Sulfated glycosaminoglycans in human vocal fold lamina propria / Glicosaminoglicanos sulfatados na lâmina própria de prega vocal humana

Abstract Introduction: The distribution, concentration and function of glycosaminoglycans in the various vocal fold tissues are still unclear. Objective: To evaluate the distribution and concentration of sulfated glycosaminoglycans in different layers of the human vocal fold according to gender and age. Methods: We used 11 vocal folds obtained from cadavers (7 men and 4 women) with no laryngeal lesion, less than 12 h after death, and aged between 35 and 98 years. The folds underwent glycosaminoglycans extraction from the cover and ligament, and post-electrophoresis analysis. Data were compared according to the layer, age and gender. Results: The concentration of dermatan sulfate was significantly higher in all layers. No differences were observed in the total concentrations of glycosaminoglycans in layers studied according to gender. It is significantly lower in the cover of individuals aged below 60 years. Conclusion: Dermatan sulfate, chondroitin sulfate, and heparan sulfate were observed in the human vocal folds cover and ligament of both genders, with the concentration of dermatan sulfate being significantly higher in all layers. Glycosaminoglycans concentration on the cover is significantly lower in individuals below 60 years compared with elderly.

Resumo Introdução: A distribuição, concentração e função dos glicosaminoglicanos nos diversos tecidos da prega vocal ainda não está esclarecida. Objetivo: Avaliar a distribuição e concentração dos glicosaminoglicanos sulfatados nas diferentes camadas da prega vocal humana de acordo com o sexo e a idade. Método: Foram usadas 11 pregas vocais obtidas de cadáveres (sete homens e quatro mulheres) sem lesão de laringe, com menos de 12 horas de óbito e entre 35 e 98 anos. As pregas foram submetidas à extração de glicosaminoglicanos da cobertura e ligamento e leitura pós-eletroforese. Os dados foram comparados segundo camada, idade e sexo. Resultados: A concentração de dermatan sulfato foi significativamente maior em todas as camadas. Não foram observadas diferenças nas concentrações totais de glicosaminoglicanos nas camadas estudadas quanto ao gênero. É significantemente menor em indivíduos abaixo de 60 anos na cobertura. Conclusão: Dermatam sulfato, condroitim sulfato e heparam sulfato foram observados na cobertura e no ligamento de pregas vocais humanas, de ambos os sexos, sendo a concentração de dermatam sulfato foi significativamente maior em todas as camadas. A concentração de glicosaminoglicanos na cobertura é significativamente menor em indivíduos abaixo de 60 anos em comparação com idosos.

Expression of heparanase in basal cell carcinoma and squamous cell carcinoma

Abstract: Background: Heparanase is an enzyme that cleaves heparan sulfate chains. Oligosaccharides generated by heparanase induce tumor progression. Basal cell carcinoma and squamous cell carcinoma comprise types of nonmelanoma skin cancer. Objectives: Evaluate the glycosaminoglycans profile and expression of heparanase in two human cell lines established in culture, immortalized skin keratinocyte (HaCaT) and squamous cell carcinoma (A431) and also investigate the expression of heparanase in basal cell carcinoma, squamous cell carcinoma and eyelid skin of individuals not affected by the disease (control). Methods: Glycosaminoglycans were quantified by electrophoresis and indirect ELISA method. The heparanase expression was analyzed by quantitative RT-PCR (qRTPCR). Results: The A431 strain showed significant increase in the sulfated glycosaminoglycans, increased heparanase expression and decreased hyaluronic acid, comparing to the HaCaT lineage. The mRNA expression of heparanase was significantly higher in Basal cell carcinoma and squamous cell carcinoma compared with control skin samples. It was also observed increased heparanase expression in squamous cell carcinoma compared to the Basal cell carcinoma. Conclusion: The glycosaminoglycans profile, as well as heparanase expression are different between HaCaT and A431 cell lines. The increased expression of heparanase in Basal cell carcinoma and squamous cell carcinoma suggests that this enzyme could be a marker for the diagnosis of such types of non-melanoma cancers, and may be useful as a target molecule for future alternative treatment.

Glycomics expression analysis of sulfated glycosaminoglycans of human colorectal cancer tissues and non-neoplastic mucosa by electrospray ionization mass spectrometry / Análise da expressão de glicosaminoglicanos sulfatados no tecido humano neoplásico colorretal e na mucosa não neoplásica por espectrometria de massa por ionização por electrospray

