Chondroitinase Activity Research Articles

Phenotypic and molecular characterization of the causative agents of edwardsiellosis causing Nile tilapia (Oreochromis niloticus) summer mortalities

Edwardsiellosis is a serious bacterial disease affecting Nile tilapia (Oreochromis niloticus), causing septicemia and mortalities. Edwardsiella tarda and Edwardsiella anguillarum were isolated from Nile tilapia summer mortality events in Egypt. Diseased fish showed hemorrhagic septicemia, skin erosions, and eye opacity. A total of 24 Edwardsiella spp. isolates were retrieved from the investigated fish specimens. Phenotypic and biochemical characteristics grouped isolates into typical Ed. tarda (n = 14 strains) and atypical Ed. tarda (n = 10 strains). The BLAST analysis of sodB gene sequencing confirmed the conventional identification of typical Ed. tarda strains (n = 14) and reidentified all the atypical strains (n = 10) as Ed. anguillarum. Isolates showed a combination of virulence factors, including biofilm formation (66.6%), hemolysis (100%), chondroitinase (50%), and proteolytic activity (20.8%). The major part of isolates showed high resistance to ampicillin, amoxicillin, gentamycin antibiotics and harbored tetA, blaCTX-M, and aadA1 resistance genes. Pathogenicity testing of isolates in O. niloticus confirmed their virulence. Challenged fish exhibited septicemic signs similar to naturally diseased fish. Infections in naturally infected tilapia triggered acute and chronic histopathological alterations. Degenerative and necrotic changes were noticed in hematopoietic organs. Granulomas were noticed in between the hepatic parenchyma. The data extracted from the study confirm that accurate identification of the causative agents of edwardsiellosis should be reliant on genetic-based approaches. Analysis of the bacterium virulence properties offers insights into establishing novel therapeutics for edwardsiellosis control. The findings refer to the need for antimicrobial sensitivity testing to minimize antimicrobial resistance and increase therapy efficacy.

YsHyl8A, an Alkalophilic Cold-Adapted Glycosaminoglycan Lyase Cloned from Pathogenic Yersinia sp. 298.

A high enzyme-yield strain Yersinia sp. 298 was screened from marine bacteria harvested from the coastal water. The screening conditions were extensive, utilizing hyaluronic acid (HA)/chondroitin sulfate (CS) as the carbon source. A coding gene yshyl8A of the family 8 polysaccharide lyase (PL8) was cloned from the genome of Yersinia sp. 298 and subjected to recombinant expression. The specific activity of the recombinase YsHyl8A was 11.19 U/mg, with an optimal reaction temperature of 40 °C and 50% of its specific activity remaining after thermal incubation at 30 °C for 1 h. In addition, its optimal reaction pH was 7.5, and while it was most stable at pH 6.0 in Na2HPO4-citric acid buffer, it remained highly stable at pH 6.0–11.0. Further, its enzymatic activity was increased five-fold with 0.1 M NaCl. YsHyl8A, as an endo-lyase, can degrade both HA and CS, producing disaccharide end-products. These properties suggested that YsHyl8A possessed both significant alkalophilic and cold-adapted features while being dependent on NaCl, likely resulting from its marine source. Yersinia is a typical fish pathogen, with glycosaminoglycan lyase (GAG lyase) as a potential pathogenic factor, exhibiting strong hyaluronidase and chondroitinase activity. Further research on the pathogenic mechanism of GAG lyase may benefit the prevention and treatment of related diseases.

