Twin-arginine signal peptide of Bacillus licheniformis GlmU efficiently mediated secretory expression of protein glutaminase

Background: Protein glutaminase specifically deamidates glutamine residue in protein and therefore significantly improves protein solubility and colloidal stability of protein solution. In order to improve its preparation efficiency, we exploited the possibility for its secretory expression mediated by twin-arginine translocation (Tat) pathway in Bacillus licheniformis. Results: The B. licheniformis genome-wide twin-arginine signal peptides were analyzed. Of which, eleven candidates were cloned for construction of expression vectors to mediate the expression of Chryseobacterium proteolyticum protein glutaminase (PGA). The signal peptide of GlmU was confirmed that it significantly mediated PGA secretion into media with the maximum activity of 0.16 U/ml in Bacillus subtilis WB600. A mutant GlmU-R, being replaced the third residue aspartic acid of GlmU twin-arginine signal peptide with arginine by site-directed mutagenesis, mediated the improved secretion of PGA with about 40% increased (0.23 U/ml). In B. licheniformis CBBD302, GlmU-R mediated PGA expression in active form with the maximum yield of 6.8 U/ml in a 25-l bioreactor. Conclusions: PGA can be produced and secreted efficiently in active form via Tat pathway of B. licheniformis, an alternative expression system for the industrial-scale production of PGA.

Estudo de mutações nas proteínas virais E, NS3 e NS4B no genoma do vírus vacinal da Febre Amarela 17D / Study of mutations in the viral proteins E, NS3 and NS4B in the genome of the yellow fever vaccine virus 17D

Resumo: A Febre Amarela (FA) é uma doença infecciosa não contagiosa, causada por um arbovírus e objeto de preocupação sanitária mundial. A principal medida de controle é a vacinação com o vírus atenuado da FA, cepa 17D, que é capaz de induzir resposta imune protetora a longo prazo com administração em dose única. Vírus atenuados são potentes vetores de expressão, pois disseminam o antígeno no hospedeiro e induzem resposta imune protetora, contribuindo para o desenvolvimento de vacinas recombinantes. Diversas estratégias foram desenvolvidas para expressão de genes heterólogos pelo vírus vacinal FA 17D. Entretanto, a inserção induz proliferação viral e imunogenicidade reduzidas. Neste trabalho foi utilizado o vírus vacinal FA 17D recombinante expressando a proteína repórter enhanced green fluorescent protein (EGFP) para avaliar o impacto de mutações específicas que possam modular a replicação viral. Mutações nas proteínas E, NS3 e NS4B foram descritas por aumentarem a proliferação viral em cultura de células e em camundongos, no genoma do vírus da dengue, sorotipos 1 e 2. As mutações em E400 (F→L), E403 (T→I), NS3439 (V→S) e NS4B54 (L→F) foram inseridas no genoma do vírus FA/EGFP, com a finalidade de caracterizar o seu efeito na proliferação viral e na indução de resposta imune humoral. O cDNA do genoma viral FA/EGFP foi utilizado para gerar vírus recombinantes carreando uma, duas ou três mutações. O estudo de proliferação viral foi realizado por cinética de infecção de células das linhagens Vero, Huh7 e C6/36. Os resultados mostram que os vírus da FA recombinantes se proliferam menos que o vírus vacinal FA 17DD. Além disso, a infectividade dos vírus mutantes em células de mamífero é diferente da infectividade em células de mosquitoOs vírus que carreiam as mutações em E400/NS3439 e E400/NS3439/NS4B54 tem a proliferação viral significativamente prejudicada em células de mamífero. Os vírus que carreiam a mutação em E400 apresentaram aumento de proliferação viral em comparação com o vírus FA/EGFP original, em células de mosquito. As diferenças entre os tipos celulares podem ter sido causadas pelas características fisiológicas das células durante a infecção viral e pelas diferenças de propriedades das proteínas virais ocasionadas pela inserção das mutações. Não foi possível recuperar partículas virais infecciosas carreando a mutação em E403. A modelagem molecular das proteínas virais mostrou diferenças discretas de carga, volume de superfície proteica e propriedade físico-química induzidas pelas mutações. Nenhuma das mutações influenciou nas interações intramoleculares. A imunogenicidade foi avaliada por imunização de camundongos das linhagens C57BL/6 e BALB/c com os vírus carreando mutações únicas e os soros foram analisados por PRNT e ELISA para obter os títulos de anticorpos neutralizantes para FA e anticorpos para GFP, respectivamente. Os soros dos camundongos imunizados com os vírus recombinantes apresentam menores títulos de anticorpos neutralizantes em comparação ao grupo imunizado com o vírus vacinal, porém não houveram alterações na indução de anticorpos para GFP. De maneira geral, as mutações em E400 e E403 produziram maiores efeitos sobre a proliferação viral e as mutações NS3439 e NS4B54, na imunogenicidade. (AU)

