The role of candidal histolytic enzymes on denture-induced stomatitis in patients living in retirement homes.

MATERIAL AND METHODS: Fifty nine elders wearing complete dentures and living in retirement homes in Curitiba (southern Brazil), were divided into two groups: group #1, 26 patients with denture-induced stomatitis and group #2, 33 patients without denture-induced stomatitis. The two groups were evaluated in relation to the degree of denture-induced stomatitis, salivary fungal loads, and secretion of some histolytic enzymes. RESULTS: Patients from group #1 showed higher degrees of colonisation by Candida albicans (p = 0.031). Candida krusei, Candida tropicalis, and Candida parapsilosis were also isolated, but there were no differences between the groups (p > 0.05). Secretory aspartyl protease (Sap) and chondroitinase did not show significant differences among the isolated Candida spp. in the two groups. Phospholipase secretion rates were higher among the strains of C. albicans from group #2 (p = 0.036). The same behaviour was not detected for non-albicans Candida species. CONCLUSIONS: The results could infer that differences in the secretion rates of candidal histolytic enzymes should not be imputed as imperative for the progress of denture-induced stomatitis.

An assay for bacterial and eukaryotic chondroitinases using a chondroitin sulfate-binding protein.

A simple, rapid, and reproducible microtiter-based chondroitinase (CSase) assay is reported here, based on the competition of chondroitin sulfate (CS) with immobilized hyaluronan (HA) for the binding of TSG-6 protein, the product of TNF-inducible gene 6. Although the catabolic reaction of bacterial and other prokaryotic CSase enzymes, often referred to as the chondroitin lyases, can be followed by tracking the generation of unsaturated bonds by the spectrophotometrical determination of the absorbance at 232 nm, no rapid, sensitive, and simple assay has been devised to date for measuring the activity of the vertebrate enzymes that cleave their substrate exclusively by hydrolysis. We provide data demonstrating that the CSase assay described here is suitable for the determination of the activities of both classes of enzymes. For the bacterial enzyme CSase ABC, both the determination of the absorbance at 232 nm and the assay based on TSG-6 binding are suitable using the same range of enzyme activities. However, for testicular hyaluronidase, considerably higher enzyme activities were needed to cleave CS than to cleave HA. Using the HA-binding domain of aggrecan for a comparison, we determined that the interaction between TSG-6 and chondroitin sulfate is uniquely suited for this CSase assay.

Composition of the peritrophic matrix of the tsetse fly, Glossina morsitans morsitans.

The three-layered peritrophic matrix of Glossina morsitans morsitans is shown, by histochemistry, to be formed of a mixture of glycosaminoglycans, glycoproteins and chitin. In all three layers the glycosaminoglycans contain GlcNAc-hexuronic and Gal-GlcNAc moieties, together with chitin. Glycosaminoglycans in layer 3 are sulphated and sulphated sites have a mean interspace distance of 53 nm - similar to the spacing of fixed charge sites in glomerular basement membrane suggesting a rôle for these sites in the filtration properties of the peritrophic matrix. O-linked oligo- saccharides are present in all three layers. Layer 1 contains the widest variety of glycoprotein oligosaccharide constructs GlcNac and alphalinked GalNAc possibly as GalNAcalpha1,3GalNAc, the latter apparently distal to Galbeta1-4GlcNAc. Lectin binding suggests that layer 2 contains GalNAcalpha1,3Galbeta1,3GlcNAc and that layer 3 contains GalNAc and Galbeta1,4GlcNAc. The evidence for N-linked oligosaccharides is more equivocal. Two dimensional electrophoresis showed that the peritrophic matrix contains a range of proteins, most of which require relatively harsh treatment for their solubilization.

Degradative enzymes of oral streptococci.