Baculovirus ODV-E66 degrades larval peritrophic membrane to facilitate baculovirus oral infection

ODV-E66 is a major envelope proteins of baculovirus occlusion derived virus (ODV) with chondroitinase activity. Here, we studied the roles of ODV-E66 during Helicoverpa armigera nucleopolyhedrovirus (HearNPV) primary infection. ODV-E66 is a late viral protein dispensable for BV production and ODV morphogenesis. Deletion of odv-e66 had a profound effect on HearNPV oral infectivity in 4th instar larvae with a 50% lethal concentration (LC50) value of 26 fold higher than that of the repaired virus, compared to in 3rd instar larvae. Calcofluor white, an agent which destroys the peritrophic membrane (PM), could rescue the oral infectivity of odv-e66 deleted HearNPV, implying the PM may be the target of ODV-E66. In vitro assays showed HearNPV ODV-E66 has chondroitinase activity. Electron microscopy demonstrated that odv-e66 deletion alleviated the damage to the PM caused by HearNPV infection. These data suggest an important role of ODV-E66 in the penetration of the PM during oral infection.

Purification and Characterization of Chondroitinase ABC from <i>Proteus vulgaris</i>, an Iraqi Clinically Isolate

Forty-eight isolates were found belonging to Proteus sp. with isolation percentage of 38 out of 125 urine samples which were collected from urinary tract infected patients. Proteus vulgaris and Proteus mirabilis represent 18.7% (9 isolates), and 81.25% (39 isolates) respectively. The production of chondroitinase enzyme was studied in a medium which contained chondroitin sulphate as substrate. One hundred per cent of P. vulgaris showed the ability to produce chondroitinase. P.v 8 isolate produced the highest enzymatic activity which reached about 150 units/ml. Chondroitinase was extracted and purified by precipitation with 60% saturation of ammonium sulphate, dialysis, and followed by column chromatography on Sephadex 6B. The specific activity increased to 5000 (units/mg), with 35.2% yield and 17.4 fold purification. Optimum pH for activity and stability was 8.0; the optimal temperature for activity was 37°C, but the stability of chondroitinase was maintained 100% at 20–40°C for 30 min. Chondroitinase activity increased to 120% and 113% when the enzyme was incubated with Mg +2 and Ca +2 respectively, while NH 4 Cl and KCl reduced the activity to 95% and 86% respectively. The human cartilage was degradable by the purified enzyme after incubation for 14 days at 37°C. Direct injection of chondroitinase at 0.1 mg/ml in knee cartilage of mice, showed changes in tissues. It may be concluded that chondroitinase enzyme may work as a virulence factor by catalysing the hydrolysis of chondroitin sulphate of cartilage and increasing the tissue permeability to invade the cartilage tissue by P. vulgaris which causes destruction of cartilage and inflammation in humans.

Insights into the virulence-related genes of Edwardsiella tarda isolated from turbot in Europe: genetic homogeneity and evidence for vibrioferrin production.

Edwardsiella tarda has long been known as a pathogen that causes severe economic losses in aquaculture industry. Insights gained on E. tarda pathogenesis may prove useful in the development of new methods for the treatment of infections as well as preventive measures against future outbreaks. In this report, we have established the correlation between the presence of virulence genes, related with three aspects typically involved in bacterial pathogenesis (chondroitinase activity, quorum sensing and siderophore-mediated ferric uptake systems), in the genome of E. tarda strains isolated from turbot in Europe and their phenotypic traits. A total of 8 genes were tested by PCR for their presence in 73 E. tarda isolates. High homogeneity was observed in the presence/absence pattern of all the strains. Positive results in the amplification of virulence-related genes were correlated with the detection of chondroitinase activity in agar plates, in vivo AHL production during fish infection and determination of type of siderophore produced by E. tarda. To the best of our knowledge, this is the first study carried out with European strains on potential virulence factors. Furthermore, we demonstrated for the first time that E. tarda produces the siderophore vibrioferrin.

