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Mucopolysaccharidoses (MPSs) are a group of genetic alterations whose effect is the progressive intralysosomal accumulation of glycosaminoglycans. Affected individuals are deficient in one or more lysosomal enzymes which, depending on the MPS, may cause coarse facial features, short stature, multiple skeletal dysplasia, joint stiffness, or developmental delay. Their diagnosis is mostly performed late or incorrectly, and it represents a challenge since it requires specialized tests only performed in major cities. This makes it difficult for patients to have access to physicians since their geographical location is distant and therefore, the use of samples collected in solid-phase represents an advantage for the study of high-risk populations. In addition, epidemiological information about rare diseases, especially in Latin America, is scarce or inconsistent. Our aim was to report the experience of 20 years of selective screening by assessing enzyme activity and reporting incidence values of MPS in Colombia. This study validated a group of fluorometric endpoint techniques in 8239 patients. The samples were dried blood spots (DBS) collected on filter paper and leukocyte extracts. Reference values in the Colombian population for α-l-iduronidase, iduronate 2-sulfatase, α-N-acetylglucosaminidase, N-acetylglucosamine-6-sulfate sulfatase, ß-galactosidase, arylsulfatase B, and ß-glucuronidase were established in leukocyte extracts, and patients reference ranges were updated in the case of DBS samples. Incidence values were calculated for each MPS and the distribution of cases across the country is also shown. This study offers very useful information for the health system, the scientific community, and it facilitates the diagnosis of these disorders. This is indispensable when seeking to develop new diagnostic or treatment approaches for patients.

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Introducción: El Síndrome Sanfilippo B es un error innato en el metabolismo lisosomal, con herencia autosómica recesiva. Se caracteriza por facie ligeramente tosca, deterioro neurológico progresivo y poca repercusión somática, provocado por mutaciones en el gen NAGLU, cuyo locus es 17q21.2. La incidencia internacionalmente es muy baja y en Cuba solo se han diagnosticado siete pacientes desde 1985. Objetivo: Describir las manifestaciones clínicas, bioquímicas y moleculares de un paciente cubano diagnosticado con Síndrome Sanfilippo B. Presentación de Caso: Se describió un paciente de 13 años, cuyas principales manifestaciones clínicas fueron: facie ligeramente tosca, sinofris, alteraciones de conducta y deterioro neurológico progresivo. El trastorno del sueño fue ocasional y frecuente las infecciones respiratorias. Se demostró la presencia de colitis ulcerativa y pólipo intestinal. Se confirmó excreción aumentada de heparán sulfato y disminución de la actividad enzimática N-acetil αD-glucosaminidasa. Se identificó la mutación c.640dupC en el gen NAGLU en homocigosis en el paciente y ambos padres resultaron ser portadores. Conclusiones: Predominaron las alteraciones de conducta, deterioro neurológico progresivo e infecciones respiratorias en el caso reportado; siendo la colitis ulcerativa y el pólipo intestinal un hallazgo no descrito anteriormente para esta enfermedad. Los estudios cromatográficos y enzimáticos resultaron positivos para Sanfilippo B. El genotipo de este paciente resultó ser homocigótico para una nueva variante alélica patogénica en el gen NAGLU. Se demostró la segregación mendeliana de la mutación en la familia(AU)

Introduction: Sanfilippo syndrome type B is an autosomal recessive lysosomal storage disease. The frequent clinical manifestations include slightly coarse facial features, progressive neurodegeneration and mild somatic repercussion caused by mutations in the NAGLU gene, whose locus is 17q21.2. The worldwide incidence is very low and only seven patients have been diagnosed in Cuba since 1985. Objective: To describe clinical, biochemical and molecular characteristics of a Cuban patient with the diagnosis of Sanfilippo Syndrome type B. Case presentation: A 13 years old patient was described. The main clinical manifestations included mild coarse facie, synophrys, behavior disturbances, and progressive neurologic deterioration. Intermittent sleep disturbance and frequent upper respiratory infections were identified. Ulcerative colitis and intestinal polyp were demonstrated. Increased excretion of heparan sulfate and very low N-acetyl α-Dglucosaminidase activity were confirmed. In addition, the presence of mutation c.640dupC in NAGLU gene was identified. The patient had homozygous genotype and both parents were heterozygous. Conclusions: Behavioral alterations, progressive neurological deterioration and respiratory infections predominated in the reported case. Other findings such as ulcerative colitis and intestinal polyps were not previously described in this disease. The chromatographic and enzymatic studies were positive for Sanfilippo type B. This patient's genotype was found to be homozygous for a novel pathogenic allelic variant in the NAGLU gene. Mendelian segregation of the mutation in the family was demonstrated(AU)

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La mucopolisacaridosis tipo III B es una enfermedad de depósito lisosomal causada por la deficiencia de la enzima N-acetil-alfa-d-glucosaminidasa, implicada en el catabolismo del heparán sulfato, que produce su acúmulo en diversos tejidos. Se presenta a un paciente de 8 años, afectado de mucopolisacaridosis tipo III B, con historia de diarrea crónica y hallazgos endoscópicos e histológicos compatibles con linfangiectasia intestinal. Tras tratamiento dietético con restricción de ácidos grasos de cadena larga y rica en triglicéridos de cadena media, presentó mejoría clínica, mantenida hasta la actualidad.La patogenia de la diarrea crónica en pacientes con mucopolisacaridosis tipo III B es aún desconocida. Debe investigarse la presencia de linfangiectasia intestinal en estos pacientes e iniciar, en caso de confirmarse, un tratamiento dietético adecuado para mejorar así su calidad de vida.

