RESUMEN
Antimicrobial resistance has been increasing globally, posing a global public health risk. It has prompted the scientific community to look for alternatives to traditional drugs. Antimicrobial Peptides (AMPs) have stood out in this context because they have the potential to control infectious diseases while causing no or little harm to mammalian cells. In the present study, three peptides, JcTI-PepI, JcTI-PepII, and JcTI-PepIII, were designed and tested for antimicrobial activity based on the primary sequence of JcTI-I, a 2S albumin with trypsin inhibitory activity from Jatropha curcas. JcTI-PepI strongly inhibited C. krusei growth, and it caused severe disruptions in cellular processes and cell morphology. C. krusei cells treated with JcTI-PepI showed indicative of membrane permeabilization and overproduction of Reactive Oxygen Species. Moreover, the yeast's ability to acidify the medium was severely compromised. JcTI-PepI was also effective against pre-formed biofilm and did not harm human erythrocytes and Vero cells. Overall, these characteristics indicate that JcTI-PepI is both safe and effective against C. krusei, an intrinsically resistant strain that causes serious health problems and is frequently overlooked. It implies that this peptide has a high potential for use as a new antimicrobial agent in the future.
Asunto(s)
Antiinfecciosos , Jatropha , Animales , Antiinfecciosos/farmacología , Chlorocebus aethiops , Humanos , Mamíferos , Pruebas de Sensibilidad Microbiana , Péptidos/farmacología , Inhibidores de Tripsina , Células VeroRESUMEN
C. albicans and C. parapsilosis are biofilm-forming yeasts responsible for bloodstream infections that can cause death. Synthetic antimicrobial peptides (SAMPs) are considered to be new weapons to combat these infections, alone or combined with drugs. Here, two SAMPs, called Mo-CBP3-PepI and Mo-CBP3-PepIII, were tested alone or combined with nystatin (NYS) and itraconazole (ITR) against C. albicans and C. parapsilosis biofilms. Furthermore, the mechanism of antibiofilm activity was evaluated by fluorescence and scanning electron microscopies. When combined with SAMPs, the results revealed a 2- to 4-fold improvement of NYS and ITR antibiofilm activity. Microscopic analyses showed cell membrane and wall damage and ROS overproduction, which caused leakage of internal content and cell death. Taken together, these results suggest the potential of Mo-CBP3-PepI and Mo-CBP3-PepIII as new drugs and adjuvants to increase the activity of conventional drugs for the treatment of clinical infections caused by C. albicans and C. parapsilosis.
RESUMEN
Christiana mennegae is a phylogenetically enigmatic taxon and represents a case in point of a species whose presence escaped the radar of the Amazon lists and the Brazilian Flora project. Here we expand its distribution by adding new records from Peru and overlooked ones from Brazil. To investigate its phylogenetic placement in the Brownlowioideae, part of the rbcL gene of the plastid and the intergenic ITS2 region were sequenced. Macro- and micro-morphological investigation of features of C. mennegae using SEM of foliar, flower, fruit and seed structures are presented. A lectotype for the name is designated here. The morphology of trichomes revealed five types of trichomes ranging from glandular to branched and unbranched and we also report stomata on the seed surface for the first time in Brownlowioideae. Christiana mennegae and C. africana were recovered as sister species in the phylogenetic analysis, albeit with low to moderate support, and more species of this and closely related genera must be sampled and analyzed in order to obtain a clearer picture of the group's affinities and relationships. We provide an update of its conservation status from Vulnerable to Least Concern. We also highlight the need for investment in the digitization of biological collections, botanical capacity building at the local level and the importance of the availability of online literature to speed the study of Amazonian plant diversity.
