Antimicrobial peptides purified from hydrolysates of kanihua (Chenopodium pallidicaule Aellen) seed protein fractions.

The kanihua (Chenopodium pallidicaule Aellen) Andean grain from the Peruvian Altiplano presents proteins of 15% to 19%. The objective was to obtain purified bioactive antimicrobial peptides (AMPs), hydrolyzed with Alcalase and Pepsin-pancreatin sequential system of protein fractions of kanihua varieties Ramis (KR) and Cupi-Sayhua (KS), and hydrolysates with different degrees of hydrolysis (DH) and percentage inhibition (IP) of the growth of E. coli, S. aureus, and C. albicans. To obtain AMPs, nutraceuticals, bio-preservatives, and novel ingredients in food design. The results showed 216 hydrolysates (1%, w/v), only 28 presented significant difference compared to controls (IP ≥ 45%, p ≤ 0.05), 4 AMPs were purified by chromatography, glutelins KS 4 h (1:10) stood out with DH 40% and IP 52% and 70% of S. aureus and C. albicans, respectively (p ≤ 0.05), showed minimum inhibitory concentration (MIC) of 95% for E. coli (p ≤ 0.05), and presented an anionic charge. In conclusion, the simulated digestion in vitro showed higher DH (7%-67%) than Alcalase (13%-54%); the majority were extensive; of 28 hydrolysates with IP ≥ 45% 4 AMPs with important IPs were obtained, and one was anionic.

Purification of Pseudomonas sp. proteases through aqueous biphasic systems as an alternative source to obtain bioactive protein hydrolysates.

Aqueous biphasic systems (ABSs) are an interesting alternative for separating industrial enzymes due to easy scale-up and low operational cost. The proteases of Pseudomonas sp. M211 were purified through ABS platforms formed by polyethylene glycol (PEG) and citrate buffer salt. Two experimental designs 23 + 4 were performed to evaluate the following parameters: molar mass of PEG (MPEG ), concentration of PEG (CPEG ), concentration of citrate buffer (CCit ), and pH. The partition coefficient (K), activity yield (Y), and purification factor (PF) were the responses analyzed. The best purification performance was obtained with the system composed of MPEG  = 10,000 g/mol, CPEG  = 22 wt%, CCit  = 12 wt%, pH = 8.0; the responses obtained were K = 4.9, Y = 84.5%, PF = 15.1, and tie-line length = 52.74%. The purified proteases of Pseudomonas sp. (PPP) were used to obtain hydrolysates of Lupinus mutabilis (Peruvian lupin cultivar) seed protein in comparison with the commercial protease Alcalase® 2.4L. A strong correlation between hydrolysis degree and radical scavenging activity was observed, and the highest antioxidant activity was obtained with Alcalase® (1.40 and 3.47 µmol Trolox equivalent/mg protein, for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and oxygen radical absorbance capacity, respectively) compared with PPP (0.55 and 1.03 µmol Trolox/mg protein). Nevertheless, the IC50 values were lower than those often observed for antioxidant hydrolysates from plant proteins. PEG/citrate buffer system is valuable to purify Pseudomonas proteases from the fermented broth, and the purified protease could be promising to produce antioxidant protein hydrolysates.

Yeasts Associated with Various Amazonian Native Fruits.

