Mutación novel en el Síndrome Sanfilippo B. Presentación de un caso / A novel mutation in Sanfilippo Syndrome type B. Case report

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)

Linfangiectasia intestinal en un paciente afectado de síndrome de Sanfilippo B / Intestinal lymphangiectasia in a patient with Sanfilippo B syndrome

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.

Cloning, expression and characterization of a new endo-β-N-acetylglucosaminidase from Streptomyces alfalfae / 生物工程学报

Endo-β-N-acetylglucosaminidase is used widely in the glycobiology studies and industries. In this study, a new endo-β-N-acetylglucosaminidase, designated as Endo SA, was cloned from Streptomyces alfalfae ACCC 40021 and expressed in Escherichia coli BL21 (DE3). The purified recombinant Endo SA exhibited the maximum activity at 35 ºC and pH 6.0, good thermo/pH stability and high specific activity (1.0×10⁶ U/mg). It displayed deglycosylation activity towards different protein substrates. These good properties make EndoSA a potential tool enzyme and industrial biocatalyst.

Relationship of protein O-GlcNAcylation levels with antitumor effect of Taxol in breast cancer / 中国生化药物杂志

Objective To study effect of Taxol on protein O-GlcNAcylation levels and investigate whether protein O-GlcNAcylation levels can affect the sensitivity of breast cancer cells to Taxol.Methods Western blot analysis was performed to examine protein O-GlcNAcylation levels and the expression of enzymes related to O-GlcNAcylation biosynthesis in Taxol treated breast cancer cells.RT-qPCR was used to analyze the effects of Taxol on OGA and OGT mRNA expression in cancer cells.The sulforhodamine B colorimetric assay was used to determine the effect of alteration of protein O-GlcNAcylation on the anti-proliferation of Taxol in breast cancer cells by adding OGA inhibitor and OGT inhibitor, respectively.Results Taxol treatment enhanced protein O-GlcNAcylation levels in dose-and time-dependent manners in breast cancer cell MDA-MB-231 ( P <0.05 ) .Taxol increased the mRNA levels of OGT and OGA after MDA-MB-231 cells were treated for 24 h(P<0.05).As OGA inhibitors increased protein O-GlcNAcylation levels, the sensitivity of MDA-MB-231 to Taxol was increased.As OGT inhibitor decreased protein O-GlcNAcylation levels, the sensitivity of MDA-MB-231 to Taxol was reduced.Conclusion Taxol treatment can enhance protein O-GlcNAcylation levels and the changes of O-GlcNAcylation levels alter the sensitivity of breast cancer cell MDA-MB-231 to Taxol.

Expression of endo-beta-N-acetylglucosaminidase H in Pichia pastoris and its applica-tion to N-glycosylation analysis / 军事医学

Objective To prepare endo-beta-N-acetylglucosaminidase H (Endo-H) expressed in Pichia pastoris, and apply it to N-glycosylation analysis .Methods One complete gene was synthesized on the basis of the cDNA sequence encoding Streptomyces plicatus reported in GenBank .The gene was cloned into the expression vector pPIC 9.The expression vector pPIC9-Endo-H was transformed into P.pastoris(JC308).The expression products were induced by methanol , puri-fied by two-step chromatography , used to analyze the glycan structures of RNaseB by the DNA sequencer assisted fluoro-phore-assisted carbohydrate electrophoresis (DSA-FACE)methods, and finally compared with peptide-N-asparagine amidase F(PNGase F).Results This enzyme expressed in P.pastoris(JC308) had the ability to hydrolyze natural or denatured high-mannose type of oligosaccharide linked by β-1,4-glycosidic bonds , but not complex-type oligosaccharide .The result of DSA-FACE showed that carbohydrate chains of Man 5 GlcNAc-Man9 GlcNAc could be obtained when RNaseB was hydrolyzed by Endo-H, and that Man5 GlcNAc2-Man9 GlcNAc2 chains became available when RNaseB was hydrolyzed by PNGase F . Conclusion Endo-H expressed in P.pastoris has bioactivity which can be used to analyze N-glycosylation with the method of DSA-FACE.