RESUMEN
BACKGROUND: Dermatophytes belonging to the Trichophyton genus are important human pathogens, but they have developed resistance to griseofulvin, the most common antifungal drug used to treat dermatophytosis. OBJECTIVE: This study was aimed to evaluate the antidermatophytic activity of synthetic peptides, as well as mechanisms of action and synergistic effect with griseofulvin. METHODS: Scanning electron microscopy (SEM), atomic force microscopy (AFM) and fluorescence microscopy (FM) were employed to understand the activity and the mechanism of action of peptides. RESULTS: Here we report that synthetic peptides at 50 µg/mL, a concentration 20-fold lower than griseofulvin, reduced the microconidia viability of T. mentagrophytes and T. rubrum by 100%, whereas griseofulvin decreased their viability by only 50% and 0%, respectively. The action mechanism of peptides involved cell wall damage, membrane pore formation and loss of cytoplasmic content. Peptides also induced overproduction of reactive oxygen species (ROS) and enhanced the activity of griseofulvin 10-fold against both fungi, suggesting synergistic effects, and eliminated the toxicity of this drug to human erythrocytes. Docking analysis revealed ionic and hydrophobic interactions between peptides and griseofulvin, which may explain the decline of griseofulvin toxicity when mixed with peptides. CONCLUSION: Therefore, our results strongly suggest six peptides with high potential to be employed alone as new drugs or as adjuvants to enhance the activity and decrease the toxicity of griseofulvin.
Asunto(s)
Antifúngicos/farmacología , Griseofulvina/farmacología , Péptidos/síntesis química , Péptidos/farmacología , Esporas Fúngicas/efectos de los fármacos , Trichophyton/efectos de los fármacos , Descubrimiento de Drogas , Farmacorresistencia Fúngica , Sinergismo Farmacológico , Humanos , Pruebas de Sensibilidad MicrobianaRESUMEN
The present work describes the synthesis, characterization, and application of a new ion-tagged iron catalyst. The catalyst was employed in the Biginelli reaction with impressive performance. High yields have been achieved when the reaction was carried out in imidazolium-based ionic liquids (BMIâ PF6, BMIâ NTf2, and BMIâ BF4), thus showing that the ionic-liquid effects play a role in the reaction. Moreover, the ion-tagged catalyst could be recovered and reused up to eight times without any noticeable loss in activity. Mechanistic studies performed by using high-resolution electrospray-ionization quadrupole-time-of-flight mass (HR-EI-QTOF) spectrometry and kinetic experiments indicate only one reaction pathway and rule out the other two possibilities under the development conditions. The theoretical calculations are in accordance with the proposed mechanism of action of the iron catalyst. Finally, the 37 dihydropyrimidinone derivatives, products of the Biginelli reaction, had their cytotoxicity evaluated in assays against MCF-7 cancer cell linages with encouraging results of some derivatives, which were virtually non-toxic against healthy cell linages (fibroblasts).
Asunto(s)
Antineoplásicos/síntesis química , Imidazoles/química , Hierro/química , Pirimidinonas/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Catálisis , Técnicas Químicas Combinatorias , Ensayos de Selección de Medicamentos Antitumorales , Espectroscopía de Resonancia por Spin del Electrón , Femenino , Fibroblastos/efectos de los fármacos , Humanos , Cinética , Estructura Molecular , Pirimidinonas/química , Pirimidinonas/farmacologíaRESUMEN
The clinical and pathological findings of intestinal obstruction caused by phytobezoars in cattle consuming Stylosanthes sp. on three farms in Mato Grosso, Brazil, are described. The morbidity varied from 3.3 to 15% and the mortality was 100%. The animals stayed on pastures with predominance of Stylosanthes sp. for at least 60 days. The overall clinical picture was apathy followed by episodes of diarrhea or reduced feces and separation from the herd. Individual clinical signs were abdominal colic characterized by anorexia, discomfort, gastrointestinal hypomotility, dehydration, increased abdominal size, sweating, vocalization, sternal or lateral recumbence with the head on the flank. The clinical course lasted from 2 to 7 days. At necropsy, duodenal or pyloric obstruction was caused by ovoid phytobezoars of 2-4cm diameter; in the area of obstruction friable intestinal tissue with intense swelling, congestion, edema, and reddish mucosa was found. The rumen, abomasum and duodenum proximal to the site of obstruction was filled with greenish liquid, and absence of food contents was observed distally to the phytobezoars. Histologically, at the site of obstruction, the duodenum exhibited diffuse necrosis of the mucosal surface, thickening of the wall by submucosal edema, neutrophilic infiltration, fibrin deposition, necrosis of smooth muscle fibers, and marked congestion or hemorrhage. Pasture with the predominance of Stylosanthes sp. is a serious problem due to the possibility of phytobezoar formation, thus leading to intestinal obstruction and high mortality rates in cattle.