Yeasts, commonly present on the surface of fruits, are of industrial interest for the production of enzymes, flavorings, and bioactive compounds, and have many other scientific uses. The Amazonian rainforest may be a good source of new species or strains of yeasts, but their presence on Amazonian fruits is unknown. The aim of this study was to identify and characterize yeasts isolated from Amazonian native fruits using molecular and phenotypic methods. In total, 81 yeast isolates were obtained from 10 fruits species. Rep-PCR showed 29 strain profiles. Using a combination of restriction-fragment length polymorphism (RFLP) of the 5.8S-ITS region and D1/D2 sequencing of the 26S rRNA gene, 16 species were identified belonging to genera Candida, Debaryomyces, Hanseniaspora, Kodamaea, Martiniozyma, and Meyerozyma. The most dominant species were Candida tropicalis, Debaryomyces hansenii, Hanseniaspora opuntiae, and Hanseniaspora thailandica. H. opuntiae and H. thailandica showed the highest number of the strain profiles. Phenotypic profiles were variable between species, and even among strains. Screening for hydrolases showed lipolytic activity in only one isolate, while proteolytic, cellulolytic and amylolytic capabilities were not detected. Yeast presence among fruits varied, with cidra (Citrus medica) and ungurahui (Oenocarpus bataua) having the highest number of species associated. This investigation broadens the understanding and possible biotechnological uses of yeast strains obtained from Amazonian native fruits.Yeasts, commonly present on the surface of fruits, are of industrial interest for the production of enzymes, flavorings, and bioactive compounds, and have many other scientific uses. The Amazonian rainforest may be a good source of new species or strains of yeasts, but their presence on Amazonian fruits is unknown. The aim of this study was to identify and characterize yeasts isolated from Amazonian native fruits using molecular and phenotypic methods. In total, 81 yeast isolates were obtained from 10 fruits species. Rep-PCR showed 29 strain profiles. Using a combination of restriction-fragment length polymorphism (RFLP) of the 5.8S-ITS region and D1/D2 sequencing of the 26S rRNA gene, 16 species were identified belonging to genera Candida, Debaryomyces, Hanseniaspora, Kodamaea, Martiniozyma, and Meyerozyma. The most dominant species were Candida tropicalis, Debaryomyces hansenii, Hanseniaspora opuntiae, and Hanseniaspora thailandica. H. opuntiae and H. thailandica showed the highest number of the strain profiles. Phenotypic profiles were variable between species, and even among strains. Screening for hydrolases showed lipolytic activity in only one isolate, while proteolytic, cellulolytic and amylolytic capabilities were not detected. Yeast presence among fruits varied, with cidra (Citrus medica) and ungurahui (Oenocarpus bataua) having the highest number of species associated. This investigation broadens the understanding and possible biotechnological uses of yeast strains obtained from Amazonian native fruits.

Obtención de hidrolizados proteicos de leguminosas usando una proteasa recombinante de Pseudomonas aeruginosa M211 / Obtaining protein hydrolysates from leguminous using a recombinant protease from Pseudomonas aeruginosa M211

El género Pseudomonas es una fuente importante de proteasas; sin embargo, su uso está restringido en la industria alimentaria. El clonaje permite aprovechar la capacidad catalítica de estas enzimas mediante su producción en microorganismos inocuos. Por otro lado, las leguminosas son fuentes ricas en proteínas, a partir de las cuales se pueden obtener compuestos con valor agregado mediante procesos de hidrólisis enzimática. En este estudio, se produjo y caracterizó una proteasa recombinante (PT4) alcalina y termoestable de Pseudomonas aeruginosa M211, para la obtención de hidrolizados proteicos de leguminosas. Para ello, el gen de la proteasa se clonó en el vector pJET1.2/blunt utilizando E. coli DHalfa como hospedero. El análisis de la secuencia nucleotídica parcial de la proteasa indicó un 99 % de similitud con Peptidasas de la Familia M4 de Pseudomonas aeruginosa. La enzima recombinante presentó un peso molecular de 80 kDa, demostró ser activa y estable en condiciones alcalinas y termófilas con un pH y temperatura óptimos de 8 y 60 °C, respectivamente, y fue inhibida por EDTA. Además, hidrolizó proteínas de semillas de Glycine max, Phaseolus lunatus, Lupinus mutabilis y Erythrina edulis, obteniéndose fracciones peptídicas menores a 40 kDa. Esta proteasa recombinante se podría utilizar en la elaboración de hidrolizados proteicos funcionales a partir proteínas de distintas fuentes y residuos agroalimentarios.

The genus Pseudomonas is an important source of proteases; however, in the food industry the use of this bacterium is restricted. Cloning allows for the use of the proteolytic activity of Pseudomonas proteases through their production in innocuous microorganisms. Leguminous are protein-rich sources from which value-added compounds can be obtained through enzymatic hydrolysis. In this study, an alkaline and thermostable recombinant protease (PT4) from Pseudomonas aeruginosa M211 was cloned and characterized in order to obtain protein hydrolysates from leguminous. Therefore, protease gene was cloned into the pJET1.2 / blunt vector using E. coli DHalpha as a host. Analysis of protease partial nucleotide sequence showed 99% homology with Peptidases M4 Family from Pseudomonas aeruginosa. The molecular weight of the recombinant enzyme was 80 kDa, it was active and stable under alkaline and thermophilic conditions, presented an optimum pH and temperature of 8 and 60 °C, respectively, and was inhibited by EDTA. In addition, it hydrolysed Glycine max, Phaseolus lunatus, Lupinus mutabilis y Erythrina edulis proteins, obtaining peptide fractions less than 40 kDa. This recombinant protease could be used in the elaboration of functional hydrolysates using protein from different sources and agricultural waste.

