Y. Michelacci - Academia.edu (original) (raw)

Papers by Y. Michelacci

Carbohydrate Research, 1981

Abstract An extra of bacteriol cells Pseudomanos sp. IFO-13309 grown medium containing 0.1% bovin... more Abstract An extra of bacteriol cells Pseudomanos sp. IFO-13309 grown medium containing 0.1% bovine cornea keratan sulfate of low sulfate content degraded exhaustively bovine cornea keratan sulfate to give 2-acetamido-2-deoxy-β-d-gluco-pyranosyl 6-sulfate-(1→ 3)- ...

Clinica Chimica Acta, 2007

The relevance of glycosaminoglycan determination in biological fluids is gradually gaining import... more The relevance of glycosaminoglycan determination in biological fluids is gradually gaining importance in the literature. Nevertheless, the results obtained by different methods vary widely. We evaluated 1,9-dimethylmethylene blue (DMB) dye-binding assays for quantification of urinary glycosaminoglycans, in comparison to densitometry after agarose gel electrophoresis. Urinary glycosaminoglycans from different mammalian species were quantified by 3 different DMB dye-binding assays. The results were compared to those obtained by densitometry after agarose gel electrophoresis of glycosaminoglycans isolated from urine samples by ion exchange chromatography. Densitometry after agarose gel electrophoresis showed glycosaminoglycan urinary concentrations of 1-20 mg/l, and glycosaminoglycan/creatinine ratios of 2-25x10(-3), for all the mammalian species here studied. A decrease with age was observed for humans, cats and horses. In comparison, DMB assays gave much higher results - up to 200 mg/l and 500x10(-3) glycosaminoglycan/creatinine ratios. These values were greatly reduced after 4-h dialysis, suggesting that low molecular weight compounds do interfere. Furthermore, urinary anions such as sulfate, phosphate and citrate, react with metachromatic dyes, such as Toluidine Blue and DMB. DMB assays, although rapid and simple, are not appropriate to quantify urinary glycosaminoglycans in normal mammalians, since other urinary components interfere with the reactions.

Molecular vision, 2007

To evaluate the acute effects of laser in situ keratomileusis (LASIK) upon the synthesis of prote... more To evaluate the acute effects of laser in situ keratomileusis (LASIK) upon the synthesis of proteoglycans (PGs) and collagen fibril organization in human corneal explants. Human corneas that had been rejected for transplants were obtained at Banco de Olhos of Hospital São Paulo. For each eye pair, one cornea was submitted to refractive surgery, and the other was used as its matched control. After surgery, the corneas were excised from the eyes and immediately placed in a Ham F-12 nutrient mixture containing (35)S-sulfate for the metabolic labeling of PGs. After 24 h incubation, PGs were extracted and identified by a combination of agarose gel electrophoresis and enzymatic degradation with protease and specific glycosaminoglycan lyases. Histopathological and birefringence analysis were performed in fixed tissue slices. A marked decrease in (35)S-sulfate incorporation in PGs was observed in corneal explants that received LASIK, especially concerning dermatan sulfate-PGs, with keratan ...

Journal of Equine Veterinary Science, 2005

The aim of the current study was to investigate if the urinary glycosaminoglycans reflect the cha... more The aim of the current study was to investigate if the urinary glycosaminoglycans reflect the changes that occur in cartilages with age, training, and osteoarthritis in horses. The glycosaminoglycans were extracted by ion exchange chromatography from urine samples of 90 sedentary and athletic horses of different ages and genders, with or without osteoarthritis. The main urinary glycosaminoglycans in horses are chondroitin sulfate, dermatan sulfate, and heparan sulfate. A marked decrease occurred with age. Trace amounts of keratan sulfate were detected by immunochemical methods, and its concentration increased linearly with age. Athletes excreted fewer glycosaminoglycans in the urine than age-matched sedentary horses (including keratan sulfate), and sedentary horses with osteoarthritis excreted fewer total glycosaminoglycans, but more keratan sulfate, than their healthy counterparts. The chondroitin sulfate excreted in the urine by foals contained a low 6-sulfated disaccharide/4-sulfated disaccharide (Di6S/Di4S) ratio, as compared with adults and aged horses. Low Di6S/Di4S ratios were also found in the chondroitin sulfate excreted by healthy racehorses and by horses with osteoarthritis. Our data show clear correlations between urinary glycosaminoglycans in horses and age, physical exercise, and osteoarthritis. The combination of qualitative, quantitative, and structural analyses of these compounds may be useful for the diagnosis and follow-up of osteoarthritis in horses.