Role of Urinary Levels of Endothelin-1, Monocyte Chemotactic Peptide-1, and N-Acetyl Glucosaminidase in Predicting the Severity of Obstruction in Hydronephrotic Neonates

PURPOSE: Antenatal hydronephrosis (AH) is found in 0.5%-1% of neonates. The aim of the study was to assess the urinary concentrations of 3 biomarkers, endothelin-1 (ET-1), monocyte chemotactic peptide-1 (MCP-1), and N-acetyl-glucosaminidase (NAG) in severely hydronephrotic neonates. MATERIALS AND METHODS: Neonates with a history of prenatal hydronephrosis were enrolled in the prospective study in 2 groups. Group 1 included neonates with severe forms of obstruction requiring surgical intervention and group 2 included neonates with milder forms of obstruction without any functional impairment. Fresh voided urinary levels of ET-1, MCP-1, and NAG were measured and their ratios to urinary Cr were calculated. RESULTS: Fourty-two neonates were enrolled into the 2 groups: group 1, 24 patients (21 male, 3 female); group 2, 18 neonates (16 male, 2 female). There were no statistically significant differences between urinary ET-1, NAG, MCP-1 values, and ET-1/Cr and NAG/Cr ratios in groups 1 and 2. The urinary MCP-1/Cr ratio was significantly higher in group 1 than in group 2. For comparison of groups 1 and 2, the cut-off values were measured as 0.5709 ng/mg (sensitivity, 75%; specificity, 67%; positive predictive value [PPV], 71%; negative predictive value [NPV], 71%), 0.927 ng/mg (sensitivity, 77%; specificity, 72%; PPV, 77%; NPV, 72%), and 1.1913 IU/mg (sensitivity, 62%; specificity, 67%; PPV, 68%; NPV, 60%) for ET-1/Cr, MCP-1/Cr, and NAG/Cr ratios, respectively. CONCLUSIONS: The urinary MCP-1/Cr ratio is significantly elevated in neonates with severe obstruction requiring surgical intervention. Based upon these results, urinary MCP-1/Cr may be useful in identification of severe obstructive hydronephrosis in neonates.

The change of the serum level of cystatin C, urinary NAG enzyme in the children affected by HSP and its clirical significale / 中国基层医药

Objective To investigate the change of the serum levels of cystatin C,urinary NAG enzyme in the children affected by HSP and its cliracal significance.Methods 60 children with HSP were divided into three groups based on urine routine and abnormal degrees of biochemícal examination results,which were kidney injury group A (KI-Group A),kidney injury group B (KI-Group B),and non-kidney injury group (non-KI-Group).Selected 28 healthy children with the same phase and the same age as the control group.A sandwich enzyme-linked immunosorbent assay(ELISA) was employed to determine the values of cystatin C,urinary NAG enzyme.Results Urinary NAG enzyme in KI-Group A (127.11 ± 15.63) ng/L and KI-Group B (132.75 ± 19.83) ng/L were significantly higher than the control group (111.36 ± 20.10) ng/L (F =7.324,P ＜ 0.05),and there was no difference between non-KI-Group (108.14 ± 13.83) ng/L and the control group.Cystatin C in KI-Group A (1.18 ±0.13) mg/L,KI-Group B (1.19 ±0.17)mg/L and non-KI-Group (1.16 ±0.11)mg/L were all significantly higher than the control group (0.79 ±0.14)mg/L (P ＜ 0.05).Conclusion Cystatin C and urinary NAG enzyme in children with HSP were significantly abnormal,and may be used as early indexes reflecting the glomerular injury.The relationship of the two above indicators had positive correlation,and the combined employment of them had the dadvantages of early detection,high sensitivity and accurate diagnosis.

Estudio de las enzimas lisosomales N-acetil-a-D-glucosaminidasa y arilsulfatasa A en adultos cubanos / Assay of lysosomal N-acetyl-a-D-glucosaminidase and arylsulfatase A activities in Cuban adults

El objetivo de este estudio fue determinar, en una muestra de individuos cubanos, un rango preliminar de valores normales de actividad específica de N-acetil- a-D-glucosaminidasa y arilsulfatasa A, enzimas deficientes en la mucopolisacaridosis tipo III B y la leucodistrofia metacromática, respectivamente. Se realizó una investigación de corte transversal, en muestras de sangre de 38 individuos adultos. Se realizó la extracción de los leucocitos y se determinó la actividad específica de las enzimas por métodos espectrofotométricos. Todos los participantes fueron caracterizados desde el punto de vista clínico como sanos para ambas enfermedades. Se obtuvieron valores medios de actividad específica de N-acetil-a-D-glucosaminidasa y arilsulfatasa A de 1,52±0,30 y 121,37±20,14 nmol/mg/h, respectivamente. No se encontraron diferencias significativas en relación a las características étnicas para ninguna de las dos enzimas (p<0,05). Este constituye el primer estudio cubano en el cual se publican rangos de actividad específica para estas enzimas en individuos cuya condición de sanos y no relacionados familiarmente con la enfermedad ha sido clínicamente demostrada. El análisis de un mayor número de muestras permitirá establecer los puntos de corte que dotarán al diagnóstico bioquímico de estas enfermedades de una mayor confiabilidad.