Novel antioxidant peptides obtained by alcalase hydrolysis of Erythrina edulis (pajuro) protein.

BACKGROUND: Oxidative reactions are responsible for the changes in quality during food processing and storage. Oxidative stress is also involved in multiple chronic diseases, such as cardiovascular and neurodegenerative disorders, diabetes, cancer, and aging. The consumption of dietary antioxidants has been demonstrated to help to reduce the oxidative damage in both the human body and food systems. In this study, the potential of Erythrina edulis (pajuro) protein as source of antioxidant peptides was evaluated. RESULTS: Pajuro protein concentrate hydrolyzed by alcalase for 120 min showed potent ABTS·+ and peroxyl radical scavenging activity with Trolox equivalent antioxidant capacity (TEAC) and oxygen radical absorbance capacity (ORAC) values of 1.37 ± 0.09 µmol TE mg-1 peptide and 2.83 ± 0.07 µmol TE mg-1 peptide, respectively. Fractionation of the hydrolyzate to small peptides resulted in increased antioxidant activity. De novo sequencing of most active fractions collected by chromatographic analysis enabled 30 novel peptides to be identified. Of these, ten were synthesized and their radical activity evaluated, demonstrating their relevant contribution to the antioxidant effects observed for pajuro protein hydrolyzate. CONCLUSIONS: The sequences identified represent an important advance in the molecular characterization of the pajuro protein, demonstrating its potential as a source of antioxidant peptides for food and nutraceutical applications. © 2018 Society of Chemical Industry.

Novel extremophilic proteases from Pseudomonas aeruginosa M211 and their application in the hydrolysis of dried distiller's grain with solubles.

Proteases are the most important group of industrial enzymes and they can be used in several fields including biorefineries for the valorization of industrial byproducts. In this study, we purified and characterized novel extremophilic proteases produced by a Pseudomonas aeruginosa strain isolated from Mauritia flexuosa palm swamps soil samples in Peruvian Amazon. In addition, we tested their ability to hydrolyze distillers dried grains with solubles (DDGS) protein. Three alkaline and thermophilic serine proteases named EI, EII, and EIII with molecular weight of 35, 40, and 55 kDa, respectively, were purified. EI and EIII were strongly inhibited by EDTA and Pefabloc being classified as serine-metalloproteases, while EII was completely inhibited only by Pefabloc being classified as a serine protease. In addition, EI and EII exhibited highest enzymatic activity at pH 8, while EIII at pH 11 maintaining almost 100% of it at pH 12. All the enzymes demonstrated optimum activity at 60°C. Enzymatic activity of EI was strongly stimulated in presence of Mn2+ (6.9-fold), EII was stimulated by Mn2+ (3.7-fold), while EIII was slightly stimulated by Zn2+ , Ca2+ , and Mg2+ . DDGS protein hydrolysis using purified Pseudomonas aeruginosa M211 proteases demonstrated that, based on glycine released, EIII presented the highest proteolytic activity toward DDGS. This enzyme enabled the release 63% of the total glycine content in wheat DDGS protein, 2.2-fold higher that when using the commercial Pronase®. Overall, our results indicate that this novel extremopreoteases have a great potential to be applied in DDGS hydrolysis. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2728, 2019.

Erythrina edulis (Pajuro) Seed Protein: A New Source of Antioxidant Peptides.

Erythrina edulis Triana ex Micheli is a protein-enriched legume traditionally used for both dietary and medicinal purposes. In this paper, protein concentrate was obtained from the seed flour. SDS-PAGE analysis revealed a high number and intensity of bands in the range between 10 and 90 kDa. Neutrase, Flavourzyme, and Alcalase were used to hydrolyze the protein concentrate at different times. By SDS-PAGE, the lower resistance of proteins to Alcalase action was observed, providing hydrolyzates with higher radical scavenging activity. The 120 min-hydrolyzate showed ORAC and TEAC values of 2.51 and 0.91 µmol Trolox equivalents/mg of protein, respectively. A fraction lower than 3 kDa and rich in hydrophobic and aromatic amino acids was demonstrated to be mainly responsible for the observed activity. E. edulis could be a new alternative in the formulation of functional foods not only for its high protein content but also for the potential biological properties of its hydrolyzates.