Modulation of the prostaglandin-endoperoxide synthase 2 gene expression by variant haplotypes: influence of the 3'-untranslated region

The inducible inflammatory enzyme cycloxigenase-2 is up-regulated in cancer, and favors tumor progression. Cycloxigenase-2 is encoded by the prostaglandin-endoperoxide synthase 2 (PTGS2) gene, which presents sequence variations in the promoter region (PR) and in the 3′-untranslated region (3′-UTR). Different PR (rs689465, rs689466, rs20417) and 3′-UTR (rs5275) variants were generated by site-directed mutagenesis, and combined in haplotypes to access expression levels using a reporter system (luciferase) in human cells (MCF-7 and HEK293FT). Luciferase activity did not differ significantly among PTGS2 PR constructs, except for pAAC (containing variant allele rs20417 C), with 40% less activity than pAAG (wild-type sequence) in MCF-7 cells (P<0.01). Despite the lack of individual significant differences, PTGS2 PR constructs enclosing rs689466 G (pAGG and pAGC) showed an approximate two-fold increase in luciferase activity when compared to those containing rs689466 A (pAAG, pGAC, pAAC and pGAG) in both cell lines (P<0.001 for MCF-7 and P=0.03 for HEK293FT). The effect of PTGS2 3′-UTR sequences varied between MCF-7 and HEK293FT: MCF-7 cells showed significant reduction (40-60%) in luciferase activity (at least P<0.01), whereas HEK293FT cells showed more diverse results, with an average 2-fold increase when combined constructs (PR and 3′-UTR) were compared to respective parental PR sequences. The contribution of 3′-UTR variant (rs5275) was not consistent in either cell line. Despite the modulation of the 3′-UTR, with variable effects of rs5275, the enhancing transcriptional effect of rs689466 G was still detectable (P<0.0001 in MCF-7 or P=0.03 in HEK293FT cells).

Improving the thermostability of Trichoderma reesei xylanase 2 by introducing disulfide bonds

Background: Xylanases are considered one of the most important enzymes in many industries. However, their low thermostability hampers their applications in feed pelleting, pulp bleaching, and so on. The main aim of this work was to improve the thermostability of Trichoderma ressei xylanase 2 (Xyn2) by introducing disulfide bonds between the N-terminal and α-helix and the ß-sheet core. Results: In this work, two disulfide bonds were separately introduced in the Xyn2 to connect the N-terminal and α-helix to the ß-sheet core of Xyn2. The two disulfide bonds were introduced by site-directed mutagenesis of the corresponding residues. The half-life of the mutants Xyn2C14­52 (disulfide bond between ß-sheets B2 and B3) and Xyn2C59­149 (disulfide bond between ß-sheets A5 and A6) at 60°C was improved by approximately 2.5- and 1.8-fold compared to that of the wild type Xyn2. In addition, the enzyme's resistance to alkali and acid was enhanced. Conclusion: Our results indicated that the connection of the N-terminal and α-helix to the ß-sheet core is due to the stable structure of the entire protein.

