Biochemical properties of Scedosporium aurantiacum extracellular elastase-like protease / 生物工程学报

Extracellular elastase-like protease is one of the key virulence proteases of Scedosporium aurantiacum. To date, little is known about this enzyme in terms of genetic information, structure, properties and virulence mechanism due to the difficulties in purification caused by its low secretion amount, high specific activity, uncompleted genome sequencing and annotation. This work investigated the gene, structure and enzymatic properties of this enzyme. The S. aurantiacum elastase-like protease from the fungal culture supernatant was analyzed through tandem mass spectrometry (MS/MS) approach, illustrating its primary structure. Bioinformatics tools were employed to predict the conserved domain and tertiary structure, the enzymatic properties were also studied. It turned out that S. aurantiacum extracellular elastase-like protease demonstrated well hydrolysis towards elastin and bovine achilles tendon collagen, with Vmax of 18.14 μg/s and 17.57 μg/s respectively, better than fish scale gelatin, with the lowest hydrolysis effect on casein. Its activity towards elastin was lower than that of the elastase from porcine pancreas, with values of Kcat/Km of 3.541 (μg/s) and 4.091 (μg/s), respectively. It was an alkaline protease, with optimal pH 8.2 and temperature 37 oC. Zn2+ promoted the enzymatic activity while Ca2+, Mg2+, Na+, elastatinal and PMSF inhibited its activity. Its sequence was similar to Paecilomyces lilacinus secreted serine protease (PDB Entry: c3f7oB_) with multiple conserved fractions each containing more than 7 amino acids, thus suitable for design of PCR primer. This study increased our knowledge on S. aurantiacum extracellular elastase-like protease in terms of structure and enzymatic properties, and may facilitate later studies on protein expression and virulence mechanism.

Association between single nucleotide polymorphisms of tropoelastin gene and aortic dissection / 中南大学学报(医学版)

OBJECTIVES@#To evaluate the relation between single nucleotide polymorphisms (SNPs) of tropoelastin gene and aortic dissection (AD) via identifying SNPs in the tropoelastin gene, and to detect the level of tropoelastin mRNA, elastin and elastic fibers.@*METHODS@#The specimens of the AD group (@*RESULTS@#Seven SNP loci of the tropoelastin gene were detected in these samples. Among them, 5 SNP loci were polymorphic. The frequency of 3 SNP loci[rs2071307 (G/A), rs34945509 (C/T) and rs17855988 (G/C)] was significantly different between the AD group and the control group (all @*CONCLUSIONS@#The polymorphisms of rs2071307 (G/A), rs34945509 (C/T), and rs17855988(G/C) in the tropoelastin gene may eventually affect the synthesis of elastic fibers and they may play an important role in the occurrence of AD.

Prevalence of scoliosis in Williams-Beuren syndrome patients treated at a regional reference center

OBJECTIVE: This study assessed the prevalence of scoliosis and the patterns of scoliotic curves in patients with Williams-Beuren syndrome. Williams-Beuren syndrome is caused by a chromosome 7q11.23 deletion in a region containing 28 genes, with the gene encoding elastin situated approximately at the midpoint of the deletion. Mutation of the elastin gene leads to phenotypic changes in patients, including neurodevelopmental impairment of varying degrees, characteristic facies, cardiovascular abnormalities, hypercalcemia, urological dysfunctions, and bone and joint dysfunctions. METHODS: A total of 41 patients diagnosed with Williams-Beuren syndrome, who were followed up at the genetics ambulatory center of a large referral hospital, were included in the study. There were 25 male subjects. The patients were examined and submitted to radiographic investigation for Cobb angle calculation. RESULTS: It was observed that 14 patients had scoliosis; of these 14 patients, 10 were male. The pattern of deformity in younger patients was that of flexible and simple curves, although adults presented with double and triple curves. Statistical analysis showed no relationships between scoliosis and age or sex. CONCLUSION: This study revealed a prevalence of scoliosis in patients with Williams-Beuren syndrome of 34.1%; however, age and sex were not significantly associated with scoliosis or with the severity of the curves. .