Caracterización de bacterias halófilas productoras de amilasas aisladas de las Salinas de San Blas en Junín / Characterization of halophilic bacteria producing amylase isolated from San Blas Salterns in Junin

El objetivo de este estudio fue caracterizar bacterias halófilas con actividad amilolítica provenientes de las Salinas de San Blas-Junín, ubicadas en los Andes peruanos aproximadamente a 4100 m de altitud. Este estudio se realizó con 34 bacterias aisladas de muestras de suelos las cuales se cultivaron en agar agua de sales (SW) 5 % conteniendo extracto de levadura 0,5 % y almidón 1 %. El 41 % de bacterias mostró la capacidad de hidrolizar almidón, éstas fueron caracterizadas mediante pruebas fisiológicas y bioquímicas convencionales. Tres bacterias fueron Gram-negativas y once Gram-positivas. El 21 % (3/14) creció en un amplio rango de concentración de sales, entre 5 y 20 %. El 14 % (2/14) de las bacterias presentó actividad lipolítica, proteolítica y nucleolítica, y el 29 % (4/14), presentó actividad proteolítica y nucleolítica. Las bacterias se identificaron mediante los perfiles de restricción de los genes ribosómicos 16S amplificados, las enzimas usadas fueron Hae III, BstU I, Hinf I y Cfo I. Los genes ribosómicos 16S de siete bacterias que presentaron perfiles de ADN diferentes se amplificaron, secuenciaron y analizaron mediante programas bioinformáticos. Del análisis fenotípico y molecular de las 14 bacterias amilolíticas se obtuvieron dos grupos, uno perteneciente al género Halomonas (3) y el otro, al género Bacillus (11). Las bacterias amilolíticas caracterizadas podrían ser de potencial uso a nivel industrial.

The aim of this study was to characterize halophilic amylolytic bacteria from San Blas Salterns-Junin, located in the Peruvian Andes at approximately 4 100 m of altitude. This study was conducted with 34 bacteria isolated from soil samples which were cultured in salt water medium (SW) 5 % containing 0,5 % yeast extract and 1 % starch. It was found that 41 % were starch-degrading bacteria, which were further characterized with conventional physiological and biochemical tests. Three bacteria were Gram-negative and eleven Gram-positive. Also, 21 % (3/14) was able to grow in a wide range of saltconcentration from 5 to 20 %. We reported that 14 % (2/14) of bacteria had all lipolytic, proteolytic and nucleolytic activity, and 29 % (4/14) had both proteolytic and nucleolytic activity. Bacteria were identified by restriction 16S ribosomal genes profiles, enzymes used were Hae III, BstU I, Hinf I and Cfo I. 16S ribosomal genes of seven isolated wich showed different DNA profiles were amplified, partial sequenced and analyzed using bioinformatic programs. By both phenotypic and molecular analysis of 14 amylolytic bacteria two groups were obtained, one belonged to the genus Halomonas (3) and the other, to the genus Bacillus (11). The characterized amylolytic bacteria could have a potential industrial use.

Clonaje y caracterización molecular in silico de un transcrito de fosfolipasa A2 aislado del veneno de la serpiente peruana Lachesis muta / Molecular cloning and characterization in silico of phospholipase A2 transcripto isolated from Lachesis muta peruvian snake venom

