GM1 gangliosidosis: a case report / Gangliosidose GM1: um relato de caso

RESUMEN La gangliosidosis GM1 es un trastorno lisosomal caracterizada por la acumulación de gangliósido GM1 (glucoesfingolípido) en el sistema nervioso central (SNC) y visceral, debido a la deficiencia de la enzima beta-galactosidase (hidrolasa lisosomal). Afecta principalmente al SNC y las vísceras y produce importantes anomalías esqueléticas, que a menudo ocurren con la presencia de linfocitos vacuolados en la muestra de la sangre periférica o médula ósea. Tiene tres formas de presentación, lo que dificulta aún más su identificación debido al amplio espectro clínico. El presente estudio tiene como objetivo describir un caso de gangliosidosis GM1 en un paciente masculino, nacido a las 38 semanas. Hasta el momento, no existe un tratamiento efectivo para la gangliosidosis GM1, es decir, el portador de la enfermedad solo recibe medidas sintomáticas y paliativas. Por tanto, el diagnóstico precoz de la enfermedad es de suma importancia, ya que su única forma de prevención, actualmente, es a través del consejo genético.

Reducción de proteínas y glucosa por reacción de Maillard en leche con lactosa hidrolisada / Protein and glucose reduction by Maillard reaction in milk with hydrolyzed lactose

Se evaluó el efecto de la temperatura sobre la desnaturalización de proteínas y la reacción de Maillard en leche entera y descremada con lactosa hidrolizada. Las leches hidrolizadas se trataron térmicamente a 100, 110, 120 y 130 °C durante un período de 1 hora y se midió la concentración de glucosa, el grado de pardeamiento y la desnaturalización de proteínas. El grado de dorado en la leche entera varió de 14.4 (100 °C) a 42.6 (130 °C). Para la leche descremada fue de 20.2 (100 °C) a 38.0 (130 °C). La concentración de glucosa en leche entera (47% p/v) y en leche descremada (41% p/v) después del tratamiento térmico (130 °C) mostró una reducción significativa en relación con el control (25 °C). El efecto de la temperatura en la desnaturalización de proteínas en leche entera y descremada en relación con el control (25 °C) fue del 100%. La leche tratada térmicamente con lactosa hidrolizada promovió la desnaturalización de proteínas con un aumento del pardeamiento característico de la reacción de Maillard, lo que afectó la calidad nutricional.

The effect of temperature in protein denaturation and Maillard reaction in whole and skim milk with hydrolyzed lactose was evaluated. Hydrolyzed milk was thermally treated at 100, 110, 120 and 130 °C over a period of 1 hour and glucose concentration, browning degree and protein denaturation were measured. The browning degree in whole milk varied from 14.42 (100 °C) to 42.63 (130 °C) and 20.21 (100 °C) to 38.03 (130 °C) in skim milk. Glucose concentration in whole milk (47% - w/v) and skim milk (41% - w/v) after heat treatment (130 °C) showed a significant reduction in relation to the control (25 °C). The temperature effect in protein denaturation in whole and skim milk in relation to the control (25 °C) was 100%. Thermally treated milk with hydrolyzed lactose promoted protein denaturation with increasing browning characteristic of the Maillard reaction, thus affecting the nutritional quality.

Clinical and CLB1 gene mutations analysis of GM1 gangliosidosis in a patient / 中华神经科杂志

Objective@#To investigate the clinical and CLB1 gene mutation characteristics of GM1 gangliosidosis patient.@*Methods@#The clinical data of one GM1 gangliosidosis patient from Children′s Hospital Affiliated to Zhengzhou University in March 2018 were reviewed and analyzed. The patient was diagnosed by gene detection and enzymatic activity.@*Results@#The patient is a 4 years and 1 month old boy, mainly presented psychomotor retrogression. His β-galactosidase activity was low (8.0 nmol·g-1·min-1). Two splice site mutations (c.458-2A(IVS4)>G and c.1068+5G(IVS10)>A) of patient′s CLB1 gene were screened by targeted next generation sequencing. The results of Sanger sequencing showed that the mutations are compound heterozygous and both are first reported. The mutation c.1068+5G(IVS10)>A was derived from patient′s mother, and the other one is de nove.@*Conclusion@#GM1 gangliosidosis is a rare neurodegenerative disease, which could be accurately diagnosed by the next generation sequencing and enzyme assay.