Genes responsible for vaginal extracellular matrix metabolism are modulated by women's reproductive cycle and menopause

Objectives To analyze the expression of genes involved in extracellular matrix (ECM) biogenesis and remodeling in vaginal tissue of women with clinically normal pelvic floor support (defined as controls) according to the phase of menstrual cycle and postmenopausal women with and without pelvic organ prolapse (POP). Materials and Methods This study examined the expression of matrix metalloproteinases (MMPs), their tissue inhibitors (TIMPs), and the Lysyl oxidase (LOX) family genes in the anterior vaginal wall of Caucasian women by real-time RT-PCR. Initially, mRNA expression was assessed in premenopausal controls in the secretory (group 1, n = 10) vs. proliferative (group 2, n = 8) phase of menstrual cycle. In addition, we compared premenopausal controls in the proliferative phase (group 2) vs. postmenopausal controls (group 3, n = 5). Finally, we analyzed postmenopausal controls (group 3) vs. postmenopausal women with advanced POP (group 4, n = 13). Results According to the phase of menstrual cycle, MMP1 was significantly reduced (p = 0.003), whereas the expression of TIMP1 and LOXL4 was significantly up-regulated during proliferative phase (both p < 0.01) when compared to the secretory phase in premenopausal control women. Regarding menopausal status/ageing, all MMPs were down-regulated, while TIMP3, TIMP4 and LOXL2 were significantly up-regulated in postmenopausal control women when compared to premenopausal controls (p = 0.005, p = 0.01 and p < 0.001, correspondingly). TIMP4 and LOXL2 mRNA levels were significantly decreased in postmenopausal POP patients compared to asymptomatic postmenopausal controls (p < 0.01 for both). Conclusions Our results indicate that ovarian cycle and age-related changes influence the expression of genes encoding proteins responsible for ECM metabolism in human vagina. Moreover, POP is associated with alteration in vaginal ECM components after menopause. .

Characterization of human adipose-derived stem cells and expression of chondrogenic genes during induction of cartilage differentiation

OBJECTIVES: Understanding the changes in chondrogenic gene expression that are involved in the differentiation of human adipose-derived stem cells to chondrogenic cells is important prior to using this approach for cartilage repair. The aims of the study were to characterize human adipose-derived stem cells and to examine chondrogenic gene expression after one, two, and three weeks of induction. MATERIALS AND METHODS: Human adipose-derived stem cells at passage 4 were evaluated by flow cytometry to examine the expression of surface markers. These adipose-derived stem cells were tested for adipogenic and osteogenic differentiation capacity. Ribonucleic acid was extracted from the cells for quantitative polymerase chain reaction analysis to determine the expression levels of chondrogenic genes after chondrogenic induction. RESULTS: Human adipose-derived stem cells were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen and successfully differentiated into adipogenic and osteogenic lineages. The human adipose-derived stem cells aggregated and formed a dense matrix after chondrogenic induction. The expression of chondrogenic genes (collagen type II, aggrecan core protein, collagen type XI, COMP, and ELASTIN) was significantly higher after the first week of induction. However, a significantly elevated expression of collagen type X was observed after three weeks of chondrogenic induction. CONCLUSION: Human adipose-derived stem cells retain stem cell characteristics after expansion in culture to passage 4 and serve as a feasible source of cells for cartilage regeneration. Chondrogenesis in human adiposederived stem cells was most prominent after one week of chondrogenic induction.

The microfibril-elastin fiber system distribution in left atrioventricular valve of the rat / Distribución del sistema de microfibrillas y fibras de elastina en la valva atrioventricular izquierda de la rata

The microfibril-elastin fiber system, an important constituent of the extracellular matrix, was studied in the rat left atrioventricular valve to investigate the interrelationship of oxytalan, elaunin and elastic fibers in left atrioventricular valve morphology. The elastin fibers forms continuous bundles observed along the length of the valve in atrial and ventricular layers and oriented parallel to endothelium. The elaunin and oxytalan fibers are distributed in the thickest fiber bundles along the length of the valve. The thinner fibers which radiated towards both the atrial and spongiosa layers, either as isolated or arborescent fiber bundles were identified as oxytalan fibers. With transmission electron microscopy elastic fibers were seen mainly in the atrial layer. The spongiosa layer was composed of elaunin and oxytalan fibers and ventricular layer showed elaunin fibers arranged in continuous bundles parallel to the endothelium. Both fibrillin and elastin were seen and identified by immunocytochemistry with colloidal gold in the left atrioventricular valve spongiosa and atrial layers. These observations allow us to suggest that the microfibril-elastin fiber system plays a role in the mechanical protection and maintenance of the integrity of the rat left atrioventricular valve.