Enzyme activity and thermostability of a non-specific nuclease from Yersinia enterocolitica subsp. palearctica by site-directed mutagenesis

Background: To identify the critical amino acid residues that contribute to the high enzyme activity and good thermostability of Yersinia enterocolitica subsp. palearctica (Y. NSN), 15 mutants of Y. NSN were obtained by site-directed mutagenesis in this study. And their enzyme activity and thermostability were assayed. Effect of several factors on the enzyme activity and thermostability of Y. NSN, was also investigated. Results: The results showed that the I203F and D264E mutants retained approximately 75% and 70% enzyme activity, respectively, compared to the wild-type enzyme. In addition to the I203F and D264E mutants, the mutant E202A had an obvious influence on the thermostability of Y. NSN. According to the analysis of enzyme activity and thermostability of Y. NSN, we found that Glu202, Ile203 and Asp264 might be the key residues for its high enzyme activity and good thermostability. Conclusions: Among all factors affecting enzyme activity and thermostability of Y. NSN, they failed to explain the experimental results well. One reason might be that the enzyme activity and thermostability of Y. NSN were affected not only by a single factor but also by the entire environment.

Asn336 is involved in the substrate affinity of glycine oxidase from Bacillus cereus

Background: Glycine oxidase (GO), a type of D-amino acid oxidase, is of biotechnological interest for its potential in several fields. In our previous study, we have characterized a new glycine oxidase (BceGO) from Bacillus cereus HYC-7. Here, a variant of N336K with increased the affinity against all the tested substrate was obtained by screening a random mutant library of BceGO. It is observed that the residue N336 is invariable between its homogeneous enzymes. This work was aimed to explore the role of the residue N336 in glycine oxidase by site-directed mutagenesis, kinetic assay, structure modeling and substrate docking. Results: The results showed that the affinity of N336H, N336K and N336R increased gradually toward all the substrates, with increase in positive charge on side chain, while N336A and N336G have not shown a little significant effect on substrate affinity. The structure modeling studies indicated that the residue Asn336 is located in a random coil between -J-18 and a-10. Also, far-UV CD spectra-analysis showed that the mutations at Asn336 do not affect the secondary structure of enzyme. Conclusion: Asn336 site was located in a conserved GHYRNG loop which adjoining to substrate and the isoalloxazine ring of FAD, and involved in the substrate affinity of glycine oxidase. This might provide new insight into the structure-function relationship of GO, and valuable clue to redesign its substrate specificity for some biotechnological application.

Parotid Incidentaloma Identified by Positron Emission/Computed Tomography: When to Consider Diagnoses Other than Warthin Tumor

Introduction Parotid gland incidentalomas (PGIs) are unexpected hypermetabolic foci in the parotid region that can be found when scanning with whole-body positron emission/computed tomography (PET/CT). These deposits are most commonly due to benign lesions such as Warthin tumor. Objective The aim of this study was to determine the prevalence of PGIs identified in PET/CT scans and to assess the role of smoking in their etiology. Methods We retrospectively reviewed all PET/CT scans performed at our center in search of PGIs and identified smoking status and standardized uptake value (SUVmax) in each case. We also analyzed the database of parotidectomies performed in our department in the previous 10 years and focused on the pathologic diagnosis and the presence or absence of smoking in each case. Results Sixteen cases of PGIs were found in 4,250 PET/CT scans, accounting for 0.4% . The average SUVmax was 6.5 (range 2.8 to 16). Cytology was performed in five patients; it was benign in four cases and inconclusive in one case. Thirteen patients had a history of smoking. Of the parotidectomies performed in our center with a diagnosis of Warthin tumor, we identified a history of smoking in 93.8% of those patients. Conclusions The prevalence of PGIs on PET/CT was similar to that reported by other authors. Warthin tumor is frequently diagnosed among PGIs on PET/CT, and it has a strong relationship with smoking. We suggest that a diagnosis other than Warthin tumor should be considered for PGIs in nonsmokers. .