ABSTRACT Objective To determine the presence of glycosaminoglycans in the extracellular matrix of connective tissue from neoplastic and non-neoplastic colorectal tissues, since it has a central role in tumor development and progression. Methods Tissue samples from neoplastic and non-neoplastic colorectal tissues were obtained from 64 operated patients who had colorectal carcinoma with no distant metastases. Expressions of heparan sulphate, chondroitin sulphate, dermatan sulphate and their fragments were analyzed by electrospray ionization mass spectrometry, with the technique for extraction and quantification of glycosaminoglycans after proteolysis and electrophoresis. The statistical analysis included mean, standard deviation, and Student’st test. Results The glycosaminoglycans extracted from colorectal tissue showed three electrophoretic bands in agarose gel. Electrospray ionization mass spectrometry showed characteristic disaccharide fragments from glycosaminoglycans, indicating their structural characterization in the tissues analyzed. Some peaks in the electrospray ionization mass spectrometry were not characterized as fragments of sugars, indicating the presence of fragments of the protein structure of proteoglycans generated during the glycosaminoglycan purification. The average amount of chondroitin and dermatan increased in the neoplastic tissue compared to normal tissue (p=0.01). On the other hand, the average amount of heparan decreased in the neoplastic tissue compared to normal tissue (p= 0.03). Conclusion The method allowed the determination of the glycosaminoglycans structural profile in colorectal tissue from neoplastic and non-neoplastic colorectal tissue. Neoplastic tissues showed greater amounts of chondroitin sulphate and dermatan sulphate compared to non-neoplastic tissues, while heparan sulphate was decreased in neoplastic tissues.

RESUMO Objetivo Determinar a presença de glicosaminoglicanos na matriz extracelular do tecido conjuntivo colorretal neoplásico e não neoplásico, tendo em vista seu papel central no desenvolvimento e na progressão dos tumores. Métodos Amostras de tecidos colorretais neoplásicos e não neoplásicos foram obtidas de 64 pacientes operados com carcinoma colorretal sem metástases a distância. As expressões de heparan sulfato, sulfato de condroitina e sulfato de dermatan e seus fragmentos foram analisadas por espectrometria de massa por ionização por electrospray, com técnica de extração e quantificação de glicosaminoglicanos após proteólise e eletroforese. Para análise estatística, utilizaram-se média, desvio padrão e teste t de Student. Resultados Em gel de agarose, os glicosaminoglicanos extraídos de tecido colorretal mostraram três bandas eletroforéticas. A espectrometria de massa por ionização por electrospray mostrou fragmentos de dissacarídeos característicos de glicosaminoglicanos e indicou sua característica estrutural. Alguns picos na espectrometria de massa por ionização por electrospray não foram caracterizados como fragmentos de açúcares, sugerindo a presença de fragmentos de proteínas estruturais dos proteoglicanos, formadas durante a purificação dos glicosaminoglicanos. A quantidade média de condroitina e dermatan aumentou no tecido neoplástico em relação ao tecido normal (p=0,01). Por outro lado, a quantidade média de heparan foi menor no tecido neoplásico em relação ao tecido normal (p=0,03). Conclusão O método empregado permitiu determinar o perfil estrutural dos glicosaminoglicanos nas amostras. Tecidos neoplásicos apresentaram maiores quantidades de sulfato de condroitina e sulfato de dermatan em comparação com os não neoplásicos, enquanto o sulfato de heparan foi encontrado em menores quantidades nos tecidos neoplásicos.

Cotransfected human chondrocytes: over-expression of IGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans

Damage to cartilage causes a loss of type II collagen (Col-II) and glycosaminoglycans (GAG). To restore the original cartilage architecture, cell factors that stimulate Col-II and GAG production are needed. Insulin-like growth factor I (IGF-I) and transcription factor SOX9are essential for the synthesis of cartilage matrix, chondrocyte proliferation, and phenotype maintenance. We evaluated the combined effect of IGF-I and SOX9 transgene expression on Col-II and GAG production by cultured human articular chondrocytes. Transient transfection and cotransfection were performed using two mammalian expression plasmids (pCMV-SPORT6), one for each transgene. At day 9 post-transfection, the chondrocytes that were over-expressing IGF-I/SOX9 showed 2-fold increased mRNA expression of the Col-II gene, as well as a 57% increase in Col-II protein, whereas type I collagen expression (Col-I) was decreased by 59.3% compared with controls. The production of GAG by these cells increased significantly compared with the controls at day 9 (3.3- vs 1.8-times, an increase of almost 83%). Thus, IGF-I/SOX9 cotransfected chondrocytes may be useful for cell-based articular cartilage therapies.

Increase of glyoosaminoglycans and metalloprotenases 2 and 9 in liver extracelllular matrix on early stages of extrahepatic cholestaiis / Aumento de glicosaminoglicanos e metaloproteinases 2 e 9 na matriz extracelular de fígado em estágios iniciais de colestase extra-hepática