Fue estudiado el sistema de fibras microfibrillas-elastina, un componente importante de la matriz extracelular, en la valva atrioventricular izquierda de rata, con la finalidad de investigar la interrelación de oxitalán, elaunin y fibras elásticas en la morfología de dicha valva. Las fibras de elastina forman paquetes continuos a lo largo de la valva en las capas atriales y ventriculares, orientadas paralelamente al endotelio. Las fibras de elaunin y oxitalán se distribuyen en haces de fibras más gruesas a lo largo de la valva. Las fibras más delgadas, las cuales se irradiaban hacia las capas atrial y esponjosa, ya sea como haces de fibras aisladas o arborescentes, fueron identificadas como fibras oxitalán. En la capa atrial a través de microscopía electrónica de transmisión se observaron principalmente fibras elásticas. La capa esponjosa estaba compuesta por fibras de elaunin y oxitalán; la capa ventricular mostró fibras de elaunin dispuestas en haces continuos paralelos al endotelio. Tanto fibrilina y elastina se observaron e identificaron por inmunocitoquímica con oro coloidal en las capas esponjosa y atrial de la valva atrioventricular izquierda. Estas observaciones nos permiten sugerir que el sistema de fibras de elastina-microfibrillas tienen participación en la protección mecánica y la mantención de la integridad de la valva atrioventricular izquierda en la rata.

Williams Syndrome: development of a new scoring system for clinical diagnosis

OBJECTIVE: To develop a scoring system based on clinical findings to assist pediatricians in the diagnosis of William syndrome and to delineate when the fluorescent in-situ hybridization test to detect the microdeletion at 7q11.23 may be needed. METHODS: The fluorescent in-situ hybridization test was performed on 20 patients presenting William syndrome suggestive clinical features. Eleven studies were selected from the literature in which there were 2 groups: patients with positive or negative fluorescent in-situ hybridization tests. Forty-two clinical characteristics were compared to those reported in the literature to determine which ones were associated with the affected patients (ie, bearing deletions) using meta-analysis. The 2-tailed Fisher exact test were used so that the frequency of findings observed in fluorescent in-situ hybridization positive and fluorescent in-situ hybridization negative patients could be compared in the present study together with the patients from the literature. We developed a scoring system based on clinical findings and their significant associations with patients with positive fluorescent in-situ hybridization tests. From themean and standard-deviation values of the data from our patients, we determined the cut-off score that that indicated the need for a fluorescent in-situ hybridization test to confirm diagnosis. RESULTS: Seventeen patients were fluorescent in-situ hybridization positive, and 3 were fluorescent in-situ hybridization negative. The more discriminative findings among fluorescent in-situ hybridization positive patients were the following: typical facies, low birth weight, feeding difficulties, constipation, supravalvar aortic stenosis, mental retardation, and friendly personality. The distribution of the points among the 20 patients ranged from 19 to 28 points with a mean value of 23.3 out of a possible total of 31 points. The cut-off score that indicated the need for a fluorescent in-situ...

OBJETIVOS: Desenvolver um sistema de pontuação (Score) baseado nos achados clínicos para auxiliar os pediatras no diagnóstico clínico da Síndrome de Williams-Beuren e na indicação do teste de hibridização in situ por fluorescência para detectar a microdeleção em 7q11.23. MÉTODOS: O teste de hibridização in situ por fluorescência foi feito em 20 acometidos pela Síndrome de Williams-Beuren, nos quais 42 achados clínicos foram estudados. Para estabelecer quais desses achados estariam associados ao teste de hibridização in situ por fluorescência positivo, realizou-se uma metanálise com 11 trabalhos da literatura em que havia dois grupos, hibridização in situ por fluorescência positivo e negativo. As freqüências dos achados presentes nos indivíduos fluorescência positivo e fluorescência negativo neste estudo foram comparadas em conjunto com os pacientes da literatura através do teste exato de Fisher. Elaboramos um sistema de pontuação (score) baseado nos achados que mostraram correlação significante (p<0,001) para os pacientes hibridização in situ por fluorescência positivo. Determinamos os valores correspondentes aos percentis baseados na média e desvio-padrão, calculados a partir dos 20 pacientes do presente trabalho. RESULTADOS: Dezessete pacientes foram hibridização in situ por fluorescência positivo e três, negativo. Os achados mais discriminativos nos hibridização in situ por fluorescência positivo (3 pontos no score) foram: fáceis típico, baixo peso ao nascimento, dificuldades alimentares, obstipação, estenose aórtica supravalvar, deficiência mental e personalidade amigável.A distribuição dos valores entre os 20 pacientes variou de 19 a 28 pontos com uma média de 23,3 pontos. CONCLUSÕES: O score elaborado permitiu propor o valor de 20 pontos para a indicação do teste de hibridização in situ por fluorescência nos pacientes com suspeita clínica de Síndrome de Williams-Beuren.