Prevalência da baixa densidade mineral óssea em mulheres na pós-menopausa tratadas de câncer de mama / Prevalence of low bone mineral density in postmenopausal breast cancer survivors

OBJETIVO: Avaliar a prevalência da baixa densidade mineral óssea (DMO) em mulheres na pós-menopausa tratadas de câncer de mama. MÉTODOS: Estudo de corte transversal que incluiu 115 mulheres tratadas de câncer de mama atendidas em Hospital Universitário do Sudeste do Brasil. Foram incluídas mulheres com amenorreia há 12 meses ou mais e 45 anos ou mais de idade, tratadas de câncer de mama e livres de doença há pelo menos 5 anos. A DMO foi mensurada pelos raios-X de dupla energia em coluna lombar (L1 a L4) e colo de fêmur. Considerou-se baixa DMO quando valores de T-score de coluna total e/ou colo de fêmur <-1,0 Score de Delphi (DP) (osteopenia e osteoporose). Por meio de entrevista, foram avaliados fatores de risco para baixa DMO. Na análise estatística, empregaram-se os testes do χ2 ou Exato de Fisher. RESULTADOS: A média de idade das pacientes foi 61,6±10,1 anos e o tempo de menopausa, 14,2±5,6 anos, com tempo médio de seguimento de 10,1±3,9 anos. Considerando coluna e colo de fêmur, 60% das mulheres tratadas de câncer de mama apresentavam baixa DMO. Avaliando os fatores de risco para baixa DMO, foi encontrada diferença significativa na distribuição percentual quanto à idade (maior porcentagem de mulheres com mais de 50 anos e baixa DMO), história pessoal de fratura prévia (11,6% com baixa DMO e nenhuma com DMO normal) e índice de massa corpórea. Maior frequência de obesidade foi observada entre mulheres com DMO normal (63%) quando comparadas àquelas com baixa DMO (26,1%; p<0,05). CONCLUSÃO: Mulheres na pós-menopausa tratadas de câncer de mama apresentaram elevada prevalência de baixa DMO (osteopenia e/ou osteoporose). .

PURPOSE: To evaluate the prevalence of low bone mineral density (BMD) in postmenopausal breast cancer survivors. METHODS: In this cross-sectional study, 115 breast cancer survivors, seeking healthcare at a University Hospital in Brazil, were evaluated. Eligibility criteria included women with amenorrhea ≥12 months and age ≥45 years, treated for breast cancer and metastasis-free for at least five years. BMD was measured by DEXA at the lumbar spine (L1-L4) and femoral neck. Low BMD was considered when total-spine and/or femoral-neck T-score values were <-1.0 Delphi Score (DP) (osteopenia and osteoporosis). The risk factors for low BMD were assessed by interview. Data were analyzed statistically by the χ2 test and Fisher's exact test. RESULTS: The mean age of breast cancer survivors was 61.6±10.1 years and time since menopause was 14.2±5.6 years, with a mean follow-up of 10.1±3.9 years. Considering spine and femoral neck, 60% of breast cancer survivors had low BMD. By evaluating the risk factors for low BMD, a significant difference was found in the percent distribution for age (higher % of women >50 years with low BMD), personal history of previous fracture (11.6% with low BMD versus 0% with normal BMD) and BMI. A higher frequency of obesity was observed among women with normal BMD (63%) compared to those with low BMD (26.1%) (p<0.05). CONCLUSION: Postmenopausal breast cancer survivors had a high prevalence of osteopenia and osteoporosis. .

"El hecho de que sean más mujeres, no garantiza nada": feminización y experiencias de las mujeres en la ginecobstetricia en México / "The fact that there are more women doesn't guarantee anything": The feminization of obstetrics and gynecology and the experiences of female medical professionals in Mexico

En el marco de la creciente feminización de la profesión médica en México, el artículo indaga sobre las características de este proceso para el caso de la ginecobstetricia. Considerando la feminización como un proceso de cambio, que se analiza cuantitativa y cualitativamente, el artículo se detiene en especial en las experiencias de las mujeres ginecobstetras, experiencias que se dan en el seno de una especialidad que, desde sus orígenes, funcionó como un dispositivo de control del cuerpo de las mujeres. Basado en una investigación etnográfica, el artículo combina fuentes estadísticas, de archivo y de observación de campo. El material que surge de las entrevistas muestra las experiencias y tensiones que viven las ginecobstetras en este contexto.