Members of the Streptococcus sanguis group (SSG) and Streptococcus milleri group (SMG) were screened for their ability to produce glycosidase, arylamidase (peptidase), protease, dextranase and glycosyltransferase activities. Species within each group produced unique patterns of activity. The most commonly produced glycosidases were beta-D-glucosidase, beta-D-galactosidase, N-acetyl-beta-D-glucosaminidase and N-acetyl-beta-D-galactosaminidase and the least commonly produced glycosidase activity was beta-fucosidase with Streptococcus intermedius (SMG) being the only species capable of producing the activity. For arylamidase activity, the most commonly produced type was lysine-arylamidase. Glycosidase and arylamidase activities were localized to particular sub-cellular fractions. alpha-galactosidase was found only in culture supernatant fluids whereas N-acetyl-beta-D-glucosaminidase was found in all fractions; the culture supernatant, cell wall, cell membrane and cytoplasm. No arylamidase activity was seen in culture supernatants. Phe-arg-arylamidase was found only in cytoplasmic fractions whereas val-pro-argarylamidase was found in cell walls, cell membranes and cytoplasmic fraction. Protease activity was measured as the degradation of bovine serum albumin (BSA) and casein. Casein was degraded by a number of strains whereas no species/strains were able to degrade BSA. Streptococcus intermedius, Streptococcus constellatus (SMG), Streptococcus mitior and Streptococcus defectivus (SSG) were the only species that produced hyaluronidase and no species produced chondroitin sulphatase. The groups were also examined for their abilities to produce glycosyltransferase and dextranase. Strep. sanguis, Streptococcus gordonii, Streptococcus mitis and Streptococcus oralis produced glucosyltransferase and, with the exception of the latter species, fructosyltransferase. No species within the SMG was capable of producing either glycosyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of glycosaminoglycan-degrading enzymes by substrate gel electrophoresis (zymography).

Substrate gel electrophoresis is of use for detecting minute amounts of hyaluronidase (HAase). In substrate gel electrophoresis, hyaluronan (HA) is impregnated in a gel. To determine the presence of degradation enzymes for other glycosaminoglycans (GAGs), the sizes of whose molecules are much smaller than that of HA, we have developed a technique by which chondroitin sulfate (CS) is chemically modified by introducing an allyl group at the reducing end for its immobilization in the gel. Enzymes with CS-degrading activity were detected on a CS-copolymerized gel in the presence or absence of sodium dodecyl sulfate. The smallest amount of chondroitinase ABC and HAase was found to be 8 microU and 0.35 mU, respectively. By zymography using HA-impregnated and modified CS-copolymerized gels human serum HAase has been shown to consist of at least two isoforms each with its own substrate specificity. Using this method, uterine tumor tissue has been shown to secrete a novel HAase which degrades HA at neutral pH, but not CS at any pH. This method was also confirmed applicable to other GAGs for determining individual GAG-degrading enzymes. In future research, it will be used to examine the regulation of each GAG species in tissue.

Characterization of proteoglycans synthesized by different layers of adult human femoral head cartilage.

Full thickness human femoral head cartilage fragments were labeled in vitro with 35S-SO4 and 3H-proline and cut tangentially to the surface in a cryostat. Sixteen-micrometer thick sections were pooled from four zones: I (0-160 microns); II (160-480 microns); III (480-960 microns); and IV (> 960 microns). The pooled sections were extracted with 4 M guanidinium chloride solvent and the extracts and 35S-labeled proteoglycans (35S-PGs) were characterized. The superficial layer gave lowest extraction yields and the deep layers gave the highest yields. The highest specific activity (dpm/mg of dry weight) of 35S-SO4 labeling was in zones II and III and that of [3H] proline in zone I. The superficial layer I also contained: (1) the highest proportions of 35S-PG monomers of small hydrodynamic size and low buoyant density; (2) an increased proportion of hydrodynamically small 35S-PG monomers; (3) the smallest proportion of endogenously reassociated 35S-PG aggregates, although similar proportions of 35S-PG monomers extracted from all layers interacted with exogenous hyaluronan (HA); and (4) the more heterogeneous population of 35S-glycosaminoglycan (GAG) side chains with respect to their size, although their chemical compositions were similar in all layers. In addition, extracted 35S-PGs had shorter GAG chains than the residual nonextracted molecules and were enriched in chondroitin-6 sulfate whereas residual/non-extracted 35S-PGs were enriched in chondroitin-4 sulfate. The extraction yields of keratan sulfate (KS)-enriched 35S-PGs also decreased with the depth of tissue, though the overall 35S-KS chain content varied little with depth of tissue, being slightly higher in the deepest zone IV.

Chondroitin sulfate depolymerase and hyaluronidase activities of viridans streptococci determined by a sensitive spectrophotometric assay.

Sensitive spectrophotometric assays for the detection of bacterial chondroitin sulfate depolymerase and hyaluronidase activities were developed by using Stains-all (1-ethyl-2-[3-(1-ethylnaphtho-[1,2-d]thiazolin-2-ylidene)-2- methylpropenyl]naphtho-[1,2-d]thiazolium bromide). Stains-all interacts with hyaluronic acid to produce a shift in the absorption spectrum with a distinct absorption peak between 620 and 660 nm, while chondroitin sulfate interacts to form a distinct shoulder between 440 and 500 nm. Assays measure undegraded substrate. A collection of 110 strains of viridans streptococci, including representatives of all the currently recognized species, was studied. Streptococcus intermedius and S. constellatus degraded hyaluronic acid, while only strains of S. intermedius, primarily isolated from brain and liver abscesses, produced chondroitin sulfate depolymerase. S. intermedius, of all the viridans streptococci, produces the widest range of glycoprotein- and glycosoaminoglycan-degrading enzymes, which may contribute to the virulence of this species.