Clinical and CLB1 gene mutations analysis of GM1 gangliosidosis in a patient / 中华神经科杂志

Objective To investigate the clinical and CLB1 gene mutation characteristics of GM1 gangliosidosis patient. Methods The clinical data of one GM1 gangliosidosis patient from Children′s Hospital Affiliated to Zhengzhou University in March 2018 were reviewed and analyzed. The patient was diagnosed by gene detection and enzymatic activity. Results The patient is a 4 years and 1 month old boy, mainly presented psychomotor retrogression. His β?galactosidase activity was low (8.0 nmol·g-1·min-1). Two splice site mutations (c.458?2A(IVS4)>G and c.1068+5G(IVS10)>A) of patient′s CLB1 gene were screened by targeted next generation sequencing. The results of Sanger sequencing showed that the mutations are compound heterozygous and both are first reported. The mutation c.1068+5G(IVS10)>A was derived from patient′s mother, and the other one is de nove. Conclusion GM1 gangliosidosis is a rare neurodegenerative disease, which could be accurately diagnosed by the next generation sequencing and enzyme assay.

Galactosialidosis: nueva mutación de novo en el gen CTSA en un paciente afecto de la forma infantil tardía / Galactosialidosis: a new "de novo" mutation in CTSA gene in a patient with late infantile galactosialidosis

La galactosialidosis (OMIM #256540) es una enfermedad metabólica lisosomal causada por mutaciones en el gen CTSA, que codifica la proteína protectora catepsina A. La pérdida de función de dicha proteína causa, secundariamente, un déficit combinado de dos enzimas, beta-galactosidasa y neuraminidasa. Se expone el caso de un paciente que presentó manifestaciones clínicas compatibles con el subtipo infantil tardío de galactosialidosis. El análisis bioquímico mostró déficits de las dos enzimas implicadas, mientras que el estudio molecular reveló dos mutaciones: una nueva mutación nunca antes descrita, p.His475Pro (c.1424 A>C), y una mutación previamente reportada, p.Arg441Cys (c.1321C>T), localizadas en los exones 15 y 14, respectivamente.

Galactosialidosis (OMIM #256540) is an autosomal recessive lysosomal storage disorder caused by mutations in the CTSAgene, which encodes the protective protein cathepsin A. The loss of function of this protein causes a secondarily deficiency of beta-galactosidase and N-acetyl-α-neuraminidase enzymes activities. We describe the clinical, biochemical and molecular analysis of a case report with a phenotype compatible with the late infantile form. The biochemical analysis reveled deficiencies of beta-galactosidase and neuraminidase activities in dried blood spot and fibroblasts and the molecular study showed two missense mutations in the CTSA gene: a previously reported mutation, p.Arg441Cys (c.1321C>T), and a novel mutation, p.His475Pro (c.1424 A>C), located in exons 14 and 15, respectively.

Oral administration of exogenous lactase in tablets for patients diagnosed with lactose intolerance due to primary hypolactasia / Administração oral de lactase exógena em comprimidos em pacientes portadores de intolerância à lactose devido à hipolactasia primária

ABSTRACT Background Primary hypolactasia is a common condition where a reduced lactase activity in the intestinal mucosa is present. The presence of abdominal symptoms due to poor absorption of lactose, which are present in some cases, is a characteristic of lactose intolerance. , Objective Evaluate the efficacy of a product containing exogenous lactase in tablet form compared to a reference product with proven effectiveness in patients with lactose intolerance. Methods Multicentre, randomized, parallel group, single-blind, comparative non-inferiority study. One hundred twenty-nine (129) adult lactose intolerance patients with hydrogen breath test results consistent with a diagnosis of hypolactasia were randomly assigned to receive the experimental product (Perlatte(r) - Eurofarma Laboratórios S.A.) or the reference product (Lactaid(r) - McNeilNutritionals, USA) orally (one tablet, three times per day) for 42 consecutive days. Results Data from 128 patients who actually received the studied treatments were analysed (66 were treated with the experimental product and 62 with the reference product). The two groups presented with similar baseline clinical and demographic data. Mean exhaled hydrogen concentration tested at 90 minutes after the last treatment (Day 42) was significantly lower in the experimental product treated group (17±18 ppm versus 34±47 ppm) in the per protocol population. The difference between the means of the two groups was -17 ppm (95% confidence interval [95% CI]: -31.03; -3.17). The upper limit of the 95% CI did not exceed the a priori non-inferiority limit (7.5 ppm). Secondary efficacy analyses confirmed that the treatments were similar (per protocol and intention to treat population). The tolerability was excellent in both groups, and there were no reports of serious adverse events related to the study treatment. Conclusion The experimental product was non-inferior to the reference product, indicating that it was an effective replacement therapy for endogenous lactase in lactose intolerance patients.