The aim of this study was to determine, in a sample of Cuban individuals, a preliminary range of normal values of N-acetyl-a-D-glucosaminidase and arylsulfatase A activity, enzymes that are deficient in mucopolysaccharidosis type III B and metachromatic leukodystrophy, respectively. A cross-sectional research was conducted. Blood samples were obtained out of 38 adult individuals. The leucocytes were extracted and the specific enzymatic activity was assayed by spectrophotometric methods. All participants were characterized from the clinical point of view as healthy for both diseases. Average values of N-acetyl-a-D-glucosaminidase and arylsulfatase A specific activities of 1.52 ± 0.30 and 121.37 ± 20.14 nmol/mg/h, respectively were obtained. There were no significant differences related to ethnicity for any of the two enzymes (p <0.05). This is the first Cuban study in which ranges of activity for these enzymes have been reported in healthy individuals whose healthy and non-familiarly related with the disease status have been clinically demonstrated. The analysis of more samples will establish the cutoff points that will increase the reliability of the biochemical diagnosis of these diseases.

Value of combined measurement of urine N-acetyl-beta-D-glucosaminidase activity and serum Cystatin C in diagnosing diabetic nephropathy in early phase / 中国医师进修杂志

Objective To explore the value of combined measurement of urine N-acetyl-beta-D-glucosaminidase (NAG) activity and serum Cystatin C in diagnosing diabetic nephropathy (DN) in early phase. Methods Sixty-two cases with type 2 diabetes (diabetic group) were divided into three groups according to their 24 hours urinary albumin excretion (24hUAE) : group A (normal albuminuria, 20 cases), group B (microalbuminuria, 22 cases) and group C (macroalbuminuria, 20 cases). Furthermore, 30 healthy people were involved in control group. 24hUAE,NAG,serum creatinine (SCr) and serum Cystatin C were measured, and endogenous creatinine clearance rate (Ccr) was calculated by Cockcroft-Gault formula. All these indexes among three groups were compared. Results The levels of urinary NAG activity and serum Cystafin C in diabetic group was significantly higher and Ccr was significantly lower than those in control group(P < 0.01). The levels of urinary NAG activity and serum cystatin C gradually increased in group A, B and C(P< 0.05 or < 0.01). While no significant difference was observed between group A and group B in the level of SCr (P > 0.05). There were significant positive correlations among the levels of urinary NAG activity, serum Cystatin C,24hUAE and SCr (P< 0.01),and all above showed negative correlations with Ccr (P<0.01). Co-detection of urinary NAG activity and serum Cystatin C had significantly higher positive rate [80.6%(50/62)] than single one [58.1%(36/62),61.3%(38/62)](P<0.05). Conclusion Co-detection of urinary NAG activity and serum Cystatin C may indicate early renal damage in DN, and it is valuable in diagnosing DN in early phase.

O-GlcNAc Modification: Friend or Foe in Diabetic Cardiovascular Disease / 당뇨병

O-Linked beta-N-acetyl glucosaminylation (O-GlcNAcylation) is a dynamic post-translational modification that occurs on serine and threonine residues of cytosolic and nuclear proteins in all cell types, including those involved in the cardiovascular system. O-GlcNAcylation is thought to act in a manner analogous to protein phosphorylation. O-GlcNAcylation rapidly cycles on/off proteins in a time scale similar to that for phosphorylation/dephosphorylation of proteins. Several studies indicate that O-GlcNAc might induce nuclear localization of some transcription factors and may affect their DNA binding activities. However, at the cellular level, it has been shown that O-GlcNAc levels increase in response to stress and augmentation of this response suppresses cell survival. Increased levels of O-GlcNAc have been implicated as a pathogenic contributor to glucose toxicity and insulin resistance, which are major hallmarks of type 2 diabetes and diabetes-related cardiovascular complications. Thus, O-GlcNAc and its metabolic functions are not yet well-understood; focusing on the role of O-GlcNAc in the cardiovascular system is a viable target for biomedical investigation. In this review, we summarize our current understanding of the role of O-GlcNAc on the regulation of cell function and survival in the cardiovascular system.