European Journal of …, 1986

... Denise S. PQ HORTON and Yara M. MICHELACCI Escola Paulista de Medicina, Departamento de Bioqu... more ... Denise S. PQ HORTON and Yara M. MICHELACCI Escola Paulista de Medicina, Departamento de Bioquimica, SZo Paulo ... sulfate 35S-labelled KS was isolated from a mixture of 35S-labelled glycosaminoglycans extracted from newborn rat and kindly supplied by Dr HB Nader. ...

Biochimica et Biophysica …, 1987

During the investigation of alternative methods for the large scale preparation of chondroitinase... more During the investigation of alternative methods for the large scale preparation of chondroitinases AC, B and C from Flavobacterium heparinum, a new chondroitinase activity was observed. This new enzyme, like the other chondroitinases, acts as an eliminase, forming unsaturated sulfated disaccharides from dermatan and chondroitin sulfates. In contrast to the chondroitinases previously described, which are endoglycosidases, this chondroitinase ABC cleaves the glycosidic linkages in an exolytic fashion, beginning at the reducing end of the substrate molecules. The oligosaccharides formed as transient products by the action of either chondroitinases or testicular hyaluronidase upon dermatan and chondroitin sulfates are also rapidly degraded by the chondroitinase ABC, regardless of their size or the presence of A-4,5 unsaturation in the terminal uronic acid residue. The maximum activity of the chondroitinase ABC occurs at 30°C and at pH 6.0-7.5. Only 15% of the activity was observed at 37°C, indicating that the enzyme is very sensitive to thermal denaturation. It is strongly inhibited by phosphate ions and is also inhibited by the unsaturated disaccharides formed.

Brazilian Journal of Medical and Biological Research, 1987

A four-step procedure was used to purify rat plasma kallikrein (RPK) with a relative molecular ma... more A four-step procedure was used to purify rat plasma kallikrein (RPK) with a relative molecular mass (Mr) of 87 kD (obtained both by gel filtration and SDS-PAGE), which indicates the purification of an alpha (intact) kallikrein, in contrast to previously described RPK preparations which had lower Mr (beta or degraded form). RPK is a neutral (pI 6.7) serine proteinase glycoprotein (15% carbohydrates) and contains (residues/mol): galactose (27), N-acetylglucosamine (24), mannose (13), glucose (13) and fucose (7). This purified alpha form of RPK has properties very similar to those of pure human and bovine kallikreins.

It has been previously shown that dextran sulfate administered to diabetic rats accumulates in th... more It has been previously shown that dextran sulfate administered to diabetic rats accumulates in the liver and kidney, and this could be due to a malfunction of the lysosomal digestive pathway. The aim of the present study was to evaluate the expression and activities of lysosomal enzymes that act upon proteins and sulfated polysaccharides in the livers of diabetic rats. Diabetes mellitus was induced by streptozotocin in 26 male Wistar rats (12 weeks old), while 26 age-matched controls received only vehicle. The livers were removed on either the 10th or the 30th day of the disease, weighed, and used to evaluate the activity, expression, and localization of lysosomal enzymes. A 50-60 % decrease in the specific activities of cysteine proteases, especially cathepsin B, was observed in streptozotocin-induced diabetes mellitus. Expression (mRNA) of cathepsins B and L was also decreased on the 10th, but not on the 30th day. Sulfatase decreased 30 % on the 30th day, while glycosidases did no...

Biological …, 1988

The isolation procedure for horse urinary kallikrein was considerably improved by the introductio... more The isolation procedure for horse urinary kallikrein was considerably improved by the introduction of two new purification steps: a) removal of mucoproteins and concentration of the urine by ultrafiltration and b) affinity chromatography on benzamidine-Sepharose conjugate. The homogeneity of the enzyme preparations, regarding their protein moiety, was demonstrated by: 1) a single symmetric peak on DEAE-Sephadex chromatography, with constant values for A280/A260 ratios, esterolytic and amidolytic specific activities; 2) a single band, although dispersed, on gel-electrophoresis at pH 8.3, also in the presence of sodium dodecyl sulfate, and 3) a unique sequence for the six amino-terminal residues. The isolated enzyme was shown to be a single chain glycoprotein (alpha-kallikrein), similar to human urinary and porcine-pancreatic kallikreins regarding the protein moiety molecular mass, amino-acid composition, and partial amino-terminal sequence; differences were found in their total sugar content and even more conspicuously in their carbohydrate composition. In contrast to porcine pancreatic beta-kallikrein, horse urinary kallikrein was not substrate-activated and unlike other alpha-kallikreins, did not present the biphasic time-course in benzoyl-L-arginine ethyl ester hydrolysis. The specificity constants (kcat/Km) for ester and 4-nitroanilide substrates were lower for horse urinary than for pancreatic beta-kallikrein and as observed with the latter enzyme, were affected by NaCl.