O objetivo deste trabalho é descrever, em três propriedades rurais no Estado de Mato Grosso, a epidemiologia, o quadro clínico e o patológico da obstrução intestinal por fitobezoares em bovinos que pastoreavam em piquetes com predomínio de Stylosanthes sp. pelo menos 60 dias. A morbidade variou de 3,3% a 15% e a mortalidade foi de 100%. O quadro clínico foi de cólica abdominal caracterizado por apatia, anorexia, inquietação, desconforto, sudorese e vocalização, seguido por episódios de diarreia ou diminuição da produção fecal, desidratação, hipomotilidade gastrintestinal, aumento do volume abdominal, decúbito esternal ou lateral com a cabeça voltada para o flanco e morte. O curso clínico foi de 2-7 dias. A principal alteração notada durante a necropsia dos bovinos foi a obstrução intestinal por fitobezoar ovoide de 2-5 cm de diâmetro, frequentemente no duodeno, próximo ao piloro, ocasionalmente em sua porção média. Na área de obstrução notou-se a necrose da parede intestinal que estava enegrecida e intensamente demarcada em relação a segmento adjacente normal, além de intenso espessamento associado a edema, congestão e hemorragia. O abomaso e os segmentos intestinais anteriores a obstrução apresentavam-se repletos de conteúdo alimentar líquido e posteriormente estava com ausência de conteúdo. O rúmen frequentemente tinha grande quantidade de conteúdo liquido a pastoso. Em todos os bovinos necropsiados múltiplas estruturas sólidas, arredondadas ou ovoides, esverdeadas (fitobezoares) foram observadas no abomaso. Estes achados indicam que pastagens com predomínio de Stylosanthes sp. predispõem a formação de fitobezoares, os quais podem levar à obstrução intestinal e morte em bovinos.
Asunto(s)
Animales , Bovinos , Fabaceae/efectos adversos , Mucosa Intestinal , Necrosis/veterinaria , Obstrucción Intestinal/veterinaria , Signos y Síntomas/veterinaria , Cólico/veterinaria , MortalidadRESUMEN
The clinical and pathological findings of intestinal obstruction caused by phytobezoars in cattle consuming Stylosanthes sp. on three farms in Mato Grosso, Brazil, are described. The morbidity varied from 3.3 to 15% and the mortality was 100%. The animals stayed on pastures with predominance of Stylosanthes sp. for at least 60 days. The overall clinical picture was apathy followed by episodes of diarrhea or reduced feces and separation from the herd. Individual clinical signs were abdominal colic characterized by anorexia, discomfort, gastrointestinal hypomotility, dehydration, increased abdominal size, sweating, vocalization, sternal or lateral recumbence with the head on the flank. The clinical course lasted from 2 to 7 days. At necropsy, duodenal or pyloric obstruction was caused by ovoid phytobezoars of 2-4cm diameter; in the area of obstruction friable intestinal tissue with intense swelling, congestion, edema, and reddish mucosa was found. The rumen, abomasum and duodenum proximal to the site of obstruction was filled with greenish liquid, and absence of food contents was observed distally to the phytobezoars. Histologically, at the site of obstruction, the duodenum exhibited diffuse necrosis of the mucosal surface, thickening of the wall by submucosal edema, neutrophilic infiltration, fibrin deposition, necrosis of smooth muscle fibers, and marked congestion or hemorrhage. Pasture with the predominance of Stylosanthes sp. is a serious problem due to the possibility of phytobezoar formation, thus leading to intestinal obstruction and high mortality rates in cattle.(AU)