A fluorimetric enzyme assay for the diagnosis of Morquio disease type A (MPS IV A).

4-Methylumbelliferyl-beta-D-galactopyranoside-6-sulphate was synthesized and used for the determination of galactose-6-sulphate sulphatase activity. Fibroblasts and leucocytes from 12 different Morquio A patients, showed 0.0-2.7% of mean normal galactose-6-sulphate sulphatase activity. Heterozygotes showed intermediate activities. The enzymatic liberation of the fluorochrome from 4-methylumbelliferyl-beta-D-galactopyranoside-6-sulphate requires the sequential action of galactose-6-sulphate sulphatase and beta-galactosidase. Normal beta-galactosidase activity caused nearly complete hydrolysis of non-fluorescing 4-methylumbelliferyl-galactoside, formed during incubation. In cell extracts with a beta-galactosidase deficiency however, a second incubation in the presence of excess beta-galactosidase is needed to avoid underestimation of galactose-6-sulphate sulphatase activity.

Diagnosis of classical Morquio's disease: N-acetylgalactosamine 6-sulphate sulphatase activity in cultured fibroblasts, leukocytes, amniotic cells and chorionic villi.

The tritiated disulphated trisaccharide 6-sulpho-N-acetylgalactosamine-glucuronic acid-6-sulpho-N-acetyl-[1-3H]galactosaminitol was prepared from chondroitin 6-sulphate for use as a substrate for N-acetylgalactosamine 6-sulphate sulphatase. The reaction product was separated on ECTEOLA cellulose rather than Dowex 1 X 2. The mean activities of normal fibroblasts, leukocytes and amniotic cells were 8.43, 2.59 and 3.14 nmol h-1 mg protein-1, respectively. Fibroblasts from five patients with classical Morquio's disease (mucopolysaccharidosis IVA; MPSIVA) and one patient with neonatal multiple sulphatase deficiency displayed activities of less than 5% of control mean. Activity in amniotic cells from a pregnancy where the fetus was affected with MPS IVA was 6% of control mean. Activity was also found to be present in normal specimens of chorionic villi (mean value 1.21 nmol h-1 mg protein-1), demonstrating the feasibility of first trimester prenatal diagnosis of MPS IVA by assay of activity in this tissue.

N-acetylgalactosamine-6-sulfate sulfatase in man. Absence of the enzyme in Morquio disease.

Human N-acetylgalactosamine-6-sulfate sulfatase (6-sulfatase) activity is measured by using as a substrate a sulfated tetrasaccharide obtained by digesting purified chondroitin-6-sulfate (C-6-S) with testicular hyaluronidase. The amount of inorganic sulfate released is measured turbidimetrically. The enzyme from human kidney has a pH optimum of 4.8; its activity is augmented by low levels of NaCl and inhibited by phosphate and high levels of NaCl. Free glucuronate, acetylgalactosamine, inorganic sulfate, polymeric C-6-S, or tetrasaccharide obtained from chondroitin-4-sulfate do not affect the enzyme activity. The method may be used for the diagnosis of Morquio disease since extracts of Morquio fibroblasts are devoid of 6-sulfatase activity.

A comparative study between a chondroitinase B and a chondroitinase AC from Flavobacterium heparinum: Isolation of a chondroitinase AC-susceptible dodecasaccharide from chondroitin sulphate B.

A chondroitinase that degrades only chondroitin sulphate B was isolated from Flavobacterium heparinum, and separated from a constitutive chondroitinase AC also present in extracts of F. heparinum. The enzyme acts only on chondroitin sulphate B, producing oligo- and tetra-saccharides, plus an unsaturated 4-sulphated disaccharide (deltaDi-4S). The oligosaccharide fraction (mol. wt. 3000) is susceptible to chondroitinase AC, producing mainly deltaDi-4S. The chondroitinase B is distinguished from chondroitinase AC by several properties, such as the effect of certain metal ions, temperature for optimal activity, and susceptibility to increasing salt concentrations. The enzyme is induced in F. heparinum by all the chondroitin sulphates, as well as by the disaccharides prepared from the chondroitins. The mechanism of induction of the enzyme and the structure of chondroitin sulphate B are discussed in relation to these results.