Mucopolysaccharidosis type IIIB is a lysosomal storage disease caused by a deficiency of the N-acetyl-alpha-d-glucosaminidase enzyme involved in the catabolism of heparan sulfate, causing its accumulation in various tissues. We present an 8-year-old patient with mucopolysaccharidosis type IIIB, with a history of chronic diarrhea and endoscopic and histological findings compatible with intestinal lymphangiectasia. After a dietary treatment with a low-fat diet supplemented with medium-chain triglyceride, our patient presents clinical improvement until today. The pathogenesis of chronic diarrhea in patients with mucopolysaccharidosis type IIIB is still unknown. The presence of intestinal lymphangiectasia in these patients should be investigated, and appropriate dietary treatment should be initiated, if confirmed, to improve their quality of life.

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N-acetylglucosaminidase produced from Lecanicillium lecanii on submerged culture displayed hydrolytic and transglycosylation activities. The highest specific activity for the enzyme was 1.87 U/mg after 120 h of culture. The chromatographic purification for a single protein fraction showed a molecular weight of 50.4 kDa and hydrolytic N-acetylglucosaminidase activity of 17.59 U/mg at 37 °C and pH 6. This enzyme was able to transglycosylate and to synthesize oligosaccharides from 2 to 6 units with a degree of acetylation between 100 and 26% employing glucose, mannose, N-acetyl-D-glucosamine and N-acetyl-D-lactosamine as donor substrates. Optimal conditions of temperature and pH were determined for both types of enzymatic activities.

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The host response observed after the application of an appropriate stimulus, such as mechanical injury or injection of neoplastic or normal tissue implants, has allowed the cataloging of a number of molecules and cells involved in the vascularization of normal repair or neoplastic tissue. Implantation of sponge matrices has been adopted as a model for the accurate quantification of angiogenic and fibrogenic responses, as they may occur during wound healing, in vivo. Such implants are particularly useful because they offer scope for modulating the environment within which angiogenesis occurs. Sponge implantation model has been optimized and adapted to characterize essential components and their roles in blood vessels formation in a variety of physiological and pathological conditions. As a direct consequence of advances in genetic manipulation, mouse models (i.e., knockouts, SCID, nude) have provided resources to delineate the mechanisms regulating the healing associated with implants. Here we outline the usefulness of the sponge implant model of angiogenesis and detailed description of the methodology.

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BACKGROUND: Glycosidases profusion in male reproductive fluids suggests a possible relationship with sperm function. Although Hexosaminidase (Hex) is the most active glycosidase in epididymal fluid and seminal plasma, as well as in spermatozoa, Glucosidase is considered a marker for epididymal function and azoospermia. OBJECTIVE: The aim of this study was to determine Hex activity in seminal plasma from patients with normal and abnormal spermograms and analyze its correlation with seminal parameters. MATERIALS AND METHODS: In this cross sectional study, seminal plasma from azoospermic, asthenozoospermic, teratozoospermic, and normozoospermic patients was analyzed for the activity of: total Hex, HexA isoform, and glucosidase. Besides, hexosamine levels were determined, and the amount of Hex was quantified by immunoblot with a specific antibody. Correlation of Hex activity with seminal parameters was also analyzed. RESULTS: Hex activity, like glucosidase, was significantly reduced in azoospermic samples (44, 49, and 60% reduction for total Hex, HexA and glucosidase, respectively). A reduced amount of Hex in azoospermic samples was confirmed by western immunoblot. Hex activity was negatively correlated with round cells in azoospermic samples and positively correlated with motility in asthenozoospermic ones. CONCLUSION: The results suggested that Hex activity was reduced in azoospermic samples and this was due to a lower amount of enzyme. The correlation to seminal parameters related to particular pathologies suggests a possible relationship of Hex with fertilizing capacity.

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Lecanicillium (Verticillium) lecanii produced chitinases using shrimp shell as inducer. Maximum production of b-N-acetylglucosaminidase was measured at 80h. Enzyme stability was obtained at temperatures ranging from 30 to 40°C and maximum activity at 50°C, pH 6.0. Enzyme activity increased with Ba2+, Co2+, Fe3+ and Zn2+. Bioassays against the phytopathogenic fungus Oidium spp. showed mycelial and germination inhibition. SDS-PAGE electrophoresis of the partially purified extract revealed four bands of 70, 58, 45 and 31kDa and this extract showed activity of b-N-acetylglucosaminidase through zymogram analysis. Chitinases produced by L. lecanii are potentially useful against phytopathogenic fungi, insects and chitosan bioconversions.

