiopatologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Neuropeptídeos/isolamento & purificação , Neurotoxinas/isolamento & purificação , Óxido Nítrico/metabolismo , Ereção Peniana/fisiologia , Pênis/inervação , Pênis/metabolismo , Ratos , Ratos WistarRESUMO
Various neurotoxins have been described from the venom of the Brazilian spider Phoneutria nigriventer, but little is known about the venoms of the other species of this genus. In the present work, we describe the purification and some structural and pharmacological features of a new toxin (PRTx3-7) from Phoneutria reidyi that causes flaccid paralysis in mice. The observed molecular mass (4627.26 Da) was in accordance with the calculated mass for the amidated form of the amino acid sequence (4627.08 Da). The presence of an alpha-amidated C-terminus was confirmed by MS/MS analysis of the C-terminal peptide, isolated after enzymatic digestion of the native protein with Glu-C endoproteinase. The purified protein was injected (intracerebro-ventricular) into mice at dose levels of 5 microg/mouse causing immediate agitation and clockwise gyration, followed by the gradual development of general flaccid paralysis. PRTx3-7 at 1 microM inhibited by 20% the KCl-induced increase on [Ca2+]i in rat brain synaptosomes. The HEK cells permanently expressing L, N, P/Q and R HVA Ca2+ channels were also used to better characterize the pharmacological features of PRTx3-7. To our surprise, PRTx3-7 shifted the voltage-dependence for activation towards hyperpolarized membrane potentials for L (-4 mV), P/Q (-8 mV) and R (-5 mV) type Ca2+ currents. In addition, the new toxin also affected the steady state of inactivation of L-, N- and P/Q-type Ca2+ currents.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Venenos de Aranha/farmacologia , Sequência de Aminoácidos , Animais , Canais de Cálcio/genética , Canais de Cálcio/fisiologia , Células Cultivadas , Eletrofisiologia , Feminino , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Ratos , Ratos Wistar , Homologia de Sequência de Aminoácidos , Venenos de Aranha/química , Venenos de Aranha/isolamento & purificação , Aranhas , Sinaptossomos/efeitos dos fármacos , TransfecçãoRESUMO
PURPOSE: We examined the significance of the CAG repeat polymorphism in the pathogenesis of cryptorchidism. MATERIALS AND METHODS: Genomic deoxyribonucleic acid (DNA) was extracted from blood samples from 42 cryptorchid boys and from 31 non-cryptorchid control subjects. In the cryptorchid group, 7 had bilateral cryptorchidism and 6 had patent processus vaginalis in the contralateral side. To determine the number of CAG repeats, the DNA was amplified by polymerase chain reaction and sequenced. RESULTS: The mean CAG repeat length in the AR gene was 22.5 (range 16 to 28) in patients and 21.5 (range 17 to 26) in controls (non-significant). Patients with bilateral cryptorchidism had a mean length of 24.3 (range 21 to 26) and patients with unilateral cryptorchidism and patent processus vaginalis in the contra lateral side had a mean of 25.2 (range 21 to 28), which was statistically different from controls (p = 0.015 and p = 0.005 respectively). CONCLUSION: CAG repeat length of the AR gene does not seem to play a major role in patients with unilateral cryptorchidism. However, in patients with bilateral undescended testis, a less functional androgen receptor through a longer polyglutamine chain may have a role in its pathogenesis. In the same way, patients with unilateral cryptorchidism a contralateral patent processus vaginalis have longer CAG repeats that might be responsible for a slower testicular descent and incomplete closure of the processus vaginalis.
Assuntos
Criptorquidismo/genética , Polimorfismo Genético/genética , Receptores Androgênicos/genética , Repetições de Trinucleotídeos/genética , Adolescente , Idoso , Estudos de Casos e Controles , Criança , Pré-Escolar , Genótipo , Humanos , Masculino , Reação em Cadeia da Polimerase , Estudos ProspectivosRESUMO
PURPOSE: We examined the significance of the CAG repeat polymorphism in the pathogenesis of cryptorchidism. MATERIALS AND METHODS: Genomic deoxyribonucleic acid (DNA) was extracted from blood samples from 42 cryptorchid boys and from 31 non-cryptorchid control subjects. In the cryptorchid group, 7 had bilateral cryptorchidism and 6 had patent processus vaginalis in the contralateral side. To determine the number of CAG repeats, the DNA was amplified by polymerase chain reaction and sequenced. RESULTS: The mean CAG repeat length in the AR gene was 22.5 (range 16 to 28) in patients and 21.5 (range 17 to 26) in controls (non-significant). Patients with bilateral cryptorchidism had a mean length of 24.3 (range 21 to 26) and patients with unilateral cryptorchidism and patent processus vaginalis in the contra lateral side had a mean of 25.2 (range 21 to 28), which was statistically different from controls (p = 0.015 and p = 0.005 respectively). CONCLUSION: CAG repeat length of the AR gene does not seem to play a major role in patients with unilateral cryptorchidism. However, in patients with bilateral undescended testis, a less functional androgen receptor through a longer polyglutamine chain may have a role in its pathogenesis. In the same way, patients with unilateral cryptorchidism a contralateral patent processus vaginalis have longer CAG repeats that might be responsible for a slower testicular descent and incomplete closure of the processus vaginalis.