Elastin ( ELN ) gene point mutation in patients with inguinal hernia

Groin hernias emerge at the myopectineal orifice of Fruchaud which is closed off by the fascia transversalis. Our previous studies showed structural and quantitative changes of the fascia transversalis elastic fibers of inguinal hernia patients and elderly people. The present study used single-strand conformation polymorphism (SSCP) elastin (analysis to investigate the 34 exons of the ELN gene of 49 inguinal hernia patients (7 females, 42 males aged 58.7 ± 19.82 years) and 75 non-herniated controls (35 females, 40 males aged 46.2 ± 14.32 years). We found that 47 patients and 24 controls had an abnormal exon 20 pattern caused by a g28197A > G missense mutation leading to an S422G amino acid substitution in the elastin hydrophobic domain. The g28197A > G allele frequency was 0.71 ± 0.045 in hernia patients and 0.21 ± 0.030 in controls and 23 patients and 7 controls were g28197A > G homozygous and 24 patients and 17 controls were heterozygous. This point-mutation showed a statistically significant association with inguinal hernia, chi-squared being 46.89 (p < 0.001) and the odds ratio 49.93 (95 percent confidence interval of @11 to 223). These results indicate that the g28197A > G mutation is involved in the genesis of inguinal hernia (possibly due to abnormal elastic fiber production) and explains impaired fascia transversalis function.

Polimorfismo do exon 20 do gene da elastina em indivíduos portadores de doença diverticular dos cólons / Polymorphism of 20 of elastin gene in individuals with colonic diverticular disease

A doença diverticular dos cólons (DDC) é relacionada à dieta, pressão intraluminal elevada, bem como alterações estruturais da parede intestinal. Pacientes com alterações genéticas do gene da elastina (ELN), como a estenose aórtica supravalvar e a cútis laxa, podem manifestar hérnia, diverticulose e disfunção urinária. Recentemente, uma mutação pontual no exon 20 do ELN foi demonstrada em pacientes com hérnia inguinal. No presente estudo é demonstrada uma mutação pontual (AGTGGT) no códon 422 do exon 20 do ELN em 5/14 pacientes com DDC e em 0/26 controles. Foi observada uma associação significativa desta mutação com o desenvolvimento da DDC. /Colonic diverticular disease (CDD) is related to diet, increased intraluminal pressure and structural changes within intestinal wall. Patients carrying genetic disorders of elastin (ELN) gene, such as supravalvular aortic stenosis and cutis laxa, may present hernias, diverticulosis and bladder dysfunction. Recently, a punctual mutation in exon 20 of ELN gene was detected in patients with inguinal hernia. Present study demonstrates a punctual mutation (AGTGGT) within codon 422 of ELN gene in 5/14 patients carrying CDD and in 0/26 among controls. This investigation demonstrated a significant association between the mutation found and CDD development...

Mutação no exon 20 do gene da elastina em mulheres com incontinência urinária de esforço decorrente de fatores extrínsecos à uretra / Mutation in exon 20 elastin women with extra urethral stress urinary incontinence

A incontinência urinária de esforço (IUE) é uma condição de alta prevalência em mulheres, representando um problema de saúde pública. Indícios de que desordens difusas do tecido conectivo, talvez de origem genética, possam estar implicadas na gênese da IUE têm sido descritos, como maior prevalência dessa afecção em mulheres com síndromes genéticas / Stress urinary incontinence (SUI) is a high prevalent condition in women and represents a public helth problem. It has been described signs that diffuse disorders of connective tissue, perhaps by genetic causes, may be involved in SUI genesis, as increased prevalence in women with others affections, such as varicose veins and hernia...