RESUMO Contexto A hipolactasia primária é uma condição muito frequente na qual há redução da atividade da lactase na mucosa intestinal.A presença de sintomas abdominais devidos à má absorção da lactose presente em alguns casos caracteriza a intolerância à lactose. Objetivo Avaliar a eficácia de um produto contendo lactase exógena em comprimidos comparativamente a de um produto comparador com eficácia comprovada em pacientes portadores de intolerância à lactose. Métodos Estudo multicêntrico, randomizado, de grupos paralelos, com investigador cego, comparativo de não-inferioridade. Cento e vinte e nove (129) pacientes adultos portadores de intolerância à lactose e teste do hidrogênio no ar expirado compatível com o diagnóstico de hipolactasia foram randomizados para receber o produto experimental (Perlatte(r) - Eurofarma Laboratórios S.A.) ou o produto comparador (Lactaid(r) - McNeil Nutritionals, EUA), por via oral (um comprimido, três vezes ao dia), durante 42 dias consecutivos. Resultados Os dados dos 128 pacientes que efetivamente receberam o tratamento do estudo foram avaliados (66 tratados com o produto experimental e 62 com o produto comparador). Os dois grupos se mostraram homogêneos quanto aos dados demográficos e clínicos basais. A média da concentração do hidrogênio expirado aos 90 minutos no teste realizado ao final do tratamento (Dia 42) foi significativamente menor no grupo tratado com o produto experimental (17±18 ppm versus 34±47 ppm na população por protocolo). A diferença entre as médias dos dois grupos foi de -17 ppm (intervalo de confiança de 95% [IC95%]: -31,03; -3,17). O limite superior do IC95% não ultrapassou a margem de não-inferioridade estipulada a priori (7,5 ppm). As análises secundárias de eficácia confirmaram a semelhança entre os tratamentos (populações por protocolo e com intenção de tratamento). A tolerabilidade foi excelente em ambos os grupos e não houve relato de eventos adversos graves relacionados ao produto. Conclusão O produto experimental se mostrou não-inferior ao produto comparador, indicando sua eficácia no tratamento substitutivo da lactase endógena em pacientes portadores de intolerância à lactose.

Permeabilization of Saccharomyces fragilis IZ 275 cells with ethanol to obtain a biocatalyst with lactose hydrolysis capacity / Permeabilização de células de Saccharomyces fragilis IZ 275 com etanol para obtenção de biocatalisador com capacidade de hidrólise de lactose

The permeabilization was used to transform microorganisms in cell biocatalysts with high enzymatic activity. The Saccharomyces fragilis IZ 275 yeast cells were permeabilized with ethanol, as permeabilizing agent. To optimize the permeabilization conditions were used the design of Box-Behnken 15 trials (3 central points). The independent variables and their levels were ethanol (29, 32 and 35%), temperature (15, 20 and 25°C) and time (15, 20 and 25 min). The answer (Y) function has beta-galactosidase activity (U mg-1). The optimum conditions for obtaining a high enzymatic activity were observed in 35% ethanol concentration, temperature 15ºC and 20 min. treatment time. The maximum activity of the enzyme beta-galactosidase obtained was 10.59 U mg-1. The permeabilization of the S. fragilis IZ 275 cells was efficient.

A permeabilização foi usada para transformar células de microrganismos em biocatalisadores com alta atividade enzimática. As células de levedura de Saccharomyces fragilis IZ 275 foram permeabilizadas com etanol, como agente permeabilizante. Para otimizar as condições de permeabilização foi utilizado o delineamento de Box-Behnken com 15 ensaios (3 repetições no ponto central) . As variáveis independentes e seus níveis foram etanol (29, 32 e 35%), temperatura (15, 20 e 25ºC) e tempo (15, 20 e 25 min.). A função resposta (Y) foi atividade de beta-galactosidase (U mg-1). As condições ótimas para a obtenção de uma alta atividade enzimática foram observadas em 35% de concentração de etanol, temperatura de 15°C e tempo de tratamento de 20 minutos. A máxima atividade da enzima beta-galactosidase obtida foi de 10.59 U mg-1. A permeabilização das células de S. fragilis IZ 275 foi eficiente.