O objetivo deste trabalho é descrever, em três propriedades rurais no Estado de Mato Grosso, a epidemiologia, o quadro clínico e o patológico da obstrução intestinal por fitobezoares em bovinos que pastoreavam em piquetes com predomínio de Stylosanthes sp. pelo menos 60 dias. A morbidade variou de 3,3% a 15% e a mortalidade foi de 100%. O quadro clínico foi de cólica abdominal caracterizado por apatia, anorexia, inquietação, desconforto, sudorese e vocalização, seguido por episódios de diarreia ou diminuição da produção fecal, desidratação, hipomotilidade gastrintestinal, aumento do volume abdominal, decúbito esternal ou lateral com a cabeça voltada para o flanco e morte. O curso clínico foi de 2-7 dias. A principal alteração notada durante a necropsia dos bovinos foi a obstrução intestinal por fitobezoar ovoide de 2-5 cm de diâmetro, frequentemente no duodeno, próximo ao piloro, ocasionalmente em sua porção média. Na área de obstrução notou-se a necrose da parede intestinal que estava enegrecida e intensamente demarcada em relação a segmento adjacente normal, além de intenso espessamento associado a edema, congestão e hemorragia. O abomaso e os segmentos intestinais anteriores a obstrução apresentavam-se repletos de conteúdo alimentar líquido e posteriormente estava com ausência de conteúdo. O rúmen frequentemente tinha grande quantidade de conteúdo liquido a pastoso. Em todos os bovinos necropsiados múltiplas estruturas sólidas, arredondadas ou ovoides, esverdeadas (fitobezoares) foram observadas no abomaso. Estes achados indicam que pastagens com predomínio de Stylosanthes sp. predispõem a formação de fitobezoares, os quais podem levar à obstrução intestinal e morte em bovinos.(AU)
Asunto(s)
Animales , Bovinos , Obstrucción Intestinal/veterinaria , Fabaceae/efectos adversos , Signos y Síntomas/veterinaria , Necrosis/veterinaria , Mucosa Intestinal , Cólico/veterinaria , MortalidadRESUMEN
Beta-ketoacyl-ACP reductase catalyzes the NADPH-dependent reduction of beta-ketoacyl-acyl carrier protein to generate beta-hydroxyacyl-acyl carrier protein and NADP+, the second step of the fatty acid elongation system type II of bacteria, plants, and apicomplexan organisms. Here, a modified and more efficient purification protocol is reported for recombinant Mycobacterium tuberculosis beta-ketoacyl-ACP reductase (MabA). The increase in alpha-secondary deuterium kinetic isotope effect values measured at pH 10 as compared to those obtained at pH 7 points to isotope- and pH-sensitive steps occurring concomitantly. Equilibrium and kinetic fluorescence studies demonstrate positive cooperativity in binding of NADPH to MabA, with two forms of free enzyme in solution. Equilibrium dialysis shows no cooperativity in acetoacetyl-CoA binding to the enzyme. Moreover, modest affinity loss occurs when the substrates bind to the monomer as compared to the dimer of MabA. A mechanism of substrate binding to MabA is proposed on the basis of the experimental data.