In the framework of an increasing feminization of the medical profession in Mexico, this article explores the characteristics of this process in the obstetrics and gynecology specialty. Understanding feminization as a process of change to be analyzed both quantitatively and qualitatively, the article focuses special attention on the experiences of female obstetrician-gynecologists within a medical specialty that has since its origins functioned as a mechanism of control over women's bodies. Based on ethnographic research, the article combines statistical and archival sources and field observation. The interviews reveal the experiences and tensions women obstetrician-gynecologists encounter in this context.

Preferential protection of domains II and III of bacillus thuringiensis cry1Aa toxin by brush border membrane vesicles / Protección preferencial de los dominios II y III de la toxina cry1Aa de bacillus thuringiensis en vesículas de membrana de borde de cepillo

The surface exposed Leucine 371 on loop 2 of domain II, in Cry1Aa toxin, was mutated to Lysine to generate the trypsin-sensitive mutant, L371K. Upon trypsin digestion L371K is cleaved into approximately 37 and 26 kDa fragments. These are separable on SDS-PAGE, but remain as a single molecule of 65 kDa upon purification by liquid chromatography. The larger fragment is domain I and a portion of domain II (amino acid residues 1 to 371). The smaller 26-kDa polypeptide is the remainder of domain II and domain III (amino acids 372 to 609). When the mutant toxin was treated with high dose of M. sexta gut juice both fragments were degraded. However, when incubated with M. sexta BBMV, the 26 kDa fragment (domains II and III) was preferentially protected from gut juice proteases. As previously reported, wild type Cry1Aa toxin was also protected against degradation by gut juice proteases when incubated with M. sexta BBMV. On the contrary, when mouse BBMV was added to the reaction mixture neither Cry1Aa nor L371K toxins showed resistance to M. sexta gut juice proteases and were degraded. Since the whole Cry1Aa toxin and most of the domain II and domain III of L371K are protected from proteases in the presence of BBMV of the target insect, we suggest that the insertion of the toxin into the membrane is complex and involves all three domains.

La superficie de la toxina Cry1Aa, en el asa 2 del dominio II contiene expuesta la leucina 371, la cual fue modificada a lisina produciendo una mutante sensible a la tripsina, L371K. Esta mutante produce dos fragmentos de 37 y 26 kDa por acción de la tripsina que son separables por SDS-PAGE, pero que a la purificación por cromatografía líquida se mantienen como una sola molécula de 65 kDa. El fragmento grande contiene al dominio I y una parte del dominio II (aminoácidos 1 al 371). El polipéptido de 26 kDa contiene la parte restante del dominio II y dominio III (aminoácidos 372 al 609). Cuando la toxina mutante fue tratada con dosis altas de jugo intestinal de Manduca sexta, ambos fragmentos fueron degradados. Sin embargo, cuando fueron incubados en VMBC de M. sexta, el fragmento de 26 kDa fue protegido preferencialmente de las proteasas intestinales. Como se ha reportado, la toxina silvestre Cry1Aa también es protegida de la degradación de las proteasas cuando es incubada en VMBC de M. sexta. Sin embargo, cuando se adicionó VMBC de ratón a la mezcla de reacción, ni la toxina Cry1Aa ni la mutante L371K mostraron resistencia a las proteasas y fueron degradadas. Dado que la toxina completa de Cry1Aa y casi todo de los dominios II y III de L371K están protegidos de proteasas en presencia de VMBC del insecto, este estudio sugiere que la inserción de la toxina en la membrana involucra los tres dominios.

