On the catalytic mechanism of polysaccharide lyases: evidence of His and Tyr involvement in heparin lysis by heparinase I and the role of Ca2+.

The structurally diverse polysaccharide lyase enzymes are distributed from plants to animals but share common catalytic mechanisms. One, heparinase I (F. heparinum), is employed in the production of the major anticoagulant drug, low molecular weight heparin, and is a mainstay of cell surface proteoglycan analysis.

Testing for urinary hyaluronate improves detection and grading of transitional cell carcinoma.

Objective: The purpose of this study is to establish a method for the diagnosis and grading of transitional cell carcinoma (TCC), which is responsible for 90% of bladder tumors, using a recently developed ultrasensitive assay for the measurement of hyaluronan (HA).

Materials And Methods: Urine samples were collected prior to surgery (cystoscopy, transurethral resection for bladder cancer (TURBT), and cystectomy) in 350 patients. After the procedure, pathologic examination revealed that 160 patients had TCC.

Glycosaminoglycan chains from alpha5beta1 integrin are involved in fibronectin-dependent cell migration.

Alpha5beta1 integrin from both wild-type CHO cells (CHO-K1) and deficient in proteoglycan biosynthesis (CHO-745) is post-translationally modified by glycosaminoglycan chains. We demonstrated this using [35S]sulfate metabolic labeling of the cells, enzymatic degradation, immunoprecipitation reaction with monoclonal antibody, fluorescence microscopy, and flow cytometry. The alpha5beta1 integrin heterodimer is a hybrid proteoglycan containing both chondroitin and heparan sulfate chains.

Sulfated glycosaminoglycans of periurethral tissue in pre- and postmenopausal women.

Objective: The objective was to determine the sulfated glycosaminoglycans (GAGs) of the extracellular matrix (ECM) in pre- and postmenopausal women.

Study Design: Periurethral tissue was obtained from 44 consecutive women who underwent surgery for urinary incontinence, for pelvic organ prolapse, or for other gynecologic benign conditions. Biopsy specimens were assessed by biochemical methods to characterize and quantify sulfated GAG.

Internalization and degradation of heparin is not required for stimulus of heparan sulfate proteoglycan synthesis.

In vitro, heparin and antithrombotic drugs specifically stimulate the synthesis of an antithrombotic heparan sulfate proteoglycan (HSPG) produced by endothelial cells. The putative heparin binding site(s) that may be related to this phenomenon were investigated. In the preceding article, using various heparin probes, it was shown that the heparin does not bind to the endothelial cell surface, but only to the extracellular matrix.

The binding of heparin to the extracellular matrix of endothelial cells up-regulates the synthesis of an antithrombotic heparan sulfate proteoglycan.

Exposure of endothelial cells to heparin and other antithrombotic drugs specifically stimulates the synthesis of an antithrombotic heparan sulfate (HS). In the present work, biotinylated heparin (BiotHep) was used to characterize the binding site(s) of heparin responsible for the stimulus in HS synthesis on endothelial cells. No differences were observed between biotinylated and non-biotinylated heparin in their ability to increase the synthesis of HS.

Development of an enzyme-linked immunosorbent assay (ELISA)-like fluorescence assay to investigate the interactions of glycosaminoglycans to cells.

Sulfated glycosaminoglycans were labeled with biotin to study their interaction with cells in culture. Thus, heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate were labeled using biotin-hydrazide, under different conditions. The structural characteristics of the biotinylated products were determined by chemical (molar ratios of hexosamine, uronic acid, sulfate and biotin) and enzymatic methods (susceptibility to degradation by chondroitinases and heparitinases).

Identification, cloning, expression and functional characterization of an astacin-like metalloprotease toxin from Loxosceles intermedia (brown spider) venom.

Injuries caused by brown spiders (Loxosceles genus) are associated with dermonecrotic lesions with gravitational spreading and systemic manifestations. The venom has a complex composition containing many different toxins, of which metalloproteases have been described in many different species of this genus. These toxins may degrade extracellular matrix constituents acting as a spreading factor.

Isolation and characterization of heparin from tuna skins.

This study characterized heparin isolated from tuna skins. Glycosaminoglycans were isolated from tuna skin after digestion using anion exchange resin. Heparin was eluted from the resin by sodium chloride gradient and was further fractionated by acetone fractionation.