Asunto(s)
Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/enzimología , 3-Oxoacil-(Proteína Transportadora de Acil) Reductasa , Acilcoenzima A/química , Acilcoenzima A/metabolismo , Oxidorreductasas de Alcohol/aislamiento & purificación , Proteínas Bacterianas/aislamiento & purificación , Catálisis , Deuterio/química , Deuterio/metabolismo , Dimerización , Hidrógeno/química , Hidrógeno/metabolismo , Cinética , Modelos Químicos , Ácidos Micólicos/síntesis química , Ácidos Micólicos/metabolismo , NADP/química , NADP/metabolismo , Unión Proteica , Protones , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Especificidad por SustratoRESUMEN
Mycobacterium tuberculosis shikimate dehydrogenase (MtbSD) catalyzes the fourth reaction in the shikimate pathway, the NADPH-dependent reduction of 3-dehydroshikimate. To gather information on the kinetic mechanism, initial velocity patterns, product inhibition, and primary deuterium kinetic isotope effect studies were performed and the results suggested a steady-state ordered bi-bi kinetic mechanism. The magnitudes of both primary and solvent kinetic isotope effects indicated that the hydride transferred from NADPH and protons transferred from the solvent in the catalytic cycle are not significantly rate limiting in the overall reaction. Proton inventory analysis indicates that one proton gives rise to solvent isotope effects. Multiple isotope effect studies indicate that both hydride and proton transfers are concerted. The pH profiles revealed that acid/base chemistry takes place in catalysis and substrate binding. The MtbSD 3D model was obtained in silico by homology modeling. Kinetic and chemical mechanisms for MtbSD are proposed on the basis of experimental data.
Asunto(s)
Oxidorreductasas de Alcohol/química , Proteínas Bacterianas/química , Mycobacterium tuberculosis/enzimología , NADP/química , Secuencia de Aminoácidos , Deuterio , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Protones , Homología de Secuencia de AminoácidoRESUMEN
Beta-ketoacyl-acyl carrier protein (ACP) reductase from Mycobacterium tuberculosis (MabA) is responsible for the second step of the type-II fatty acid elongation system of bacteria, plants, and apicomplexan organisms, catalyzing the NADPH-dependent reduction of beta-ketoacyl-ACP to generate beta-hydroxyacyl-ACP and NADP(+). In the present work, the mabA-encoded MabA has been cloned, expressed, and purified to homogeneity. Initial velocity studies, product inhibition, and primary deuterium kinetic isotope effects suggested a steady-state random bi-bi kinetic mechanism for the MabA-catalyzed reaction. The magnitudes of the primary deuterium kinetic isotope effect indicated that the C(4)-proS hydrogen is transferred from the pyridine nucleotide and that this transfer contributes modestly to the rate-limiting step of the reaction. The pH-rate profiles demonstrated groups with pK values of 6.9 and 8.0, important for binding of NADPH, and with pK values of 8.8 and 9.6, important for binding of AcAcCoA and for catalysis, respectively. Temperature studies were employed to determine the activation energy of the reaction. Solvent kinetic isotope effects and proton inventory analysis established that a single proton is transferred in a partially rate-limiting step and that the mechanism of carbonyl reduction is probably concerted. The observation of an inverse (D)2(O)V/K and an increase in (D)2(O)V when [4S-(2)H]NADPH was the varied substrate obscured the distinction between stepwise and concerted mechanisms; however, the latter was further supported by the pH dependence of the primary deuterium kinetic isotope effect. Kinetic and chemical mechanisms for the MabA-catalyzed reaction are proposed on the basis of the experimental data.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Mycobacterium tuberculosis/enzimología , 3-Oxoacil-(Proteína Transportadora de Acil) Reductasa , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/genética , Concentración de Iones de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética/métodos , Estructura Molecular , Oxidación-Reducción , Especificidad por Sustrato , TemperaturaRESUMEN
Tuberculosis (TB) poses a major worldwide public health problem. The increasing prevalence of TB, the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, the causative agent of TB, and the devastating effect of co-infection with HIV have highlighted the urgent need for the development of new antimycobacterial agents. Analysis of the complete genome sequence of M. tuberculosis shows the presence of genes involved in the aromatic amino acid biosynthetic pathway. Experimental evidence that this pathway is essential for M. tuberculosis has been reported. The genes and pathways that are essential for the growth of the microorganisms make them attractive drug targets since inhibiting their function may kill the bacilli. We have previously cloned and expressed in the soluble form the fourth shikimate pathway enzyme of the M. tuberculosis, the aroE-encoded shikimate dehydrogenase (mtSD). Here, we present the purification of active recombinant aroE-encoded M. tuberculosis shikimate dehydrogenase (mtSD) to homogeneity, N-terminal sequencing, mass spectrometry, assessment of the oligomeric state by gel filtration chromatography, determination of apparent steady-state kinetic parameters for both the forward and reverse directions, apparent equilibrium constant, thermal stability, and energy of activation for the enzyme-catalyzed chemical reaction. These results pave the way for structural and kinetic studies, which should aid in the rational design of mtSD inhibitors to be tested as antimycobacterial agents.