Context Cholestasis produces hepatocellular injury, leukocyte infiltration, ductular cells proliferation and fibrosis of liver parenchyma by extracellular matrix replacement. Objective Analyze bile duct ligation effect upon glycosaminoglycans content and matrix metalloproteinase (MMPs) activities. Methods Animals (6-8 weeks; n = 40) were euthanized 2, 7 or 14 days after bile duct ligation or Sham-surgery. Disease evolution was analyzed by body and liver weight, seric direct bilirubin, globulins, gamma glutamyl transpeptidase (GGT), alkaline phosphatase (Alk-P), alanine and aspartate aminotransferases (ALT and AST), tissue myeloperoxidase and MMP-9, pro MMP-2 and MMP-2 activities, histopathology and glycosaminoglycans content. Results Cholestasis caused cellular damage with elevation of globulins, GGT, Alk-P, ALT, AST. There was neutrophil infiltration observed by the increasing of myeloperoxidase activity on 7 (P = 0.0064) and 14 (P = 0.0002) groups which leads to the magnification of tissue injuries. Bile duct ligation increased pro-MMP-2 (P = 0.0667), MMP-2 (P = 0.0003) and MMP-9 (P<0.0001) activities on 14 days indicating matrix remodeling and establishment of inflammatory process. Bile duct ligation animals showed an increasing on dermatan sulfate and/or heparan sulfate content reflecting extracellular matrix production and growing mitosis due to parenchyma depletion. Conclusions Cholestasis led to many changes on rats’ liver parenchyma, as so as on its extracellular matrix, with major alterations on MMPs activities and glycosaminoglycans content. .

Contexto Colestase produz lesão hepatocelular, infiltração leucocitária, proliferação de células ductulares e fibrose do parênquima hepático por matriz extracelular. Objetivo Analisar os efeitos da ligação do ducto biliar sobre conteúdo de glicosaminoglicanos e atividade de metaloproteinases de matriz (MMP). Métodos Animais (6-8 semanas; n = 40) foram eutanasiados 2, 7 ou 14 dias após ligação do ducto biliar ou falsa ligação. A evolução da doença foi analisada por peso corporal e do fígado, concentrações séricas de bilirrubina direta, globulinas, gama glutamil transpeptidase (GGT), fosfatase alcalina (Alk-P), alanina e aspartato aminotransfesases (ALT e AST), alterações teciduais de mieloperoxidase e metaloproteinases (MMP-9, pro MMP-2 e MMP-2), histopatologia e conteúdo de glicosaminoglicanos. Resultados A colestase causou dano celular com elevação dos níveis séricos de globulinas, GGT, Alk-P, ALT e AST. Houve também infiltração leucocitária observada pelo aumento na atividade de mieloperoxidase nos grupos 7 (P = 0,0064) e 14 dias (P = 0,0002) o que leva ao aumento das lesões no tecido. Ligação do ducto biliar aumentou as atividades de pro MMP-2 (P = 0,0677), MMP-2 (P = 0,0003) e MMP-9 (P<0,0001) aos 14 dias indicando remodelamento da matriz e estabelecimento de processo inflamatório. Animais com ligação do ducto biliar mostraram um aumento do conteúdo de dermatam sulfato e/ou heparam sulfato refletindo a produção de matriz extracelular e aumento de mitose devido a depleção do parênquima hepático. Conclusões Colestase causou várias mudanças no parênquima hepático de ratos, bem como em sua matriz extracelular, com importantes alterações na atividade ...

Effect of JJYMD-C, a novel synthetic derivative of gallic acid, on proliferation and phenotype maintenance in rabbit articular chondrocytes in vitro

Tissue engineering encapsulated cells such as chondrocytes in the carrier matrix have been widely used to repair cartilage defects. However, chondrocyte phenotype is easily lost when chondrocytes are expanded in vitro by a process defined as “dedifferentiation”. To ensure successful therapy, an effective pro-chondrogenic agent is necessary to overcome the obstacle of limited cell numbers in the restoration process, and dedifferentiation is a prerequisite. Gallic acid (GA) has been used in the treatment of arthritis, but its biocompatibility is inferior to that of other compounds. In this study, we modified GA by incorporating sulfamonomethoxine sodium and synthesized a sulfonamido-based gallate, JJYMD-C, and evaluated its effect on chondrocyte metabolism. Our results showed that JJYMD-C could effectively increase the levels of the collagen II, Sox9, and aggrecan genes, promote chondrocyte growth, and enhance secretion and synthesis of cartilage extracellular matrix. On the other hand, expression of the collagen I gene was effectively down-regulated, demonstrating inhibition of chondrocyte dedifferentiation by JJYMD-C. Hypertrophy, as a characteristic of chondrocyte ossification, was undetectable in the JJYMD-C groups. We used JJYMD-C at doses of 0.125, 0.25, and 0.5 µg/mL, and the strongest response was observed with 0.25 µg/mL. This study provides a basis for further studies on a novel agent in the treatment of articular cartilage defects.