Presence and significance of Bacillus thuringiensis Cry proteins associated with the Andean weevil Premnotrypes vorax (Coleoptera: Curculionidae)

The Andean weevil Premnotrypes vorax represents an important cause of damage to Colombian potato crops. Due to the impact of this plague on the economy of the country, we searched for new alternatives for its biological control, based on the entomopathogenic bacteria Bacillus thuringiensis. A total of 300 B. thuringiensis strains obtained from potato plantations infested with P. vorax were analyzed through crystal morphology, SDS-PAGE, PCR and bioassays. We used site- directed mutagenesis to modify the Cry3Aa protein. Most of the B. thuringiensis isolates had a bipyramidal crystal morphology. SDS-PAGE analyses had seven strains groups with σ-endotoxins from 35 to 135 kDa. The genes cry 2 and cry 1 were significantly more frequent in the P. vorax habitat (PCR analyses). Three mutant toxins, 1 (D354E), 2 (R345A, ∆Y350, ∆Y351), and 3 (Q482A, S484A, R485A), were analyzed to assess their activity against P. vorax larvae. Toxicity was low, or absent, against P. vorax for isolates, wild type cry 3Aa and cry 3Aa mutants. The genetic characterization of the collection provides opportunities for the selection of strains to be tested in bioassays against other insect pests of agricultural importance, and for designing Cry proteins with improved insecticidal toxicity. Rev. Biol. Trop. 57 (4): 1235-1243. Epub 2009 December 01.

El gorgojo andino Premnotrypes vorax es una causa importante de daño en los cultivos colombianos de este tubérculo. Debido al impacto que esta plaga tiene sobre la economía del país, nos interesamos en buscar alternativas nuevas para el control biológico de P. vorax, basadas en la bacteria entomopatógena Bacillus thuringiensis. Se recolectaron un total de 300 cepas de B. thuringiensis a partir de plantaciones de papa infestadas con P. vorax, las cuales fueron analizadas por medio de la morfología del cristal, SDS-PAGE, PCR y ensayos biológicos. La mayoría de los aislamientos de B. thuringiensis presentaron cristales bipiramidales. Los análisis de SDS-PAGE indicaron la presencia de siete grupos de cepas con σ- endotoxinas que variaban entre 35 a 135 kDa. Las pruebas con PCR demostraron que los genes cry 2 y cry 1 fueron significativamente más frecuentes en el medioambiente de P. vorax. Además, se utilizó la mutagénesis sitio-dirigida para modificar la proteína Cry3Aa. Se analizaron tres toxinas mutantes, 1 (D354E), 2 (R345A, ∆Y350, ∆Y351), y 3 (Q482A, S484A, R485A), para determinar su actividad contra larvas de P. vorax. Los ensayos de toxicidad señalaron escasa, o nula, actividad hacia P. vorax tanto para las cepas, la toxina Cry3Aa de referencia y las proteínas Cry3Aa mutantes. La caracterización genética de la colección puede proveer oportunidades para la selección de cepas que pueden evaluarse por medio de bioensayos contra otros insectos-plaga de importancia agrícola, y para el diseño de proteínas Cry con actividad toxica mejorada.

Detección de una mutación no estándar en el proto-oncogen ret por mutagénesis dirigida / Detection of a non-standard mutation in the ret protoncogene by site directed mutagenesis

MEN2A is an autosomic dominant disease, characterized by medullary thyroid cancer, pheochromocytoma and parathyroid hyperplasia. Mutations in the ret proto-oncogene are associated with this disease, with almost 100% of penetrance. The gene, situated on chromosome 10q11.2, codes for a transmembrane protein with a tyrosinkinase-like receptor function. Mutations that affect its extracellular domain, stimulate spontaneous homodimerization and elevate the basal tyrosinkinase activity. The codon 634 of the gene is considered a hot-spot site, since it is mutated in 85% of the MEN2A families. Our group developed in 2002 an indirect and costless strategy to detect alterations in this site. We present a family suspected of having MEN2A. We applied our PCR based indirect strategy on the DNA of the index patient and found that there was no mutation in that site. Posterior sequencing of exon 10 and 11 confirmed that the mutation affecting this family was in codon 611. Thus, we developed a new costless family-specific strategy based on mutagenic PCR and enzymatic cuts to diagnose all the family members. A seven-year old boy with this mutation was preventively thyroidectomized. In this way, combining the indirect methodology for codon 634 previously developed by our group, and a posterior family-specific mutation detection strategy, we were able to diagnose and intervene presymptomatically the family members, avoiding sending all the samples to foreign centers.