Objetivo. Aislar y caracterizar in silico un transcrito del gen de fosfolipasa A2 (PLA2) aislado del veneno de Lachesis muta de la Amazonía peruana. Materiales y métodos. Se amplificó el transcrito del gen sPLA2 mediante la técnica de RT-PCR a partir de RNA total utilizando cebadores específicos, el producto de DNA amplificado se insertó en el vector pGEM para su posterior secuenciación. Mediante análisis bioinformático de la secuencia nucleotídica se determinó un marco de lectura abierta de 414 nucleótidos que codifica 138 aminoácidos, incluyendo16 aminoácidos del péptido señal, el peso molecular y el pI fueron de 13 976 kDa y 5,66 respectivamente. Resultados. La secuencia aminoacídica denominada Lm-PLA2- Perú, contiene Asp49, así como Tyr-28, Gly-30, Gly-32, His-48, Tyr52, Asp99 importantes para la actividad enzimática. La comparación de Lm-PLA2-Perú con las secuencias aminoacídicas de los bancos de datos mostró 93 por ciento de similitud con las sPLA2 de Lachesis stenophrys y más del 80 por ciento con otras sPLA2 de venenos de la familia Viperidae. El análisis filogenético de la secuencia nucleotídica del transcrito del gen sPLA2 indica que Lm-PLA2-Perú se agrupa con otras sPLA2 [Asp49] ácidas previamente aisladas del veneno de Bothriechis schlegelii con un 89 por ciento de identidad. El modelaje tridimensional de Lm-PLA2-Perú, presenta una estructura característica de sPLA2 del Grupo II formada por tres hélices-α, una lámina-β, una hélice corta y un lazo de unión con calcio. Conclusión. La secuencia nucleotídica corresponde al primer transcripto del gen de PLA2 clonado a partir del veneno de la serpiente Lachesis muta, que habita en la selva del Perú.

Objective. Isolate and characterize in silico gene phospholipase A2 (PLA2) isolated from Lachesis muta venom of the Peruvian Amazon. Material and methods. Technique RT-PCR from total RNA was using specific primers, the amplified DNA product was inserted into the pGEM vector for subsequent sequencing. By bioinformatic analysis identified an open reading frame of 414 nucleotides that encoded 138 amino acids including a signal peptide of 16 aminoacids, molecular weight and pI were 13 976 kDa and 5.66 respectively. Results. The aminoacid sequence was called Lm-PLA2-Peru, contains an aspartate at position 49, this aminoacid in conjunction with other conserved residues such as Tyr-28, Gly-30, Gly-32, His-48, Tyr52, Asp99 are important for enzymatic activity. The comparison with the amino acid sequence data banks showed of similarity between PLA2 from Lachesis stenophrys (93 percent) and other PLA2 snake venoms and over 80 percent of other sPLA2 family Viperidae venoms. A phylogenetic analysis showed that Lm-PLA2-Peru grouped with other acidic [Asp49] sPLA2 previously isolated from Bothriechis schlegelii venom showing 89 percent nucleotide sequence identity. Finally, the computer modeling indicated that enzyme had the characteristic structure of sPLA2 group II that consisted of three α-helices, a β-wing, a short helix and a calcium-binding loop. Conclusion. The nucleotide sequence corresponding to the first transcript of gene from PLA2 cloned of Lachesis muta venom, snake from the Peruvian rainforest.

[Molecular cloning and characterization in silico of phospholipase A(2) transcript isolated from Lachesis muta peruvian snake venom]. / Clonaje y caracterización molecular in silico de un transcrito de fosfolipasa A(2) aislado del veneno de la serpiente peruana Lachesis muta.

OBJECTIVE: Isolate and characterize in silico gene phospholipase A(2) (PLA(2)) isolated from Lachesis muta venom of the Peruvian Amazon. MATERIAL AND METHODS: Technique RT-PCR from total RNA was using specific primers, the amplified DNA product was inserted into the pGEM vector for subsequent sequencing. By bioinformatic analysis identified an open reading frame of 414 nucleotides that encoded 138 amino acids including a signal peptide of 16 aminoacids, molecular weight and pI were 13,976 kDa and 5.66 respectively. RESULTS: The aminoacid sequence was called Lm-PLA(2)-Peru, contains an aspartate at position 49, this aminoacid in conjunction with other conserved residues such as Tyr-28, Gly-30, Gly-32, His-48, Tyr52, Asp99 are important for enzymatic activity. The comparison with the amino acid sequence data banks showed of similarity between PLA(2) from Lachesis stenophrys (93%) and other PLA(2) snake venoms and over 80% of other sPLA(2) family Viperidae venoms. A phylogenetic analysis showed that Lm-PLA(2)-Peru grouped with other acidic [Asp(49)] sPLA(2) previously isolated from Bothriechis schlegelii venom showing 89 % nucleotide sequence identity. Finally, the computer modeling indicated that enzyme had the characteristic structure of sPLA(2) group II that consisted of three α-helices, a ß-wing, a short helix and a calcium-binding loop. CONCLUSION: The nucleotide sequence corresponding to the first transcript of gene from PLA(2) cloned of Lachesis muta venom, snake from the Peruvian rainforest.