Two novel dermonecrotic toxins LiRecDT4 and LiRecDT5 from brown spider (Loxosceles intermedia) venom: from cloning to functional characterization.

Loxoscelism (the condition produced by the bite of brown spiders) has been reported worldwide, but especially in warmer regions. Clinical manifestations include skin necrosis with gravitational spreading while systemic loxoscelism may include renal failure, hemolysis and thrombocytopenia. The venom contains several toxins, of which the best biochemically and biologically studied is the dermonecrotic toxin, a phospholipase-D.

Hyaluronidases in Loxosceles intermedia (Brown spider) venom are endo-beta-N-acetyl-d-hexosaminidases hydrolases.

In studying Loxosceles venom, we detected degradation of purified hyaluronic acid (HA) and hydrolysis of purified chondroitin sulphate (CS) while neither dermatan sulphate, heparin or heparan sulphate were affected. In addition, with HA-degrading kinetic assays, we show that a hydrolase enzyme was involved in the HA cleavage. By use of the Reissig colorimetric reaction, we found that venom hyaluronidase is an endo-beta-N-acetyl-d-hexosaminidase that generates terminal N-acetylglucosamine residues upon cleavage of HA.

The effect of brown spider venom on endothelial cell morphology and adhesive structures.

Spiders of the Loxosceles genus have been responsible for severe clinical cases of envenomation worldwide. Accidents involving brown spiders can cause dermonecrotic injury, hemorrhage, hemolysis, platelet aggregation and renal failure. Histological findings of animals treated by venom have shown subendothelial blebs, vacuoles and endothelial cell membrane degeneration of blood vessel walls, as well as fibrin and thrombus formation.

Molecular cloning and functional characterization of two isoforms of dermonecrotic toxin from Loxosceles intermedia (brown spider) venom gland.

Brown spider (Genus Loxosceles) bites are normally associated with necrotic skin degeneration, gravitational spreading, massive inflammatory response at injured region, platelet aggregation causing thrombocytopenia and renal disturbances. Brown spider venom has a complex composition containing many different toxins, of which a well-studied component is the dermonecrotic toxin. This toxin alone may produce necrotic lesions, inflammatory response and platelet aggregation.

Sulfated glycosaminoglycans of the periurethral tissue in women with and without stress urinary incontinence, according to genital prolapse stage.

Objective: The objective was to determine sulfated glycosaminoglycans (GAG) of the extracellular matrix (ECM) in women with and without stress urinary incontinence according to genital prolapse stage.

Study Design: Periurethral tissue was obtained from 30 women who underwent surgery for urinary incontinence, for pelvic organ prolapse, or for other benign gynecologic conditions. Biopsy specimens were assessed by biochemical methods to characterize and quantify sulfated glycosaminoglycans.

Urinary hyaluronan as a marker for the presence of residual transitional cell carcinoma of the urinary bladder.

Objective: The purpose of this report is to evaluate the value of urinary hyaluronan (HA) as a maker of residual transitional cell carcinoma (TCC).

Patients And Methods: Urine samples were collected from 83 patients hospitalized for transurethral resection (TUR). Patient ages ranged from 36 to 86 years.

Structural and hemostatic activities of a sulfated galactofucan from the brown alga Spatoglossum schroederi. An ideal antithrombotic agent?

The brown alga Spatoglossum schroederi contains three fractions of sulfated polysaccharides. One of them was purified by acetone fractionation, ion exchange, and molecular sieving chromatography. It has a molecular size of 21.

Fucan inhibits Chinese hamster ovary cell (CHO) adhesion to fibronectin by binding to the extracellular matrix.

In recent years, sulfated fucans have emerged as an important class of natural biopolymers. In this study, the anti-adhesive activity of a fucan from the brown seaweed Spatoglossum schröederi was analyzed using tumorigenic cells: wild-type Chinese hamster ovary cells (CHO-K1) and the mutant type deficient in xylosyltransferase (CHO-745). Fibronectin (FN) was used as substrate for cell attachment.

Genetic polymorphism in the sulfotransferase SULT1A1 gene in cancer.