Lecanicilium (Verticillium) lecanii produjo quitinasas mediante el uso de caparazón de camarón como inductor. La máxima producción de b-N-acetylglucosaminidasa se obtuvo a las 80h. Se observó estabilidad de la enzima en el intervalo de temperatura entre 30 y 40°C y su actividad máxima a los 50°C, pH 6,0. La actividad enzimática se incrementó con Ba2+, Co2+, Fe3+ and Zn2+. El bioensayo contra el hongo fitopatógeno Oidium spp. mostró inhibición micelar y de la germinación. La electroforesis SDS-PAGE del extracto parcialmente purificado mostró cuatro bandas de 70, 58, 45 y 31kDa y este extracto mostró actividad de b-N-acetylglucosaminidasa a través de un análisis de zimograma. Las quitinasas producidas por L. lecanii pueden ser potencialmente utilizadas contra hongos fitopatógenos, insectos y en la bioconversión del quitosán.

Lecanicillium (Verticillium) lecanii producido quitinases utilizou exoesqueleto do camarão como inductor. Produção máxima de b-N-acetilglucosaminidasa foi obtido em 80h. A estabilidade de enzima estava em o intervalo de temperaturas de 30 - 40°C e os niveis máximos de atividade enzimática foram obtidos em 50°C, pH 6,0. A atividade de enzima foi aumentada com Ba2+, Co2+, Fe3+ e Zn2+. O Bioensaios contra fungo fitopatógenos Oidium spp. mostrou inibição miceliar e germinação. O electroforesis SDS-PAGE do extrato parcialmente purificado revelou quatro faixas de 70, 58, 45 e 31kDa e este extrato apresentou atividade b-N-acetilglucosaminidasa através de uma análise zimograma. O quitinases produzido por L. lecanii são potencialmente capaz de ser utilizado contra fungos fitopatógenos, insetos e bioconversions de quitosano.

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The effect of G protein modulators and cyclic AMP (cAMP) on N-acetylglucosaminidase (NAGase) production was investigated during 84 h of growth of a Trichoderma harzianum strain in chitin-containing medium. Caffeine (5 mM), N6--2'-O-dibutyryladenosine 3'5'-cyclic monophosphate sodium salt (dBcAMP) (1 mM) and 3-isobutyl-1-methylxanthine (IBMX) (2 mM) decreased extracellular NAGase activity by 80%, 77% and 37%, respectively. AlCl3/KF (100 µM/10 mM and 200 µM/ 20 mM) decreased the activity by 85% and 95%, respectively. Cholera (10 µ/mL) and pertussis (20 µ/mL) toxins also affected NAGase activity, causing a decrease of approximately 75%. Upon all treatments, protein bands of approximately 73 kDa, 68 kDa and 45 kDa had their signals diminished whilst a 50 kDa band was enhanced only by treatment with cholera and pertussis toxins. N-terminal sequencing analysis identified the 73 kDa and 68 kDa proteins as being T. harzianum NAGase in two different truncated forms whereas the 45 kDa band comprised a T. harzianum endochitinase. The 50 kDa protein showed sequence similarity to Coriolus vesicolor cellobiohydrolase. The above results suggest that a signaling pathway comprising G-proteins, adenylate cyclase and cAMP may be involved in the synthesis of T. harzianum chitinases.

O efeito de cAMP e de moduladores de proteínas G sobre a produção de N-acetilglicosaminidase (NAGase) foi investigado durante o crescimento de Trichoderma harzianum em meio contendo quitina. Cafeína (5 mM), dBcAMP (1mM) e IBMX (2 mM) provocaram diminuições na atividade extracelular de NAGase em 80%, 77% e 37%, respectivamente. Por outro lado, a presença de AlCl3/KF nas concentrações de 100 µM/10 mM e 200 µM/ 20 mM causou decréscimo na atividade em 85% e 95%, respectivamente. A toxina do cólera (10 µ/mL) e a toxina pertussis (20 µ/mL) também afetaram a atividade de NAGase, causando um decréscimo de aproximadamente 75%. Análises eletroforéticas mostraram que todos os tratamentos citados causaram diminuição no sinal de bandas correspondendo a polipeptídios de 73 kDa, 68 kDa e 45 kDa, enquanto uma banda de 50 kDa foi intensificada apenas com tratamento com as toxinas do cólera e pertussis. Análises de sequenciamento N-terminal permitiram a identificação das proteínas de 73 kDa e 68 kDa como sendo NAGase de T. harzianum em duas formas diferentemente processadas enquanto a banda de 45 kDa correspondeu a uma endoquitinase de T. harzianum. A proteína de 50 kDa mostrou similaridade de sequência com uma celobiohidrolase de Coriolus vesicolor. Os resultados sugerem que uma via de sinalização composta por proteínas G, adenilato ciclase e cAMP possa estar envolvida na produção de quitinases T. harzianum.