Serotyping of Avibacterium paragallinarum isolates from Peru.

This study appears to represent the first serotyping study of 24 isolates of Avibacterium paragallinarum obtained from different regions of Peru during 1998-2008. All isolates were characterized as beta-nicotinamide adenine dinucleotide dependent. According to the Page scheme, modified by Blackall, it was found that eight isolates were classified as serogroup A, seven isolates as serogroup B, and five isolates as serogroup C, while four isolates could not be serotyped. Further serotyping, following the same scheme but using rabbit antiserum raised against Argentinean strains of the three serogroups, allowed allocation of these four unclassified isolates to serogroup B. These results suggest that some of the Peruvian B isolates appear to be similar to the previously described variant B isolates from Argentina. Therefore, inactivated vaccines used in Peru should include the three recognized serogroups (A, B, and C), with the addition of at least one of these variant B isolates. Cross-protection trials are needed to compare the protection conferred by vaccines containing traditional B serovar strains to the protection by experimental vaccines containing variant B serovar isolates from Peru.

Expression and substrate specificity of a recombinant cysteine proteinase B of Leishmania braziliensis.

The cysteine proteinase B of Leishmania parasites is an important virulence factor. In this study we have expressed, isolated and characterized for the first time a recombinant CPB from Leishmania braziliensis, the causative agent of mucocutaneous leishmaniosis. The mature region of the recombinant CPB shares a high percentage identity with its Leishmania mexicana CPB2.8 (rCPB2.8DeltaCTE) counterpart (76.36%) and has identical amino acid residues at the S(1), catalytic triad and S'(1) subsites. Nevertheless, when the kinetics of substrate hydrolysis was measured using a combinatorial library of internally quenched fluorescent peptides based upon the lead sequence Abz-KLRSSKQ-EDDnp, significant differences were obtained. These results suggest that the differences in substrate utilization observed between the L. mexicana and L. braziliensis CPs must be related to amino acid modifications outside the core of the active site cleft. Moreover, a potent inhibitor with Pro at P1 and high affinity for L. braziliensis recombinant CPB showed less affinity to L. mexicana CPB 2.8, which preferred Phe, Leu, and Asn at the same position.

Amplified 16S ribosomal DNA restriction analysis for identification of Avibacterium paragallinarum.

A molecular technique based on the restriction fragment length polymorphism of the 16S ribosomal genes amplified by a polymerase chain reaction (PCR), referred to as amplified 16S ribosomal DNA restriction analysis (ARDRA), was designed to identify 19 Avibacterium paragallinarum strains isolated from infraorbital sinus and nasal turbinate bone samples of broiler chickens, breeders, and laying hens from different regions of Peru. The 16S rDNA was amplified by PCR using a pair of bacterial universal primers and restriction analysis of 16S rDNA sequences was done to select endonucleases with the highest number of cutting points inside the 16S rDNA. The DNA patterns with DdeI and RsaI endonucleases were identical for the 19 A. paragallinarum strains, but differed from those obtained for Ornithobacterium rhinotracheale, a bacterium with a high genetic and phenotypic resemblance to A. paragallinarum, as well as from Escherichia coli, a bacterium associated with infectious coryza. The ARDRA method could prove to be valuable for molecular identification of A. paragallinarum, a microorganism implicated in respiratory diseases in commercial birds.

Mutación F508 en pacientes diagnosticados con fibrosis quística del Instituto de Salud del Ni±o / Mutation F508 in patients diagnosed with cystic fibrosis of the Institute of Health of the Child

El objetivo de este trabajo fue detectar la mutación delta F508 en el gen CFTR en pacientes diagnosticados con Fibrosis Quística (FQ) del Servicio de Neumología del Instituto Especializado de Salud del Ni±o. En el estudio participaron once pacientes diagnosticados con FQ, cuyos padres firmaron un consentimiento informado. Se extrajo ADN genómico de muestras de sangre y se amplificó el exón 10 del gen CFTR por la reacción en cadena de lapolimerasa con cebadores específicos. El polimorfismo en el producto amplificado se determinó usando la enzima Mbol y electroforesis en geles de agarosa. Se identificaron cuatro pacientes heterocigotos con la mutación delta F508, lafrecuencia alélica estimada fue de18.2 por ciento para la mutación delta F508 en la población con FQ estudiada. Se concluye que la mutación delta F508 fue detectada en 4 de los 11 pacientes con diagnóstico de FQ.