Evaluation of the metabolism of glycosaminoglycans in patients with interstitial cystis

Introduction: Painful bladder syndrome/interstitial cystitis (PBS/IC) pathogenesis is not fully known, but evidence shows that glycosaminoglycans (GAG) of bladder urothelium can participate in its genesis. The loss of these compounds facilitates the contact of urine compounds with deeper portions of bladder wall triggering an inflammatory process. We investigated GAG in urine and tissue of PBS/IC and pure stress urinary incontinence (SUI) patients to better understand its metabolism. Materials and Methods: Tissue and urine of 11 patients with PBS/IC according to NIDDK criteria were compared to 11 SUI patients. Tissue samples were analyzed by histological, immunohistochemistry and immunofluorescence methods. Statistical analysis were performed using t Student test and Anova, considering significant when p < 0.05. Results: PBS/IC patients had lower concentration of GAG in urine when compared to SUI (respectively 0.45 ± 0.11 x 0.62 ± 0.13 mg/mg creatinine, p < 0.05). However, there was no reduction of the content of GAG in the urothelium of both groups. Immunofluorescence showed that PBS/IC patients had a stronger staining of TGF-beta, decorin (a proteoglycan of chondroitin/dermatan sulfate), fibronectin and hyaluronic acid. Conclusion: the results suggest that GAG may be related to the ongoing process of inflammation and remodeling of the dysfunctional urothelium that is present in the PBS/IC. .

Análise da composição do matriz extracelular das camadas urotelial e muscular da bexiga de mulheres em diferentes idades / Analysis of the extracellular matriz of urothelial and muscle layers of the bladder in women at different ages

Acomplacência da bexiga depende de músculos lisos, fibras colágenas, fibras elásticas e suas relações. O objetivo deste trabalho é determinar a composição da matriz extracelular em amostras de bexigas normais através de análise bioquímica de colágeno e glicosaminoglicanos em amostras obtidas de mulheres em diferentes grupos de idade, analisando separadamente as camadas urotelial e muscular. Avaliamos 17 amostras de bexiga divididas em três grupos: infância (N=5), menacme (N=6) e pós-menopausa (N=6). As bexigas foram analisadas para concentração de GAG total e colágeno e para análise qualitativa de GAG por eletroforese em gel de agarose. Na camada muscular, não houve diferença entre os grupos tanto para GAG quanto para colágeno. Na camada urotelial, a análise da concentração de colágeno não mostrou diferença entre os grupos, mas a concentração de GAG no grupo da pós-menopausa (0.21 +- 0.12 ug de ácido hexurônico/mg de tecido seco) apresentou diferença em relação aos grupos do menacme (1.78 +- 1.62 ug de ácido hexurônico/mg de tecido seco) e da infância (2.29 +- 1.32 ug de ácido hexurônico/mg de tecido seco). Nosso trabalho concluiu que a concentração de GAG está substancialmente diminuída na cadama urotelial da bexiga de mulheres na pós-menopausa.

Bladder compliance is dependent on smooth muscle, collagen fibers, elastic fiber and their ratios. The luminal surface of the urothelium is covered by an adhering glycosaminoglycan (GAG) layer. The aim of this study was to determine the composition of the extracellular matrix (ECM) in normal samples of women bladders through biochemistry analysis of collagen and GAG on samples obtained from individuals from different age groups, analyzing separately the urothelial and muscular layers. We studied samples taken from bladders of 17 patients divided in three different groups: childhood (N=5), menacme (N=6) and menopause (N=6). Bladders were analyzed for total GAG and collagen concentration per mg dry tissue and for the contents of GAG species, as determined by agarose electrophoresis and reported as the percent of total sulfated GAG. In muscular layer, collagen and GAG concentration showed no difference between groups. In urothelial layer, collagen concentration showed no difference between groups but GAG concentration in menopause (0.21 +- 0.12 ug hexuronic acid/mg dry tissue) was different from menacme (1.78 +- 1.62 ug hexuronic acid/mg dry tissue) and childhood (2.29 +- 1.32 ug hexuronic acid/mg dry tissue). There was no difference between sulfated GAG in three groups. In conclusion, GAG concentration in urothelial layer was substantially lower in menopause women.

Composição da matriz extracelular do ligamento cardinal de mulheres na pós-menopausa com e sem prolapso uterino / Composition of extracellular matrix of the cardinal ligament in postmenopausal women with and without uterine prolapse

O prolapso uterino tem sua incidência aumentada na pós-menopausa. O objetivo deste estudo é identificar as alterações na matriz extracelular do ligamento cardinal associadas à menopausa e ao prolapso uterino. Ligamento cardinal de três diferentes grupos de mulheres, pré-menopausa, prolapso uterino e pós-menopausa, foram identificados e biopsiados durante 57 histerectomias abdominais ou vaginais. As amostras foram processadas por métodos bioquímicos para caracterização e quantificação de glicosaminoglicanos sulfatados e colágeno. As concentrações relativas de glicosaminoglicanos foram obtidas por eletroforese. Procedimentos histológicos foram feitos para identificar fibras elásticas (Weigert), distribuição de colágeno (Picro Sirius) e decorin (imunohistoquímica). Nossos resultados mostraram aumento na concentração de GAG de 72,2%, redução na concentração de colágeno de 37% e diminuição de 22% de fibras elásticas no grupo de prolapso uterino quando comparado ao grupo da pós-menopausa (p<0,05, p<0,04 e p<0,05 respectivamente). As concentrações relativas de glicosaminoglicanos sulfatados para condroitin sulfato, heparan sulfato e dermatan sulfato não mostraram diferenças entre os três grupos. A organização do colágeno foi similar entre os três grupos e a marcação do decorin pareceu estar diminuída no grupo de prolapso uterino. Nossos resultados indicam alterações no metabolismo do tecido conjuntivo. O ligamento cardinal da mulher na pós-menopausa possui uma matriz extracelular mais densa. Esta alteração não ocorre na mulher com prolapso uterino.