Occupational asthma induced by beat-galactosidase

Galactosidase is generated from Aspergillus oryzae, which is widely used for antidiarrhea medicine to infants. Antibiotics and digestives were reported as a causative allergen inducing occupational asthma. Galatosidase-induced occupational asthma has not been reported yet. A forty-year-old female has suffered from rhinorrhea, sneezing, and nasal obstruction 1 year after handling galactosidase at obstetric and pediatric hospital, and then dyspnea appeared later. Skin prick test with inhalent allergens, beta-galactosidase, and Aspergillus oryzae showed strong positive reaction to beta-galactosidase only. Immunoinhibition test with beta-galactosidase and A. oryzae revealed inhibition to beta-galactosidase only. Bronchial provocation test with beta-galactosidase showed the dual asthmatic response. With these results, we confirmed that the patient has beta-galactosidase-induced occupational asthma and rhinitis.

Prolonged expression of senescence markers in mice exposed to gamma-irradiation

Although ionizing radiation is known to induce cellular senescence in vitro and in vivo, its long-term in vivo effects are not well defined. In this study, we examined the prolonged expression of senescence markers in mice irradiated with single or fractionated doses. C57BL/6 female mice were exposed to 5 Gy of gamma-rays in single or 5, 10, 25 fractions. At 2, 4, and 6 months after irradiation, senescence markers including mitochondrial DNA (mtDNA) common deletion, p21, and senescence-associated beta-galactosidase (SA beta-gal) were monitored in the lung, liver, and kidney. Increases of mtDNA deletion were detected in the lung, liver, and kidney of irradiated groups. p21 expression and SA beta-gal staining were also increased in the irradiated groups compared to the non-irradiated control group. Increases of senescence markers persisted up to 6 months after irradiation. Additionally, the extent of mtDNA deletion and the numbers of SA beta-gal positive cells were greater as the number of radiation fractions increased. In conclusion, our results showed that ionizing radiation, especially that delivered in fractions, can cause the persistent upregulation of senescence marker expression in vivo. This should be considered when dealing with chronic normal tissue injuries caused by radiation therapy or radiation accidents.

Achados oculares em pacientes com mucopolissacaridoses / Ocular findings in patients with mucopolysaccharidosis

OBJETIVO: O objetivo deste estudo foi determinar a prevalência e gravidade das complicações oculares em pacientes com mucopolissacaridoses (MPS). MÉTODOS: Vinte e nove pacientes com diagnóstico de mucopolissacaridoses foram estudados. Foram avaliados: idade, sexo, acuidade visual, presença de estrabismo, erros refrativos, exame de fundo de olho, pressão intraocular, espessura corneal central e ultrassonografia ocular. RESULTADOS: Foram avaliados três pacientes com MPS I (12 por cento), 11 pacientes com MPS II (37,9 por cento), um paciente com MPS III (3,4 por cento) e 14 pacientes com MPS VI (48,3 por cento). A média de idade foi de 9,5 anos (DP 5,5). Observou-se hipermetropia em 88,5 por cento (23 pacientes) e astigmatismo em 51,7 por cento (15 pacientes). A média da acuidade visual corrigida foi de 0,45 logMAR (DP 0,68). A média do equivalente esférico foi +3,57 D (DP 2,46) e da pressão intraocular foi 17 mmHg (DP 3,9). Os achados mais comuns foram: espessamento palpebral 24,1 por cento (7 pacientes); opacidade da córnea, 55,2 por cento dos casos (16 pacientes); atrofia do nervo óptico, 23,1 por cento (6 pacientes); dobras radiais na retina 24 por cento (7 pacientes). O fundo de olho não foi examinado em 3 pacientes devido à opacidade de córnea. A média da espessura do complexo esclera-retina-coroide (ERC) medida por ultrassom foi de 1,78 mm (DP 0,51). CONCLUSÃO: Os achados oftalmológicos mais proeminentes foram espessamento palpebral, diminuição da acuidade visual, hipermetropia moderada, opacidade da córnea, dobras radiais na retina perimacular e atrofia do nervo óptico.