To detect the delta F508 mutation in CFTR gene in patients diagnosed with cystic fibrosis (CF) from Neumology Service at Instituto Especializado de Salud del Ni±o, eleven individuals diagnosed with CF were taken, the parents of the patients previously signed an informed consent. Genomic DNA from blood samples was extracted and it was amplified exon 10 of CFTRgene by polymerase chain reaction with specific primers. Polymorphisms in the amplified products were analyzed using Mbol enzyme and agarose gel electrophoresis. Four heterozygote patients with the delta F508 mutation were detected. Allelic frequency was estimated in 18.2 per cent for the delta F508 mutation in the studied CF population. In conclusion, the delta F508 mutation was detected in 4 of 11 CF patients.

Distribution of paraoxonase-1 gene polymorphisms and enzyme activity in a Peruvian population.

Paraoxonase-1 (PON1) is a serum esterase associated with high density lipoproteins and capable of detoxifying toxic metabolites of organophosphorus (OP) compounds. Two major polymorphisms have been described in the coding region of the PON1 gene at positions 192 and 55 and at least five in the 5'-regulatory region, the most important at position -108. Depending on the substrate, PON1 192 Q/R polymorphism can affect PON1 enzymatic activity. In the present study, we have determined the distribution of the PON1 192 Q/R and -108 C/T polymorphisms in a Peruvian population and compared the distribution of these polymorphisms with those of other world populations. PON1 phenotype and enzyme activity also were measured as they can influence the population resistance to the toxicity of OP compounds. The genotype distribution at position 192 was: QQ = 0.236, QR = 0.607, and RR = 0.157; and distribution at position -108 was: CC = 0.315, CT = 0.596, and TT = 0.089. The frequencies of the high activity R and C alleles were 0.461 and 0.613, respectively. The frequency of the PON1 192 Q allele was significantly lower than that of American, Caucasian-American, European-Brazilian, and Costa Rican samples. Outside the American continent, the frequency of this allele was lower than for all European countries, Thais, and Indians, but higher than for Chinese or Japanese. Regarding the toxicological importance of these polymorphisms, it was inferred that PON1 phenotyping (assessment of the R alloform) and genotyping (determination of the PON1 -108TT genotype) could be helpful as individual markers of susceptibility. PON1 phenotyping may be useful in further epidemiological studies involving agriculture workers occupationally exposed to OP compounds in developing countries.

Intraspecies genetic variability of Ornithobacterium rhinotracheale in commercial birds in Peru.

Strains of the bacterium Ornithobacterium rhinotracheale (ORT), a causal agent of respiratory diseases in birds, were microbiologically isolated, identified, and molecularly characterized. Blood-enriched culture media and biochemistry tests were used for microbiologic identification. Polymerase chain reaction (PCR) and repetitive extragenic palindromic PCR (rep-PCR) techniques were used for molecular identification and characterization, respectively, of the microorganism. ORT strains were isolated in enriched media from the trachea and air sacs of broilers, breeders, and layers from several geographic zones of Peru. Of the original 75 strains isolated from 75 clinical samples from which ORT was recovered during 1998-2000, 25 were selected for further study based on ORT as the primary pathogenic isolate (no other pathogens were detected). Selected isolates were molecularly identified and characterized by PCR using specific primers designed from the conserved zones of the 16S ribosomal genes. Primers used for the identification of ORT produced a specific fragment of 784 base pair (bp), which did not appear in Haemophilus paragallinarum or Pasteurella multocida, microorganisms with similar morphologic and biochemical characteristics that produce dinical signs identical to those of ORT. All 25 strains of ORT tested with rep-PCR had a genetic profile similar to that of ORT American Type Culture Collection 51463, indicating the presence of only one genotype in the ORT strains studied.