Uterine prolapse has increase of incidence after menopause. The aim of this study was to identify the changes in extracellular matrix of cardinal ligaments associated to menopause and uterine prolapse. Cardinal ligament of 3 different groups (pre-menopause, menopause and uterine prolapse) are identified and biopsied during 57 women's abdominal or vaginal hysterectomy. Biopsy specimens were assessed by biochemical methods to characterize and quantify sulfated glycosaminoglycans and collagen. Relative concentrations of GAG were obtained by electrophoresis. Histological procedures are made to identify elastic fibers (Weigert) collagen distribution (Picro sirius) and decorin (immunohistochemistry). Our results showed increase in GAG concentration 72.2% in uterine prolapse group compared to menopause group (p<0.05). Collagen concentration was 37% lower in uterine prolapse group compared to menopause group (p<0.04). Relative concentration of GAG: heparan sulfate, chondroitin sulfate and dermatan sulfate showed no differences among three groups. Elastic fibers showed a significant reduction of aproximately 22% uterine prolapse group compared to menopause group (p<0.05). Collagen organization was similar in three groups and the staining pattern of decorin seemed to be decreased in uterine prolapse group. Our results indicate changes in connective tissue metabolism. Cardinal ligament in postmenopausal women has a denser extracellular matrix. This change is not observed in women with uterine prolapse.

Changes in Glycogen and Glycosaminoglycan Levels in Hepatocytes of Iduronate-2-Sulfatase Knockout Mice before and after Recombinant Iduronate-2-Sulfatase Supplementation

PURPOSE: Mucopolysaccharidosis II (MPS II) is a lysosomal storage disorder caused by a deficiency of iduronate-2 sulfatase (IdS), which is involved in the degradation of glycosaminoglycan (GAG). In this study, the frequency of fasting hypoglycemia in patients with MPS II was investigated and changes in accumulation of glycogen and GAG in the hepatocytes of IdS-knockout (KO) mice were evaluated before and after recombinant IdS enzyme replacement therapy (ERT). MATERIALS AND METHODS: Plasma glucose levels were evaluated after an 8-hour fast in 50 patients with MPS II. The IdS-KO mice were divided into three groups (group 2; saline, group 3; 0.15 mg/kg of IdS, and group 4; 0.5 mg/kg of IdS); wild-type mice were included as controls (group 1). ERT was initiated intravenously at four weeks of age, and continued every week until 20 weeks of age. RESULTS: The mean glucose level after an 8-hour fast was 94.1 +/- 23.7 mg/dL in the patients with MPS II. Two (4%) out of 50 patients had fasting hypoglycemia. For the mice, GAG in the lysosomes nearly disappeared and glycogen particles in the cytoplasm were restored to the normal range in group 4. CONCLUSION: Glucose metabolism in patients with MPS II appeared to function well despite hepatocytic GAG accumulation and hypothetical glycogen depletion. A higher dose of IdS infusion in MPS II mice led to disappearance of lysosomal GAG and restoration of glycogen to the cytoplasm of hepatocytes.

Biochemical evaluation in human saliva with special reference to ovulation detection.

Aim : The aim of the present study was to investigate the level of salivary sialic acids and glycosaminoglycans with reference to salivary hormones during the normal menstrual cycle. Settings and Design: Fifty women volunteers were selected for the present study. Materials and Methods : Saliva was collected from 50 women and ovulation was detected in women with normal menstrual cycles through basal body temperature (BBT), ultrasound and salivary ferning. Samples were divided into five categories, as prepubertal (6-9 years), pre-ovulatory phase (6-12 days), ovulatory phase (13-14 days), postovulatory phase (15-26 days) and menopause (above 45 years). Each sample was subjected to evaluation of the sialic acids and glycosaminoglycans along with salivary hormones. Results : The result revealed that the ovulatory phase has increased sialic acid and glycosaminoglycans during the menstrual cycle when compared with that of the other phases. Consequently, an increased level of hormones such as luteinizing hormone and estrogen during the ovulatory period when compared to that of the pre-ovulatory and postovulatory periods appeared to be noteworthy. Statistically, analysis was performed using one way-ANOVA (LSD; post hoc method) to determine the significance as P < 0.001, 0.01, 0.05 in between the reproductive phases of the menstrual cycle. Conclusion : This study concluded that saliva-specific carbohydrates in the ovulatory saliva make the possibility to develop a biomarker for detection of ovulation by non-invasive methods.