Cytosolic sulfotransferases are enzymes that catalyze the conjugation of sulfate groups to a variety of xenobiotic and endogenous substrates. A mutation in the SULT1A1 gene has been associated with decreased sulfotransferase activity. We studied 125 cancer patients and 100 healthy controls from Brazil matched by age and gender.

Concentration of hyaluronic acid in primary open-angle glaucoma aqueous humor.

We compared the concentration of hyaluronic acid in the aqueous humor of primary open-angle glaucoma (POAG) patients and non-glaucomatous patients. Aqueous humor samples were obtained from 22 patients just before trabeculectomy for clinically uncontrolled POAG (POAG group). Aqueous humor (0.

Identification and partial characterisation of hyaluronidases in Lonomia obliqua venom.

By studying Lonomia obliqua (caterpillar) venom we were able to detect a lytic activity on purified hyaluronic acid. The venom hydrolyses purified chondroitin sulphate, but was unable to degrade either heparan sulphate or dermatan sulphate. Moreover, through purified hyaluronic acid-degrading kinetic assays, we observed that this lytic activity was caused by a hydrolase rather than lyase enzyme.

Sulfated glycosaminoglycans of the vagina and perineal skin in pre- and postmenopausal women, according to genital prolapse stage.

The aim of this study was to analyze the amount and types of sulfated glycosaminoglycans (GAGs) of the extracellular matrix (ECM) in the posterior vaginal wall and perineal skin in menacme and postmenopausal women, according to genital prolapse stage. Samples of vaginal tissue and perineal skin were obtained from 40 women who underwent vaginal surgery. Sulfated glycosaminoglycans were extracted by extensive tissue maxatase digestion, submitted to electrophoresis on agarose gel, and their concentrations were determined by densitometry.

Heparan sulfate and control of endothelial cell proliferation: increased synthesis during the S phase of the cell cycle and inhibition of thymidine incorporation induced by ortho-nitrophenyl-beta-D-xylose.

The effect of xylosides on the synthesis of [35S]-sulfated glycosaminoglycans by endothelial cells in culture was investigated. Ortho-nitrophenyl-beta-D-xylose (10(-3)M) produces a dramatic enhancement on the synthesis of heparan sulfate and chondroitin sulfate secreted to the medium (20- and 100-fold, respectively). Para-nitrophenylxyloside, at the same concentration, produces an enhancement of only 37- and 3-fold of chondroitin sulfate and heparan sulfate, respectively.

Comparison of practical methods for urinary glycosaminoglycans and serum hyaluronan with clinical activity scores in patients with Graves' ophthalmopathy.

Objective: Immunosuppressive treatment of Graves' opthalmopathy (GO) should be restricted to patients with active eye disease, but assessing disease activity is difficult. Several methods to evaluate GO activity have been introduced, but none of them is satisfactory. Glycosaminoglycans (GAGs) are complex polysaccharides that participate on the pathogenesis of GO and attempts to correlate its local increase to urinary GAGs (uGAGs) or serum hyaluronan (sHA) have been made, but the available techniques are labourious, time-consuming and difficult for routine use.

Heparins and heparinoids: occurrence, structure and mechanism of antithrombotic and hemorrhagic activities.

The correlation between structure, anticlotting, antithrombotic and hemorrhagic activities of heparin, heparan sulfate, low molecular weight heparins and heparin-like compounds from various sources that are in used in clinical practice or under development is briefly reviewed. Heparin-like molecules composed exclusively of iduronic acid 2-O-sulfate residues have weak anticlotting activities, whereas molecules that contain both iduronic acid 2-O sulfate, iduronic acid and small amounts of glucuronic acid, such as heparin, or mixed amounts of glucuronic and iduronic acids (mollusk heparins) possess high anticlotting and anti-Xa activities. These results also suggest that a proper combination of these elements might produce a strong antithrombotic agent.

Glycosaminoglycan profile in bladder and urethra of castrated rats treated with estrogen, progestogen, and raloxifene.

Objective: This study was undertaken to evaluate the action of conjugated equine estrogens alone, medroxyprogesterone, the combination of these estrogens with progestogens, and of raloxifene on the glycosaminoglycan profile in the bladder and urethra of adult oophorectomized rats in comparison with noncastrated rats.

Study Design: Sixty adult rats, of which 50 were submitted to bilateral oophorectomy, were studied. After 4 days, the latter were assigned to five groups of 10 animals each.