The purpose of the present work was to investigate the effects of mechanical epithelial debrideme... more The purpose of the present work was to investigate the effects of mechanical epithelial debridement upon glycosaminoglycan synthesis by human corneal explants. Corneal explants were maintained under tissue culture conditions for 2-72 days and the glycosaminoglycans synthesized 35 in 24 h were metabolically labeled by addition of S-sulfate to the culture medium. These compounds were isolated from the tissue explants and analyzed by a combination of agarose gel electrophoresis and enzymatic degradation with specific mucopolysaccharidases. The glycosaminoglycans synthesized by isolated epithelial cells and by corneas previously submitted to epithelial cell debridement were compared to controls. Keratan sulfate (26 kDa) and dermatan sulfate (43 kDa) were the main corneal glycosaminoglycans, each one corresponding to about 50% 35 35 of the total. Nevertheless, the main S-labeled glycosaminoglycan was S-dermatan sulfate 35 35 (73%), with smaller amounts of S-keratan sulfate (15%) and S-heparan sulfate (12%), suggesting a lower synthesis rate for keratan sulfate. The main glycosaminoglycan synthesized by isolated epithelial cells was heparan sulfate. The removal of epithelial layer caused a decrease in heparan sulfate labeling and induced the synthesis of dermatan sulfate by stromal cells. This increased synthesis of dermatan sulfate suggests a relationship between epithelium and stroma and could be related to the corneal opacity that may appear after epithelial cell debridement.

The objective of the present study was to investigate the expression and activities of lysosomal ... more The objective of the present study was to investigate the expression and activities of lysosomal enzymes that act upon proteins and sulfated polysaccharides in diabetic rat kidney. Cathepsins, glycosidases and sulfatases were studied on the 10th (DM-10) and on the 30th (DM-30) day of streptozotocin-induced diabetes mellitus (DM). The activity of cathepsin B, the main kidney cysteine protease, was decreased both in DM-10 and DM-30. Gel filtration chromatography of urinary proteins has shown the prevalence of low molecular weight peptides in normal and DM-10 urine, in contrast to the prevalence of high molecular weight peptides and intact proteins in DM-30. These results show that the decrease in lysosomal proteases could explain, at least in part, the increased albuminuria detected by radial immunodiffusion (RID), due to the excretion of less degraded or intact albumin. Concerning sulfated polysaccharides, the activities of β-glucuronidase, N-acetylβ-D-glucosaminidase, and N-acetyl-β-D-galactosaminidase were also decreased in DM-30, while aryl sulfatases did not vary. Increased toluidine blue metachromatic staining of the tissue suggests that the lower activities of glycosidases could lead to intracellular deposition of partially digested molecules, and this could explain the decreased urinary excretion and increased tissue buildup of these molecules. The main morphological changes observed in kidney were proximal convoluted tubules with thinner walls and thinner brush border. Immunohistochemistry revealed that most of cathepsin B was located in the brush border of proximal tubular cells, highlighting the involvement of proximal convoluted tubules in diabetic nephropathy.

Comparative Biochemistry and Physiology …, 1989

The Journal of laboratory and clinical medicine, 1992

We have investigated the effects of nontoxic doses of vitamin A on the hepatic contents of collag... more We have investigated the effects of nontoxic doses of vitamin A on the hepatic contents of collagen and sulfated glycosaminoglycans (SGAGs) in rats chronically treated with CCl4. When the animals were treated with this retinoid before the intoxication with CCl4, liver collagen level was significantly reduced as compared with that in rats that received only CCl4 (3.31 +/- 0.40 vs 5.00 +/- 0.61 mg/gm wet liver, mean +/- SD, respectively), although no significant differences were found for the relative proportion of type III collagen related to type I collagen. The absolute increment in the total amount of liver SGAG in the vitamin A--pretreated group was followed by a more important increase in the concentration of dermatan sulfate as compared with the CCl4 group (dermatan sulfate-to-heparan sulfate ratio: 1.15 for the CCl4 group vs 1.70 for the vitamin A--pretreated group). A significant proportion of the dermatan sulfate from this last group was of higher molecular weight when compa...

Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas, 1994

1. Two proteoglycans, PG1 and PG2, have been isolated from shark cartilage. Both are highly polyd... more 1. Two proteoglycans, PG1 and PG2, have been isolated from shark cartilage. Both are highly polydisperse and large (molecular mass: 1-10 x 10(6) Daltons) and contain chondroitin sulfate and keratan sulfate side chains, but PG2 is somewhat smaller than PG1 and contains less keratan sulfate. 2. Monoclonal antibodies were raised against PG1. Many antibodies were obtained and one of them, MST1, was subcloned and further characterized. This monoclonal antibody reacts with PG1 and PG2 from shark cartilage and also with aggrecan from bovine trachea cartilage. Chondroitinase AC-treated proteoglycans react with MST1, indicating that the antibody does not recognize chondroitin sulfate. MST1 also recognizes aggrecan from human cartilage and a proteoglycan from bovine brain (neurocan) but it does not recognize proteoglycans from rat Walker tumor, fetal calf muscle and decorin from human myoma. 3. Using MST1 we were able to demonstrate that both PG1 and PG2 aggregate with hyaluronic acid.