Glycosaminoglycan distribution in the rat uterine cervix during the estrous cycle

OBJECTIVE: To analyze the amount of glycosaminoglycans in the uterine cervix during each phase of the rat estrous cycle. DESIGN: Based on vaginal smears, forty female, regularly cycling rats were divided into four groups (n = 10 for each group): GI - proestrous, GII - estrous, GIII - metaestrous and GIV - diestrous. Animals were sacrificed at each phase of the cycle, and the cervix was immediately removed and submitted to biochemical extraction and determination of sulfated glycosaminoglycans and hyaluronic acid. The results were analyzed by ANOVA followed by the Bonferroni post-hoc test. RESULTS: The uterine cervix had the highest amount of total sulfated glycosaminoglycans and dermatan sulfate during the estrous phase (8.90 ± 0.55 mg/g of cetonic extract, p<0.001; and 8.86 ± 0.57 mg/g of cetonic extract, p<0.001). In addition, there was more heparan sulfate at the cervix during the proestrous phase (0.185 ± 0.03 mg/g of cetonic extract) than during any other phase (p<0.001). There were no significant changes in the concentration of hyaluronic acid in the uterine cervix during the estrous cycle. CONCLUSION: Our data suggest that the amount of total sulfated glycosaminoglycans may be influenced by hormonal fluctuations related to the estrous cycle, with dermatan sulfate and heparan sulfate being the glycosaminoglycans most sensitive to hormonal change.

Avaliação dos glicosaminoglicanos do tecido periuretral de pacientes com e sem prolapso genital / Evaluation of glycosaminoglycans of periurethral tissue in patients with and without pelvic organ prolapse

OBJETIVOS: Caracterizar e quantificar os subtipos de glicosaminoglicanos sulfatados (GAGs) existentes no tecido peri-uretral de pacientes com e sem prolapso genital. METODOS: Foram incluídas 35 pacientes que se submeteram a cirurgia vaginal para correção de distopias genitais e/ou incontinência urinária de esforço ou por outra condição benigna. As pacientes foram avaliadas por anamnese padronizada, exame físico e urodinâmico e agrupadas segundo a existência do prolapso genital. Durante o procedimento cirúrgico, amostras de aproximadamente 1,0 x 1,0 cm do tecido periuretral foram retiradas para avaliação. Os GAGs foram extraídos do tecido por proteólise e precipitação por ácido tricloroacético e caracterizados por eletroforese em gel de agarose. A quantificação foi feita por meio de densitometria a 525 nm do gel corado com azul de toluidina. Compararam-se os dados pela análise de variância (ANOVA). RESULTADOS: Nos grupos estudados, houve maior predomínio de dermatam sulfato (DS), em torno de 85 por cento do total de GAGs, seguido do condroitim sulfato (CS) e do heparam sulfato (HS). Observou-se aumento significativo dos GAGs totais, do DS e do HS em mulheres com prolapso genital. Não se observou diferença significante com relação ao CS. CONCLUSÃO: Este estudo demonstrou diferenças na matriz extracelular do tecido periuretral com aumento de GAGs totais, DS e HS nas mulheres com prolapso genital.

OBJECTIVE: To characterize and quantify periurethral tissue sulphated glycosaminoglycans (GAGs) in women with and without pelvic organ prolapse. STUDY DESIGN: Periurethral tissue was obtained from 35 women who underwent surgery for pelvic organ prolapse, for stress urinary incontinence, or for other gynecological benign conditions. Patients were submitted to a clinical history, physical and urodynamic examination and were divided in two groups according to genital prolapse. The standard biopsy with 1.0 x 1.0 cm was taken from periurethral tissue during surgery and assessed by biochemical methods. The GAGs were obtained by proteolysis and precipitated by trichloroacetic acid. The relative concentration of sulfated GAGs was determined by densitometry of toluidine blue stained gel using a spectrophotometer with a 525 nm wavelength. Data were compared using analysis of variance (ANOVA). RESULTS: In the two groups dermatan sulphate (DS) was the predominant glycosaminoglycan (85 percent), followed by chondroitin sulphate (CS) and heparan sulphate (HS). Women with pelvic organ prolapse had significantly more total GAGs, DS and HS. Differences in CS were not observed. CONCLUSIONS: This study showed altered biochemical characteristics in the extracellular matrix of periurethral tissue and also accumulation of GAGs, DS and CS, in women with pelvic organ prolapse.

Perfil de glicosaminoglicanos sulfatados no útero de camundongas durante o ciclo estral / Profile of sulphated glycosaminoglycans content in the murine uterus during the different phases of the estrous cycle