Assuntos
Adolescente , Idoso , Criança , Pré-Escolar , Humanos , Masculino , Criptorquidismo/genética , Polimorfismo Genético/genética , Receptores Androgênicos/genética , Repetições de Trinucleotídeos/genética , Estudos de Casos e Controles , Genótipo , Reação em Cadeia da Polimerase , Estudos ProspectivosRESUMO
The proteomes of the venoms of the Brazilian wandering "armed" spiders Phoneutria nigriventer, Phoneutria reidyi, and Phoneutria keyserlingi, were compared using two-dimensional gel electrophoresis. The venom components were also fractionated using a combination of preparative reverse phase HPLC on Vydac C4, analytical RP-HPLC on Vydac C8 and C18 and cation exchange FPLC on Resource S at pH 6.1 and 4.7, or anion exchange HPLC on Synchropak AX-300 at pH 8.6. The amino acid sequences of the native and S-pyridyl-ethylated proteins and peptides derived from them by enzymatic digestion and chemical cleavages were determined using a Shimadzu PPSQ-21(A) automated protein sequencer, and by MS/MS collision induced dissociations. To date nearly 400 peptides and proteins (1.2-27 kDa) have been isolated in a pure state and, of these, more than 100 have had their complete or partial amino acid sequences determined. These sequences demonstrate, as might be expected, that the venoms of P. reidyi and P. keyserlingi (Family: Ctenidae) both contain a similar range of isoforms of the neurotoxins as those previously isolated from P. nigriventer which are active on neuronal ion (Ca(2+), Na(+) and K(+)) channels and NMDA-type glutamate receptors. In addition two new families of small (3-4 kDa) toxins, some larger protein (>10 kDa) components, and two serine proteinases of the venom of P. nigriventer are described. These enzymes may be responsible for some of the post-translational modification observed in some of the venom components.
Assuntos
Neurotoxinas/química , Venenos de Aranha/química , Aranhas , Sequência de Aminoácidos , Animais , Brasil , Feminino , Moscas Domésticas/efeitos dos fármacos , Dose Letal Mediana , Masculino , Camundongos , Dados de Sequência Molecular , Neurotoxinas/isolamento & purificação , Neurotoxinas/toxicidade , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/toxicidade , Proteínas/química , Proteínas/isolamento & purificação , Proteínas/toxicidade , Proteoma , Alinhamento de Sequência , Venenos de Aranha/isolamento & purificação , Venenos de Aranha/toxicidadeRESUMO
AIMS: The aim of this work was to purify and characterize antibacterial compounds produced by Lactobacillus murinus strain L1. METHODS AND RESULTS: Antagonistic activity was observed in a deferred agar-spot assay against spoilage and pathogenic bacteria, but not against lactobacilli. The inhibitory activity occurred between pH 3.0 and 5.0, and was heat stable. The active compounds were purified by gel filtration chromatography and two peaks of antibacterial activity were observed using Bacillus cereus ATCC 11778 and Shigella sonnei ATCC 11060 as indicator strains. Two active low molecular weight compounds were responsible for this phenomenon and UV spectroscopy, gas chromatography and mass spectrometry were used to characterize them. One of them is lactic acid, while the other is a mono-substituted aromatic ring apparently constituted by group residues of m/z 192 linked in tandem to phenylalanine. CONCLUSIONS: Lactobacillus murinus produces at least two low molecular weight compounds active against B. cereus and Sh. sonnei. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first purification of a new broad-spectrum antibacterial compound from Lact. murinus which inhibits various pathogenic and food spoilage bacteria without acting on other lactobacilli. Using it as a biotechnological control agent of bacterial spoilage may be a promising possibility for the food industry.
Assuntos
Antibacterianos/isolamento & purificação , Lactobacillus/metabolismo , Animais , Bacillus cereus/efeitos dos fármacos , Cromatografia Gasosa/métodos , Cromatografia em Gel/métodos , Meios de Cultura , Fezes/microbiologia , Concentração de Íons de Hidrogênio , Espectrometria de Massas/métodos , Peso Molecular , Peptídeo Hidrolases/metabolismo , Ratos , Ratos Wistar , Shigella sonnei/efeitos dos fármacos , Espectrofotometria Ultravioleta/métodos , TemperaturaRESUMO
The crystalline structures of the superionic high-temperature copper selenides Cu(2-x)Se (0 < x < 0.25) produced using mechanical alloying were investigated using X-ray diffraction (XRD). The measured XRD patterns showed the presence of peaks corresponding to the crystalline superionic high-temperature alpha-Cu(2)Se phase in the as-milled sample, and its structural data were determined by means of a Rietveld refinement procedure. After heat treatment in argon at 473 K for 90 h, this phase transforms to the superionic high-temperature alpha-Cu(1.8)Se phase, whose structural data were also determined by Rietveld refinement. In this phase, a very low occupation of the trigonal 32(f) sites ( approximately 3%) by Cu ions is found. In order to explain the evolution of the phases in the samples, two possible mechanisms are suggested: (i). the high mobility of Cu ions in superionic phases and (ii). the important diffusive processes in the interfacial component of samples produced by mechanical alloying.