Research in Veterinary Science

The synovial membrane (SM) presents itself with distinctive characteristics during arthroscopic p... more The synovial membrane (SM) presents itself with distinctive characteristics during arthroscopic procedures in cases of osteoarthritis (OA) as well as osteochondritis dissecans (OCD) in horses. Most of the arthroscopic findings of the SM are limited to a description of a nonspecific inflammation state. In the present study, the macroscopic and histological aspects of the SM in OA and OCD horses were compared to those of healthy horses. The expression of interleukin (IL) in SM was also investigated. Besides, the concentrations of ILs and keratan sulfate (KS) in the synovial fluid (SF), and the molecular weights of the SF hyaluronic acid (HA) were also determined and correlated to the macroscopic and histological aspects of SM. This study included 10 healthy horses (control group), 12 horses with OA, and 12 with OCD. Macroscopic scores of the SM were higher in the OA group in comparison to the control and OCD groups. However, histological scores between OA and OCD were not different, and both were higher than the control group. Only in the OA group, there was a correlation between macroscopic and histological aspects of the SM, especially between volume and quantity of villi with perivascular inflammatory cells and synovial proliferation. The OA group has shown decreased expression of IL-10 in the SM, lower IL-10 and KS, and higher IL-1β and IL-6 in the SF in comparison to the control and OCD groups. There was a significant negative correlation between the macroscopic aspect of the SM and the molecular weights AH in the OA group. There was no correlation between the macroscopic aspect of the SM and all dosages in the OA and OCD group. In the OA joints, the evaluation of the shape of the SM during arthroscopy promotes a better indicator for joint inflammatory or tissue repair processes, while in the osteochondritic joints, investigation of the histological aspects are recommended to rule out an incipient OA development process. Both are helpful and should be considered to guide the postoperative treatment.

Carbohydrate Research, 1986

The dermatan sulfates from hog, rat, rabbit, and beef liver, hog, rat, beef, and dog spleen, and ... more The dermatan sulfates from hog, rat, rabbit, and beef liver, hog, rat, beef, and dog spleen, and hog skin were isolated and submitted to structural analysis. All of them migrated as single bands, close to the standard position for dermatan sulfate in agarose-gel electrophoresis. In polyacrylamide gel, however, each dermatan sulfate showed a characteristic electrophoretic migration-pattern: one, two, or three polydisperse bands, corresponding to different molecular weights, were obtained for the dermatan sulfates according to their origins. Chemical analysis showed that all of the dermatan sulfates here described are hybrid polymers composed of D-glucuronic and L-iduronic acid-containing disaccharide units. The relative position of these units in the polymer chains and the presence of 6-sulfated disaccharides were determined with the aid of chondroitinases B and AC from Flavobacterium heparinum. These studies show that each dermatan sulfate has a unique structure as regards the molecular weight, the presence of 6-sulfated disaccharide units, and also the relative amount and position of glucuronic and iduronic acid residues in the chains. These findings suggests a tissue- and species-specificity for the dermatan sulfates.

Brazilian Journal of Medical and Biological Research, 2001

In many tumors, the amount of chondroitin sulfate in the extracellular matrix has been shown to b... more 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 Dglucuronic 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.

The aim of the present study was to characterize the urinary excretion of glycosaminoglycans in k... more The aim of the present study was to characterize the urinary excretion of glycosaminoglycans in kittens and adult healthy cats, as well as in cats with a low urinary tract disease, the feline urologic syndrome (FUS). The main urinary glycosaminoglycan in cats was found to be chondroitin sulfate, with smaller amounts of dermatan sulfate and heparan sulfate. There was no difference in the urinary glycosaminoglycan concentration with sex, but a marked decrease occurred with age, due to chondroitin sulfate. Trace amounts of keratan sulfate were also detected in the urine of kittens, but not of healthy adult cats. Dermatan sulfate and heparan sulfate were the only glycosaminoglycans found in the urinary tract and kidney, and chondroitin sulfate was the only glycosaminoglycan found in the plasma. These data suggest that the main urinary glycosaminoglycan chondroitin sulfate is of systemic origin and filtered in the kidney, while the minor components dermatan sulfate and heparan sulfate may come from the urinary tract. The urinary glycosaminoglycan concentration was greatly decreased in animals with FUS, as compared to normal adults. We hypothesize that these low glycosaminoglycan levels reflect a damage to the bladder surface, resulting in absorption and/or degradation of the endogenous urinary glycosaminoglycans.

Journal of Biological Chemistry, 1973