OBJETIVOS: Quantificar glicosaminoglicanos sulfatados (GAGs) no útero de camundongas durante o ciclo estral. MÉTODOS: Utilizaram-se quatro grupos de camundongas virgens com 100 dias de idade (n= 10 cada) conforme a fase ciclo estral: proestro, estro, metaestro e diestro. Amostras da porção média dos cornos uterinos foram preparadas para observação em microscopia de luz (H/E e Alcian blue + PAS). Os GAGs foram extraídos e caracterizados por eletroforese em gel de agarose. Os dados foram analisados pelo teste t de Student não pareado. RESULTADOS: A microscopia de luz, os GAGs sulfatados apresentam-se em todas as camadas do útero, em especial no endométrio, entre as fibras colágenas, na membrana basal e ao redor dos fibroblastos. A análise bioquímica mostrou haver dermatam sulfato (DS), condroitim sulfato (CS) e heparam sulfato (HS) durante todas as fases do ciclo estral. Não houve separação eletroforética clara entre DS e CS, de modo que estes dois GAGs foram considerados em conjunto (DS+CS) (proestro = 0,854 ± 0,192; estro = 1,073 ± 0,254; metaestro = 1,003 ± 0,255; e diestro = 0,632 ± 0,443 μg/mg). Os resultados de HS foram: proestro = 0,092 ± 0,097; estro = 0,180 ± 0,141; metaestro = 0,091 ± 0,046; e diestro = 0,233 ± 0,147 μg/mg. A concentração DS+CS apresentou-se maior no estro (ação estrogênica) e a do HS no diestro (ação progestagênica). CONCLUSÃO: Os GAGs no útero de camundongas sofrem alterações durante as fases do ciclo estral, refletindo o constante processo de renovação, sendo modulados pelos hormônios sexuais.

OBJECTIVE: Identification and quantitation of sulphated glycosaminoglycans (GAGs) in the uterus of female mice during the estrous cycle. METHODS: Four groups (n = 10 each) of virgin, 100-day old female mice were assembled according to the estrous cycle phase: proestrus, estrus, metaestrus and diestrus. Samples of the median portion of uterine horns were processed for light microscopy examination (H/E and Alcian blue + PAS). The GAGs were extracted and characterized by agarose gel electrophoresis. Data were analyzed by the unpaired Student's t-test. RESULTS: At light microscopy GAGs appear in all layers of the uterus, especially in the endometrium, between collagen fibers, in the basal membrane and around fibroblasts. Biochemical analyses disclosed presence of dermatan sulphate (DS), chondroitin sulphate (CS and heparan sulphate (HS) during all estral cycle phases. There was no clear electrophoretic separation between DS and CS, thus these two GAGs were considered together (DS+CS) (proestrus = 0.854 ± 0.192; estrus = 1.073 ± 0.254; metaestrus = 1.003 ± 0.255; diestrus = 0.632 ± 0.443 μg/mg). HS was as follows: proestrus = 0.092 ± 0.097; estrus = 0.180 ± 0.141; metaestrus = 0.091 ± 0.046; diestrus = 0.233 ± 0.147 μg/mg. The uterine content of DS+CS peaked at estrus (estrogenic action) and that of HS at diestrus (progestagen action). CONCLUSION: Due to a constant turnover process, there are definite alterations in the uterine profile of GAGs content during the estrous cycle in mice, which may be modulated by female sex hormones.

Distribution of small proteoglycans and glycosaminoglycans in humerus-related articular cartilage of chickens

The expression of components present in the cartilaginous extracellular matrix is related to development, gender, and genotype, as well as to the biomechanical properties of each type of cartilage. In the present study, we analyzed small proteoglycans and glycosaminoglycans present in different cartilages of the chicken wing after extraction with guanidine hydrochloride or papain. Quantitative analysis of glycosaminoglycans showed a larger amount in humeral cartilage (around 200 mg/g tissue) than in articular cartilage of the radius and ulna, with 138 and 80 mg/g tissue, respectively. Non-collagenous proteins isolated were predominantly from cartilage in the proximal regions of the humerus and radius. D4 fractions obtained by ultracentrifugation were separated by DEAE-Sephacel and Octyl-Sepharose chromatography and analyzed by SDS-PAGE. Two bands of 57 and 70-90 kDa were observed for all samples treated with ß-mercaptoethanol. Immunoblotting of these proteins was positive for the small proteoglycans fibromodulin and decorin, respectively. Apparently, the 57-kDa protein is present in macromolecular complexes of 160 and 200 kDa. Chondroitin sulfate was detected in all regions. HPLC analysis of the products formed by chondroitinase AC and ABC digestion mainly revealed ß-D-glucuronic acid and N-acetyl ß-D-galactosamine residues. The 4-sulfation/6-sulfation ratio was close to 3, except for the proximal cartilage of the radius (2.5). These results suggest functional differences between the scapula-humerus, humerus-ulna, and humerus-radius joints of the chicken wing. This study contributes to the understanding of the physiology of cartilage and joints of birds under different types of mechanical stress.