PURPOSE: The objective of this study was to determine the prevalence and severity of ocular complications in patients with mucopolysaccharidosis (MPS). METHODS: Twenty-nine patients with diagnosis of mucopolysaccharidosis were studied. Age, gender, visual acuity, presence of strabismus, refractive error, fundus examination, intraocular pressure, central corneal thickness and ocular echography were assessed for each individual. RESULTS: There were three patients with MPS I (12 percent), eleven patients with MPS II (37.9 percent), one patient with MPS III (3.4 percent) and fourteen patients with MPS VI (48.3 percent). Mean age was 9.5 years (ranged from 1.2 to 20 years, DP 5.5). Refraction was available in 26 patients, from which 88.5 percent (23 patients) were hyperopic, and 53.8 percent (14 patients) presented astigmatism. Best corrected visual acuity was available in 18 patients and the mean was 0.45 logMAR (DP 0.68). The mean spherical equivalent was +3.57 D (SD 2.46) and intraocular pressure was 17 mmHg (SD 3.9). The most common findings were: eyelid thickening in 24.1 percent (7 patients); corneal opacity in 55.2 percent of cases (16 patients); optic nerve atrophy in 23.1 percent (6 patients); and radial folds in the retina in 24 percent (7 patients). The fundus was examined in 26 out of 29 patients because corneal opacity avoided the exam in 3 of them. The average thickness of the complex sclera-retina-choroid (SRC was 1.78 mm (SD 0.51). CONCLUSION: The most prominent ophthalmologic findings were eyelid thickening, decreased visual acuity, high hyperopia, corneal opacity, perimacular radial folds in the retina and optic nerve atrophy.

Whey upgrading by enzyme biocatalysis

Whey is a co-product of processes for the production of cheese and casein that retains most of the lactose content in milk. World production of whey is estimated around 200 million tons per year with an increase rate of about 2 percent/per year. Milk production is seasonal, so surplus whey is unavoidable. Traditionally, whey producers have considered it as a nuisance and strategies of whey handling have been mostly oriented to their more convenient disposal. This vision has been steadily evolving because of the upgrading potential of whey major components (lactose and whey proteins), but also because of more stringent regulations of waste disposal. Only the big cheese manufacturing companies are in the position of implementing technologies for their recovery and upgrading, so there is a major challenge in incorporating medium and small size producers to a platform of whey utilization, conciliating industrial interest with environmental protection within the framework of sustainable development. Within this context, among the many technological options for whey upgrading, transformation of whey components by enzyme biocatalysis appears as prominent. In fact, enzymes are green catalysts that can perform a myriad of transformation reactions under mild conditions and with strict specificity, so reducing production costs and environmental burden. This review pretends to highlight the impact of biocatalysis within a platform of whey upgrading. Technological options are shortly reviewed and then an in-depth and critical appraisal of enzyme technologies for whey upgrading is presented, with a special focus on newly developed enzymatic processes of organic synthesis, where the added value is high, being then a powerful driving force for industrial implementation.

Actividad &beta-galactosidasa asociada con la senescencia en fibroblastos del estroma ovárico in vitro / Senescence-associated &beta-galactosidase activity in the in vitro ovarian stromal fibroblasts / Atividade &beta-galactosidase associada com a senescência em fibroblastos do estroma ovariano in vitro

Introducción: Un campo de investigación creciente de la biología es la senescencia celular, mecanismo que ha sido asociado -bajo determinadas circunstancias- con la transformación maligna. Teniendo en cuenta la elevada incidencia de cáncer ovárico y su génesis preferencial a partir del epitelio superficial del ovario, así como la posibilidad de ocurrencia de una transición epitelio-mesenquimática, se evaluó, tanto el crecimiento in vitro de los fibroblastos del estroma cortical, como la actividad a pH 6 de la β-galactosidasa, enzima cuya expresión ha sido clásicamente considerada como marcador de senescencia replicativa. Metodología: 48 muestras de fibroblastos de la corteza ovárica provenientes de donantes sin antecedentes de cáncer fueron cultivadas en forma seriada hasta el final de su vida replicativa. Mediante el método quimioluminiscente, en cada pase fue cuantificada la actividad β-galactosidasa a pH 6. Como control se utilizaron cultivos de células del epitelio superficial ovárico de las mismas donantes. La actividad enzimática fue también evaluada en fibroblastos previamente inducidos a senescencia con peróxido de hidrógeno. Resultados: Las lecturas de actividad enzimática, analizadas en conjunto con la capacidad replicativa, indican que los cultivos de fibroblastos alcanzaron el estado senescente hacia los pases 4-5, lo que también ocurrió con las células epiteliales. Los fibroblastos inducidos a senescencia mostraron valores variables de actividad enzimática. Conclusiones: La semejanza entre los fibroblastos y las células epiteliales en cuanto al inicio de la senescencia podría estar relacionado con la transición epitelio-mesenquimática que ha sido descrita como factor de riesgo de cáncer derivado del epitelio superficial ovárico. Valores bajos de actividad β-galactosidasa podrían sugerir que, en algunos casos, ocurrió inactivación de las vías de respuesta al estrés oxidativo.