b-n-acetylglucosaminidase production by Lecanicillium (Verticillium) lecanii ATCC 26854 by solid-state fermentation utilizing shrimp shell / Producción de b-n-acetilglucosaminidasa de lecanicillium (verticillium) lecanii ATCC 26854 por cultivo sólido fermentado utilizando caparazón de camarón / Produção de b-n-acetilglucosaminidasa de lecanicillium (verticillium) lecanii ATCC 26854 em fermentação em estado sólido utilizou exoesqueleto do camarão

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.

Relationship between the ende mic arsenism and the liver,renal damage / 中国地方病学杂志

Objective To explore the relationship between the endemic arsenism and the liver,renal damage.Methods Some permanent residents were selected as investigated subjects who lived at 3 villages in Datong in Shanxi Province,an arseniasis-endemic areas,These objects were divided into arsenic poisoning and control group on the basis of Diagnosis Standard for Endemic Arsenism(WS/T 211-2001).Then blood and urine samples were collected in the surveyed people.Serum glutamate pyruvic transaminase(ALT)were detected by Enzyme-linked immunosorbent assay as the indicator of the impaired hepatic function.The microdosis albumen (mAlb)and acetylglucosaminidase(NAG)in urine were detected by end-point method and alkaline picric acid as the renal damage indicators.Results A total of 661 people investigated,of which 144 cases were arsenic poisoning patients.The rates of abnormal liver function were significant hisher in arsenic poisoning group[10.42% (15/144)]than that in control[5.22%(27/517)],and both wag significant[X2=5.107,P＜0.05;OR=2.11,95%CI (1.09-4.08)].The geometric mean of mAlb/Ucr was 2.16 mg/g Cr in control,and 2.31 mg/g Cr in arsenic poisoning group,and both was not significant(t=-1.71,P＞0.05).The geometric mean of NAG waft higher in arsenic poisoning group(2.43 U/g Cr)than that in the control(2.22 U/g Cr),and both was significant(t=-3.55, P＜0.05).Conclusions The damage of the liver and renal function were related with endemic arsenism,and NAG is the early indicators suggesting impaired renal function due to endemic arsenism.

Clinical Significance of Combined Detecting of Urine N - Acetyl - D - Glucosaminidase and ?2 Microglobulin in Early Diagnosis of Anaphylatoid Purpura Renal Injuries / 实用儿科临床杂志

0.05) ,but the differences reached statistical significance compared the positive ratios of two index together to urine NAG and ?2 - MG (X2 = 4.41,7.28 P

N-Acetyl-beta-D-glucosaminidase in acute myocardial infarction

The objective of the study was to investigate whether the lysosomal enzyme, N-Acetyl-beta-D-glucosaminidase (NAG) activity is increased in plasma of patients with acute myocardial infarction (AMI) and to determine if there is any association between plasma levels of NAG and severity of myocardial infarction (MI). NAG activity in plasma was monitored in 69 patients with AMI and 135 normal healthy subjects using a spectrofluorimetric method. A modified Aldrich ST elevation score was used to gauge the severity of MI in terms of size of the infarct. Plasma NAG levels in AMI patients and normal healthy subjects were found to be 10.92+/-7.5 U/l and 6.8+/-2.2 U/l, respectively. These two mean value when compared by Student's t-test were significantly different P = 0.0001. No statistically significant differences in NAG activity were observed in patients in terms of gender, age, location of infarct, time from onset of chest pain to blood sampling in the hospital and size of the infarct.

The Dynamic Changes of Renal Function in Premature Infants after Asphyxia / 中华围产医学杂志

Objective To study changes of urinary protein in premature infants after asphyxia in order to explore influence of asphyxia on the renal function. Methods Microalbumin(mAlb),retinal-bindingprotein (RBP) ,N-acety-?-D-aminoglucosidase in urine and serum urea nitrogen(BUN),creatinine(Cr) were performed in 56 normal premature infants and 49 asphyxia ones with immunoturbidimetric method, ELISA method, rate method , enzymic method and picric acid method when they were 1,4,7 day age after born. Results (1)With ages increasing urinary mAlb took on decreasing trend in the same gestation age but there was no different while with the gestation age increasing in the same ages urinauy mAlb was decreased significantly (P