Las mucopolisacaridosis / The mucopolysaccharidosis

Las mucopolisacaridosis (MPS) son errores innatos del metabolismo de los glicosaminoglicanos (GAG), que ocurren por déficit de alguna de las enzimas que degradan estas moléculas en los lisosomas. Esto conduce al depósito intralisosomal progresivo de GAG en diferentes tejidos, lo que explica el carácter multistémico de estas patologías. Las MPS, se presentan con una frecuencia aproximada de 1 caso en 10.000 a 25.000 recién nacidos vivos y son de herencia autosómica recesiva, salvo la MPS II o enfermedad de Hunter, que se hereda ligada al cromosoma X. Las características clínicas más frecuentes de las MPS son la presencia de rasgos faciales toscos, macrocefalia, opacidades corneales, disostosis múltiple, talla baja, valvulopatía mitroaórtica, hepatoesplenomegalia, hernias umbilical e inguinales, con o sin retraso del desarrollo sicomotor y con un deterioro neurológico progresivo. Salvo para las formas menos severas de MPS I, hasta ahora no hay un tratamiento efectivo para estas patologías, por lo que el daño sistémico progresivo produce la muerte entre los fines de la primera y de la cuarta década de la vida. En esta revisión se discuten las características clínicas de las MPS con las particularidades de cada fenotipo, el modo de confirmar el diagnóstico y los avances recientes en su tratamiento.

Diagnosis of mucopolysaccharidoses: how to avoid false positives and false negatives.

OBJECTIVE: This paper advocates a complete procedure, which includes both quantitative and qualitative analysis of urinary GAGs in the diagnosis of MPS in a clinically suspected population. METHODS: Urine samples from 219 clinically suspected mucopolysaccharidoses (MPS) patients and 91 controls were analysed using a combination of methods. Quantitation of isolated urinary glycosaminoglycans (GAGs) were carried out using acid alcian blue complex formation method and qualitative urinary GAG analysis by multisolvent sequential thin layer chromatography RESULTS: Of the 219 patients analysed, 131 were confirmed to be suffering from MPS. Quantitation of urinary GAGs alone would have missed 60 low GAG excreting MPS patients and misdiagnosed 26 high GAG excreting nonMPS as MPS patients. Further qualitative analysis and enzyme estimation were needed to identify these 60 low GAG excreting MPS patients and 26 high GAG excreting non MPS patients. CONCLUSION: These results emphasize that quantitation of urinary GAGs alone cannot diagnose MPS patients, it should be coupled with qualitative analysis and enzyme estimations for differential/definitive diagnosis.

Morphological alterations of the parotid gland of rats maintained on a liquid diet

Esse trabalho teve por objetivo analisar as alterações morfológicas das glândulas parótidas de ratos submetidos a uma dieta líquida. Trinta e seis animais foram divididos aleatoriamente em dois grupos. O grupo controle recebeu dieta sólida, e o grupo experimental recebeu dieta líquida. Os animais foram sacrificados 8, 15 e 30 dias após o início da experimentação. As glândulas foram incluídas em parafina e analisadas no microscópio de luz. Os resultados mostraram uma redução estatisticamente significante no peso das glândulas parótidas dos animais do grupo experimental quando comparado aos dos animais do grupo controle nos períodos de 15 e 30 dias. A alteração morfológica mais importante foi a evidenciação de vacúolos no citoplasma das glândulas parótidas dos animais alimentados com dieta líquida. Os vacúolos citoplasmáticos reagiram negativamente às técnicas de coloração específicas para glicoproteínas e mucopolissacarídeos (PAS e Alcian blue). Concluiu-se que a dieta líquida causou atrofia das glândulas parótidas nos períodos experimentais de 15 e 30 dias.

Galactosaminoglycans from normal myometrium and leiomyoma

In many tumors, the amount of chondroitin sulfate in the extracellular matrix has been shown to be elevated when compared to the corresponding normal tissue. Nevertheless, the degree of chondroitin sulfate increase varies widely. In order to investigate a possible correlation between the amount of chondroitin sulfate and tumor size, several individual specimens of human leiomyoma, a benign uterine tumor, were analyzed. The glycosaminoglycans from eight tumors were extracted and compared with those from the respective adjacent normal myometrium. The main glycosaminoglycan found in normal myometrium was dermatan sulfate, with small amounts of chondroitin sulfate and heparan sulfate. In leiomyoma, both dermatan sulfate and chondroitin sulfate were detected and the total amounts of the two galactosaminoglycans was increased in all tumors when compared to normal tissue. In contrast, the heparan sulfate concentration decreased in the tumor. To assess the disaccharide composition of galactosaminoglycans, these compounds were incubated with bacterial chondroitinases AC and ABC. The amounts of L-iduronic acid-containing disaccharides remained constant, whereas the concentration of D-glucuronic acid-containing disaccharides increased from 2 to 10 times in the tumor, indicating that D-glucuronic acid-containing disaccharides are responsible for the elevation in galactosaminoglycan concentration. This increase is positively correlated with tumor size

Histochemical study in the epididymis of the dog canis familiaris

Para la determinación de glucosaminoglicanos en el epidídimo del perro fueron usadas reacciones histoquímicas. En los tres segmentos del epitelio ductal la determinación de mucosustancias neutras reveló reacciones medias. No obstante, la presencia de gránulos PAS positivos en el epitelio, indicarían una probable secreción glucoproteica de las células principales. Las determinaciones histoquímicas confirman las interpretaciones previas