Introduction: A growing biological research field is the cellular senescence, a mechanism that has been associated, under certain circumstances, with malignant transformation. Given the high incidence of ovarian cancer and its main origin from the ovarian surface epithelium, as well as the possibility that an epithelial-mesenchymal transition occurs, we evaluated both the in vitro growth of stromal fibroblasts from the ovarian cortex and their β-galactosidase activity at pH 6, enzyme whose expression is considered as a marker of replicative senescence. Methods: 48 samples of ovarian cortical fibroblasts from donors without a history of cancer were serially cultured until the end of their replicative life. β-galactosidase activity at pH 6 was quantified in each passage by the chemiluminiscent method. As control, we used ovarian epithelial cell cultures from the same donors. The enzyme activity was also evaluated in fibroblasts previously induced to senescence by exposure to hydrogen peroxide. Results: The analysis of the enzyme activity and the replicative capacity taken together showed that the fibroblast cultures reached the senescent state at passages 4-5, as what happened with the control epithelial cells. Fibroblasts induced to senescence showed high variability in the values of enzymatic activity. Conclusions: The similarity between both types of cells in reaching the senescent state deserves to be taken into account in relation to the epithelial-mesenchymal transition that has been proposed to explain their behavior in the genesis of cancer arising from ovarian surface epithelium. Low β-galactosidase activity values at pH 6 would suggest possible inactivation of the response pathways to oxidative stress.

Introdução: um campo de pesquisa crescente da biologia é a senescência celular, mecanismo que tem sido associado, sob determinadas circunstancias, com a transformação maligna. Tendo em conta a elevada incidência de câncer ovariano e sua gênese preferencial a partir do epitélio superficial do ovário, assim como a possibilidade de ocorrência de uma transição epitélio-mesenquimática, se avaliou, tanto o crescimento in vitro dos fibroblastos do estroma cortical, como a atividade a pH 6 da β-galactosidase, enzima cuja expressão tem sido classicamente considerada como marcador de senescência replicativa. Metodologia: 48 amostras de fibroblastos do córtex ovariano provenientes de doadores sem antecedentes de câncer foram cultivadas em forma seriada até o final de sua vida replicativa. Mediante o método quimioluminescente, em cada passe foi quantificada a atividade β-galactosidase a pH 6. Como controle utilizou-se cultivos de células do epitélio superficial ovariano das mesmas doadoras. A atividade enzimática foi também avaliada em fibroblastos previamente induzidos a senescência com peróxido de hidrogeno. Resultados: as leituras da atividade enzimática, analisada em conjunto com a capacidade replicativa, indicam que os cultivos de fibroblastos alcançaram o estado senescente para os passes 4-5, o que também ocorreu com as células epiteliais. Os fibroblastos induzidos a senescência mostraram valores variáveis de atividade enzimática. Conclusões:as semelhanças entre os fibroblastos e as células epiteliais em quanto ao inicio da senescência poderia estar relacionado com a transição epitélio-mesenquimática que tem sido descrita como fator de risco de câncer derivado do epitélio superficial ovariano. Valores baixos de atividade β-galactosidase poderiam sugerir que, em alguns casos, ocorreu inativação das vias de resposta ao estresse oxidativo.

Population analysis of the GLB1 gene in South Brazil

Infantile GM1 gangliosidosis is caused by the absence or reduction of lysosomal beta-galactosidase activity. Studies conducted in Brazil have indicated that it is one of the most frequent lysosomal storage disorders in the southern part of the country. To assess the incidence of this disorder, 390 blood donors were tested for the presence of two common mutations (1622-1627insG and R59H) in the GLB1 gene. Another group, consisting of 26 GM1 patients, and the blood donors were tested for the presence of two polymorphisms (R521C and S532G), in an attempt to elucidate whether there is a founder effect. The frequencies of the R59H and 1622-1627insG mutations among the GM1 patients studied were 19.2 percent and 38.5 percent, respectively. The frequency of polymorphism S532G was 16.7 percent, whereas R521C was not found in the patients. The overall frequency of either R59H or 1622-1627insG was 57.7 percent of the disease-causing alleles. This epidemiological study suggested a carrier frequency of 1:58. Seven different haplotypes were found. The 1622-1627insG mutation was not found to be linked to any polymorphism, whereas linkage disequilibrium was found for haplotype 2 (R59H, S532G) (p < 0.001). These data confirm the high incidence of GM1 gangliosidosis and the high frequency of two common mutations in southern Brazil.