El síndrome de MEN2A es una enfermedad autosómica dominante que se caracteriza por el desarrollode cáncer medular de tiroides, feocromocitoma e hiperplasia de paratiroides. Mutaciones en elret proto-oncogén se asocian con MEN2A, con una penetrancia cercana al 100%. El gen se encuentra en elcromosoma 10q11.2 y codifica para una proteína transmembrana con función de receptor del tipo tirosina quinasa.Mutaciones que afectan el dominio extracelular de la proteína estimulan la dimerización espontánea del receptory un aumento de la actividad de tirosina quinasa basal. El codón 634 codifica para una cisteína, y es consideradoun sitio hot-spot por encontrarse mutado en el 85% de las familias con MEN2A. Para este sitio, nuestro grupo desarrolló en 2002 una metodología de detección indirecta y económica. Ante una familia sospechada de MEN2A, se aplicó esta estrategia, que reveló un codón 634 sano. Por posterior secuenciación se confirmó que el paciente índice portaba una mutación en el codón 611. Se desarrolló una nueva estrategia familiaespecífica por PCR mutagénica, que permitió diagnosticar en nuestro país a todos los integrantes de la familiacon costos accesibles. Un niño en el cual se halló la mutación, fue tiroidectomizado preventivamente, y a lafecha goza de buena salud. De esta manera, combinando la estrategia de detección de mutaciones en el sitiohot-spot y un posterior diseño de otra metodología familia-específica se pudo diagnosticar e intervenir preventivamente a la familia, sin enviar todas las muestras al extranjero.

Mutation of highly conserved arginine residues disrupts the Structure and Function of Annexin V

Background. Annexins are a family of structurally related proteins that bind to phospholipid membranes in a Ca²+-dependent manner. Annexins are characterized by highly conserved canonical domains of approximately 70 amino acids. Anexin V contains four such domains. Each of these domains has a highly conserved arginine (R). Methods. To evaluate the role of the conserved arginines in the molecular structure of annexin V, negatively charged amino acids were substituted for arginines at positions R43, R115, R199, and R274 using site-directed mutagenesis. Results. Mutants R199D and R274E were rapidly degraded when expressed in bacteria, and were not further characterized. R43E exhibited an electrophoretic mobility similar to the wild-type protein, while R115E migrated significantly in a slower fashion, suggesting a less compact conformation, R43E and R115E exhibited much grater susceptibility to proteolytic digestion than the wild type. While Ca²+-dependence for phospholipid binding was similar in both mutants (half-maximal 50-80 µ; Ca²+), R43E and R115E exhibited a phospholipid affinities of the annexins, a phospholipid-dependent clotting reaction, the activated partial thromboplastin time (aPTT), was significantly prolonged by the wild-type protein and mutants R115E and R115A. The aPTT was unaffected by R43E. Conclusions. Our data suggest that mutation of these highly conserved arginine residus in each of the four canonical domains of annexin have differential effects on the phospholipid binding tertiary structure, and proteolytic susceptibility of annexin V. The site I mutation , R43E, produced a large decrease in phospholipid affinity associated with an increase in proteolytic susceptibility. The site II mutation, R115E, produced a small change in phospholipid binding but a significant modification of electrophoretic mobility. Our data suggest that highly conserved arginine residues are required to stabilize the tertiary structure of ammexin V by establishing hydrogen bonds and ionic bridges

Caracterización molecular en pacientes con enfermedad granulomatosa crónica por deficiencia en p47 phox / Molecular characterization in patients with chronic granulomatous disease due to p47phox deficiency