Asunto(s)
Oxidorreductasas de Alcohol/aislamiento & purificación , Proteínas Bacterianas/aislamiento & purificación , Mycobacterium tuberculosis/enzimología , Oxidorreductasas de Alcohol/antagonistas & inhibidores , Oxidorreductasas de Alcohol/química , Antibacterianos/uso terapéutico , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Cromatografía Liquida/métodos , Diseño de Fármacos , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Genoma Bacteriano , Infecciones por VIH/complicaciones , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/enzimología , Cinética , Espectrometría de Masas/métodos , Mycobacterium tuberculosis/química , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Tuberculosis Resistente a Múltiples Medicamentos/complicaciones , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/enzimologíaRESUMEN
Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of nucleosides and deoxynucleosides, generating ribose 1-phosphate and the purine base, which is an important step of purine catabolism pathway. The lack of such an activity in humans, owing to a genetic disorder, causes T-cell impairment, and drugs that inhibit this enzyme may have the potential of being utilized as modulators of the immunological system to treat leukemia, autoimmune diseases, and rejection in organ transplantation. Here, we describe kinetics and crystal structure of human PNP in complex with 7-methyl-6-thio-guanosine, a synthetic substrate, which is largely used in activity assays. Analysis of the structure identifies different protein conformational changes upon ligand binding, and comparison of kinetic and structural data permits an understanding of the effects of atomic substitution on key positions of the synthetic substrate and their consequences to enzyme binding and catalysis. Such knowledge may be helpful in designing new PNP inhibitors.
Asunto(s)
Guanosina/análogos & derivados , Purina-Nucleósido Fosforilasa/química , Purina-Nucleósido Fosforilasa/metabolismo , Tionucleósidos/metabolismo , Catálisis , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Guanosina/metabolismo , Guanosina/farmacología , Humanos , Cinética , Ligandos , Fosforilación , Unión Proteica , Conformación Proteica , Purinas/metabolismo , Ribosamonofosfatos/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Tionucleósidos/farmacología , Tionucleótidos/metabolismoRESUMEN
Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for human PNP causes T-cell deficiency as the major physiological defect. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant tissue rejection, psoriasis, rheumatoid arthritis, lupus, and T-cell lymphomas. Human PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation. In addition, bacterial PNP has been used as reactant in a fast and sensitive spectrophotometric method that allows both quantitation of inorganic phosphate (P(i)) and continuous assay of reactions that generate P(i) such as those catalyzed by ATPases and GTPases. Human PNP may therefore be an important biotechnological tool for P(i) detection. However, low expression of human PNP in bacterial hosts, protein purification protocols involving many steps, and low protein yields represent technical obstacles to be overcome if human PNP is to be used in either high-throughput drug screening or as a reagent in an affordable P(i) detection method. Here, we describe PCR amplification of human PNP from a liver cDNA library, cloning, expression in Escherichia coli host, purification, and activity measurement of homogeneous enzyme. Human PNP represented approximately 42% of total soluble cell proteins with no induction being necessary to express the target protein. Enzyme activity measurements demonstrated a 707-fold increase in specific activity of cloned human PNP as compared to control. Purification of cloned human PNP was achieved by a two-step purification protocol, yielding 48 mg homogeneous enzyme from 1L cell culture, with a specific activity value of 80 Umg(-1).