Expression of β-catenin in oxidative stress-induced premature senescence of human skin fibroblasts / 中华皮肤科杂志

Objective To observe the changes of β-catenin expression in human skin fibroblasts (HSFs) after induced by oxidative stress, and to explore its possible roles in oxidative stress-induced premature senescence (SIPS) of HSFs. Methods Fibroblasts were isolated from the foreskin of a child and subjected to a primary culture. The fibroblasts of second to fourth passage were treated with various concentrations of H2O2 for 2 hours to establish an optimized model of stress-induced premature senescence, β-galactosidase assay kit was used to detect the activity of β-galactosidase in H2O2rinduced HSFs, RT-PCR and Western blot to measure the mRNA and protein expressions of β-catenin in control and senescent HSFs. Results Premature senescence of HSFs could be induced by the treatment with H2O2 of 150 μmol/L for 2 hours. The proportion of β-galactosidase-positive cells was (2.97 ± 0.25)% in control HSFs and (37.67 ± 1.53)% in senescent HSFs (P< 0.01). A significant increase was observed in the β-catenin/GAPDH protein ratio and β-catenin/GAPDH mRNA ratio in control HSFs compared with the senescent HSFs (0.62 ± 0.03 vs. 0.31 ± 0.01, t = 14.97, P < 0.01; 0.59 ± 0.04 vs. 0.29 ± 0.30, t = 10.06, P < 0.01). Conclusions The two-hour treatment with H2O2 of 150 μmol/L could induce the premature senescence of HSFs, and there is a notable decrease in the expression of β-catenin in prematurely senescent HSFs induced by oxidative stress, implying that β-catenin is an important target gene for the regulation of skin aging.

Spontaneous senescence in malignant tumor cell lines / 肿瘤

Objective:To explore whether spontaneous sene-scence widely existed in malignant tumor cells. Methods:Sene-scence-associated beta-galactosidase (SA-β-Gal) staining kit was used to detect the activity of SA-β-Gal in ten different malignant tumor cell lines before and after serum deprivation. Results:SA-β-Gal was expressed in some cells of 10 malignant tumor cell lines during exponential growth phase without any treatment. However, the percentage of senescent cells was significantly different among them, the lowest expression was observed in HeLa cell line (0.65%), and the highest expression was seen in HepG2 cell line (3.69 %, F=13.006, P= 0.000). Furthermore, not all the SA-β-Gal positive aging cells were polyploid cells. After 24-h serum deprivation, the number of SA-β-Gal positive cells was significantly increased (P=0.001). Conclusion:These findings indicate that immortal malignant tumor cell lines could undergo spontaneous senescence but the level was different between various cell lines. Short-term serum de-privation significantly increased the percentage of aging cells indicating that serum deprivation-induced cell senescence may be a rapid, easy, and effective way for anti-tumor therapy.

Late-onset Krabbe's Disease (Globoid Cell Leukodystrophy): A case report

Krabbe's disease is a rare autosomal recessive disorder characterized by hemiplegia, paraplegia, ataxia, cortical blindness, and peripheral neuropathy. This disease is caused by deficiency of the lysosomal enzyme galactocerebroside beta-galactosidase(GALC), resulting in demyelination of white matter of brain and peripheral nerve. We reported a 38-year-old female developed a slowly progressive weakness of lower extremities and gait disturbance since age of 10. Neurological examination revealed spastic weakness of both lower extremities, hyperactive deep tendon reflexes and intrinsic muscle atrophy of both hands and feet. Electrophysiologic study showed uniform demyelinating sensorimotor peripheral neuropathy. T2-weighted brain MRI (magnetic resonance imaging) findings revealed symmetric high signal intensity along the bilateral corticospinal tract. The diagnosis of Krabbe's disease was confirmed by finding of markedly reduced GALC activity in leukocyte. We recommended to consider Krabbe's disease in the diagnosis of patients affecting both central and peripheral nervous system.