El sistema NAOPH oxidasa es un complejo enzimático transportador de electrones localizado en la membrana de las células fagocíticas. De este sistema hacen parte varias proteínas; un flavocitocromo b558' el cual está conformado por una cadena b (gp91-phox) y una cadena a (p22-phox) y poral menos 3 proteínas citosólicas (p47-phox, p67- phox, p40-phox). Una alteración gen ética en cualquiera de estas proteínas causa el síndrome de Enfermedad Granulomatosa Crónica (EGC). La caracterización de las mutaciones de los pacientes con EGC ha sido fundamental para dilucidar la estructura y función de los componentes del sistema NAOPH oxidasa. En el caso de la p47-phox, se han obtenido hallazgos importantes que la hacen un modelo interesante para estudiar el mecanismo molecular involucrado en regular la expresión y función bioquímica de este sistema. En los pacientes con defecto en la p47-phox investigados hasta ahora, se ha hallado una deleción del dinucleótido GT al comienzo del exón 2 , siendo la mayoría de ellos homocigóticos para esta deleción, la cual posiblemente se debe a eventos de recombinación entre el gen p47 -phox normal y un seudogen recientemente descrito. En el diagnóstico de pacientes no homocigóticos, cualquier mutación encontrada en el análisis del ONA (gONA o cONA) puede representar un cambio sufrido por el seudogen. Por lo tanto, para la identificación precisa del defecto genético es necesario separar el gen normal del seudogen y analizar las secuencias en forma individual. Los pacientes no homocigóticos posiblemente deben tener una segunda mutación en el alelo tipo silvestre diferente a la deleción GT. De otro lado, a través de mutagénesis sitio-dirigida se pueden modificar algunos de los aminoácidos o dominios de la p47-phox, los cuales pueden ser esenciales para su funcionamiento y su relación con la EGC. Con esta metodología, es posible introducir cambios en un gen cuya secuencia es totalmente conocida, el cual es amplificado; las mutantes así generadas pueden dar información acerca de la estructura y función de los genes analizados, observando su efecto sobre la función. De esta manera se puede determinar lo importante que puede ser un cambio estructural en la función de esta proteína.

NADPH oxidase system is an enzymatic electron transport complex localized in the membrane of phagocytic cells. Several proteins belong to this system: A flavocytochrome b558, formed by a b chain (gp91.phox) and an a chain (p22.phox) and, at least, 3 cytosolic proteins (p47.phox, p67.phox and p40 phox). Genetic alteration in any of these proteins causes the syndrome of Chronic Granulomatous Disease (CGD). Characterization of mutations in patients with CGD has been fundamental to elucidate the structure and function of NADPH oxidase system ComponentS. Several findings make p47.phoX an interesting model to study the molecular mechanism involved in regulating the expreSSion and bioChemical function ofthis system. So far, in patients with p47.phoX defect a deletion of dinucleotide GT has been foUnd at the beginning of exon 2; most of them are homocygotic for this deletion which is probably due to recombinant events between normal p47.phoX gen and a recently described pseudogen. Any mutation found when diagnosing non.homocygotic patients (gDNA or cDNA) may represent a pseudogen change. Therefore, for precise identification of the genetic defect it is necessary to separate the normal gen from the pseudogen and to analyze individual sequences. Non.homocygotic patients posibly have a second mutation in the wild type allele different fron GT deletion. On the other hand, through site. oriented mutagenesis it is posible to modify some of the aminoacids or domains of p47.phoX, which may be essential for its function and relationship with CGD. With this method010gy it is possible to introduce changes in a gen whoSe sequence is thoroughly known and which is amplified; mutants So generated can give information concerning the structure and function of the analyzed genes, observing their effect on function. In this way the importance of a structural change on the function of a protein can be determined.

Protein engineering as a powerful tool for the chemical modification of enzymes

This article discusses the techniques of site-specific mutagenesis and protein engineering and their application in the study of enzyme active sites and the mechanism of enzyme action. Particular emphasis is given to beta-lactamase

Chemical modification of allosteric properties of enzymes

Chemical modification is usually employed to study enzyme active sites. Valuable information can also be obtained, however, when this technique is used to probe allosteric sites. This approach is discussed in this article, and it is exemplified in chemical modification studies of the allosteric enzyme phosphofructokinase