Alterações moleculares do gene da elastina nos pacientes portadores de hérnia inguinal / Molecular alterations of elastin gene in inguinal hernia patients

O objetivo do presente estudo foi verificar a ocorrência de mutações do gene da elastina nos pacientes portadores de hérnia inguinal. Estudou-se o DNA genômico de 19 pacientes com hérnia inguinal e 16 controles. Os 34 exons do gene foram amplificados e foi realizada a análise conformacional de fita simples. Observou-se bandas anormais no exon 20. O seqüenciamento do exon 20 revelou uma dupla substituição de bases no códon 404 em 78 por cento e 50 por cento dos pacientes idosos e adultos com hérnia inguinal respectivamente e a substituição de uma base no códon 422 em todos os pacientes com hérnia inguinal, em todos os indivíduos controles idosos e em 37,5 por cento dos indivíduos controles adultos. É possível que estas mutações possam estar relacionadas à produção de moléculas de elastina alteradas e desempenhem papel na gênese da hérnia inguinal.The present study was designed to verify the presence of elastin gene mutations from genomic DNA in patients with inguinal hernia. Nineteen adult patients with diagnosed inguinal hernia and 16 controls were analyzed. All 34 exons were amplified and mutations were sought by single strand conformation polymorphism. DNA amplification revealed an abnormal band in exon 20. Sequencing showed a double base substitution in codon 404 in 78 per cent of the elderly hernia patients and in 50 per cent of the younger hernia group. It also revealed a single base substitution in codon 422 in all inguinal hernia patients, in all the elderly controls as well as in 37,5 per cent of the younger controls. It is possible that these elastin mutations may be responsible for the production of abnormal elastic fibers, thus playing a role in inguinal hernia genesis...

Uso da análise de Fish para diagnóstico em 11 pacientes com síndrome de Williams-Beuren / Use of Fish analysis to diagnosis approach of 11 Brazilian patients with Williams-Beuren syndrome

A síndrome de Williams-Beuren (SWB) é uma rara síndrome de deleçäo de genes contíguos que feta múltiplos sistemas. A doença é causada por uma deleçäo submicroscópica na regiäo 7q11.23, que é detectada pela análise de Fish em 90-95 por cento dos pacientes. Relatamos o estudo clínico e a análise citogenética por Fish realizados em 11 pacientes (6M:5F), todos esporádicos. Fácies típicos de "duende", atraso de desenvolvimento/retardo mental, anomalias dentárias e esqueléticas estavam presentes em todos os pacientes; anomalias oculares em dez (91 por cento); cardiopatias congênitas em oito (73 por cento), sendo metade, estenose supravalvar aórtica; baixa estatura em seis (54 por cento); hipercalcemia transitória em quatro (36 por cento); hérnia inguinal em três (27 por cento) e anomalias renais em dois (18 por cento). Analisanmos a hemizigosidade do gene da elastina por Fish, utilizando a sonda LSI Williams Syndrome Region DNA Fish (Vysis) nas células interfásicas e metafásicas. O paciente sem deleçäo apreentava fácies e comportamento tópico, exceto cardiopatia. A análise pelo Fish do locus da elastina é um teste útil para confirmaçäo diagnóstica da SWB, o que auxilia no seguimento clínico adequado e na prevençäo das complicaçöes

Williams syndrome and the elastin gene in Thai patients.

Williams syndrome (WS) has long been known as a complex disorder of dysmorphic facial features, described as elfin face, mental retardation or learning disability, loquacious personality, and supravalvular aortic stenosis. The etiology is now known to be due to deletion of the elastin gene (ELN) on long arm of chromosome 7. Thai patients were previously reported by clinical diagnosis. This study reports the first two cases of WS with ELN deletion diagnosed by fluorescent in situ hybridization (FISH) technique. Clinically, hyperacusis is a common finding in WS associated with otitis media. Neither of the patients had hyperacusis, but one of them had bilateral sensorineural hearing loss, which to our knowledge, has never been reported.