A Case of Krabbe Disease with Infantile Spasm

Krabbe disease is a rare autosomal recessive disorder clinically characterized by retardation in motor development, prominent spasticity, seizures, and optic atrophy. Pathologically, there are many globoid cells in the white matter, in addition to the lack of myelin and the presence of severe gliosis. Hence Krabbe disease is known as globoid cell leukodystrophy. Biochemically, the primary enzymatic deficiency in Krabbe disease is galactocerebroside beta-galactosidase. Patients with Krabbe disease can be subdivided into the early-onset type and late-onset type, according to the onset of clinical manifestations. Most patients with early-onset type die before their second birthday. We describe a girl with Krabbe disease associated with uncontrolled seizures, which was confirmed with biochemical study and MRI. The clinical findings of this patient included hyperirritability, scissoring of the legs, flexion of arm, and clenching of the fists, and generalized tonic seizures. EEG showed hypsarrhythmia, and MRI demonstrated degenerative white matter changes in bilateral periventricular white matter, posterior rim of internal capsule, basal ganglia and brain stem on T2W1 and FLAIR image. The diagnosis was based on clinical features of progressive neurologic deterioration in conjunction with low galactocerebroside beta-galactosidase activity.

Additive Estrogenic Activities of the Binary Mixtures of Four Estrogenic Chemicals in Recombinant Yeast Expressing Human Estrogen Receptor

To evaluate the estrogenic activities of several chemicals such as 17 beta-estradiol (E2), rho-nonylphenol, bisphenol A, butylparaben, and combinations of these chemicals, we used recombinant yeasts containing the human estrogen receptor [Saccharomyces cerevisiae ER + LYS 8127]. We evaluated E2 was most active in the recombinant yeast assay, followed by rho-nonylphenol, bisphenol A, butylparaben. The combinations of some concentrations of 17-estradiol as a strong estrogen and bisphenol A or butylparaben as a weak estrogen showed additive estrogenic effects. Also, the combinations of some concentrations of nonlyphenol and butylparaben and combination of butylparaben and bisphenol A showed additive effects in the estrogenic activity. Therefore, the estrogenic activities of the combinations of two chemicals were additive, not synergistic.

Study of GE7-transferring system mediated ?-galactosidase gene transfer in a rat model of ovarian tumor by intraarterial route / 中华妇产科杂志

Objective To investigate the efficiency and targeting of the GE7 system mediated gene transfer in the chemically induced ovarian tumor by intraarterial route. Methods Animal models were chemically induced by 7,12-dimethylbenz [ a] anthracene ( DMBA ) . GE7-polylysine/p-galactosidase ( ?-gal)/polylysine-HA20 complexes were constructed. Fifteen rats with induced ovarian tumor were divided into three groups, the complexes were injected through ovarian artery and tail vein, respectively. The tumor, heart, liver, spleen, lung and kidney were obtained at 72 h. X-gal staining was used to check expression of ?-gal. Results ?-gal was expressed strongly and well-distributed in tumor in the first group, and stained blue in the liver, spleen, lung and kidney, but much weaker than that of tumor. In the second group, ?-gal was expressed in the tumor,and weakly expressed in the liver, none was detected in the other organs. In the third group ?-gal was detected in the lung slightly, none was detected in the tumor and other organs. Conclusions When GE7 complexes were administrated through ovarian artery, the introduced gene expressed preferentially in ovary. This result was the basis of intraarterial administration of therapeutic gene to treat ovarian cancer.

Transfection of SV 40 Large T Antigen into Corneal Endothelial Cells

The coeneal endothelium is essential for the maintenance of normal corneal hydration, thickness, and transparency. However, corneal endothelial cells are incapable of significant proliferation in vivo. As we age, the density of corneal endothelial (CEN) cells gradually decreases. The goal of our study is to explore the possibility of enhancing the proliferation of corneal endothelial cells by introduction of SV 40 large T antigen, a transforming protein. To this end, introduction of protein into CEN cells was assessed by liposome assisted beta-galactosidase transfection in vivo, ex vivo, and in vivo. In all cases, cells treated with liposome-protein complex have shown dramatic blue stain in beta-galactosidase activity staining. This result convinced us that we could artificially introduce a foreign protein into a cell. To ascertain where SV 40 large T antigen is localized in the cell, purified SV 40 large T antigen was transfected into the cells using liposome and its presence was determined immunohistochemically. We show that the liposome delivered SV 40 large is localized in the nucleus and mitotic figures which may suggest its functional activity.