Biological and Serological Characterization of a Non-gliding Strain of Tenacibaculum maritimum Isolated from a Diseased Puffer Fish Takifugu rubripes

Tenacibaculum maritimum is a Gram-negative, gliding marine bacterium that causes tenacibaculosis, an ulcerative disease of marine fish. The bacterium usually forms rhizoid colonies on agar media. We isolated T. maritimum that formed slightly yellowish round compact colonies together with the usual rhizoid colonies from a puffer fish Takifugu rubripes suffering from tenacibaculosis, and studied the biological and serological characteristics of a representative isolate of the compact colony phenotype, designated strain NUF1129. The strain was non-gliding and avirulent in Japanese flounder Paralichthys olivaceus in immersion challenge test and showed lower adhesion ability to glass wall in shaking broth culture and to the body surface of flounder. It lacked a cell-surface antigen commonly detected in gliding strains of the bacterium in gel immunodiffusion tests. SDS-PAGE analysis showed different polypeptide banding patterns between NUF1129 and gliding strains. Like gliding strains, NUF1129 exhibited both chondroitinase and gelatinase activities, which are potential virulence factors of the bacterium. These results suggest that some cell-surface components related to gliding and adhesion ability are implicated in the virulence of T. maritimum.

Characterization of Edwardsiella tarda rpoN: roles in σ70 family regulation, growth, stress adaption and virulence toward fish

Edwardsiella tarda EIB202, a Gram-negative pathogen with strong virulence, is an opportunistic pathogen capable of causing edwardsiellosis with high mortality to fish. Alternative sigma factor 54 (RpoN) is an important regulator of virulence and stress resistance genes in many bacterial species and mainly responsible for transcription of genes in nitrogen utilization. In this study, the in-frame rpoN deletion mutant was constructed to analyze the function of RpoN in Edwardsiella tarda firstly. Compared to the wild-type and complemented strain rpoN (+), the ΔrpoN was impaired in terms of the ability to survive under oxidative stress, osmotic stress and acid resistance, as well as the growth in Luria-Bertani medium, demonstrating essential roles of RpoN in stress resistance and nitrogen utilization. In addition, the ΔrpoN displayed markedly decreased biofilm formation and chondroitinase activity and was attenuated in virulence reflected in the increased median lethal dose value and extended infection cycle. Real-time polymerase chain reaction revealed that the expression levels of σ(70) class changed in varying degrees in the rpoN mutant. Especially, the expression levels of rpoS and fliA were down-regulated 4.1-fold and 7.9-fold in stationary phase in comparison with the wild type, respectively. Furthermore, two differential expression genes, znuA and flhC, were detected in the wild type and ΔrpoN using the method of differential display reverse transcription PCR.

Virulent and nonvirulent Flavobacterium columnare colony morphologies: characterization of chondroitin AC lyase activity and adhesion to polystyrene

Colony morphology variants of fish pathogenic Flavobacterium columnare were studied to clarify the role of colony morphology change in the virulence of the bacterium. Typical rhizoid colony (Rz) variants are virulent and moderately adherent, nonrhizoid rough (R) colony variants are nonvirulent and highly adherent, and soft colony (S) variants are nonvirulent and poorly adherent. Chondroitin AC lyase activity, adhesion to polystyrene at different temperatures and after modification of bacterial surface, and lipopolysaccharide (LPS) profiles of the variants were studied. The chondroitinase activity was significantly higher in the virulent, rhizoid variants than in the rough variants of the same strain. Temperature significantly increased the adhesion of rhizoid variants up to 20°C. Modification of bacterial surface suggested that adhesion molecules contain both carbohydrates and proteins. LPS did not differ between the variants of the same strain. The results suggest that in Fl. columnare both rhizoid colony morphology and high chondroitinase activity are needed for virulence and that temperature may promote the adhesion of the virulent variants to surfaces at fish farms. New information is produced on the virulence mechanisms of Fl. columnare and the reasons behind the survival of the bacterium at fish farms.

Baculovirus Envelope Protein ODV-E66 Is a Novel Chondroitinase with Distinct Substrate Specificity

Chondroitin sulfate is a linear polysaccharide of alternating D-glucuronic acid and N-acetyl-D-galactosamine residues with sulfate groups at various positions of the sugars. It interacts with and regulates cytokine and growth factor signal transduction, thus influencing development, organ morphogenesis, inflammation, and infection. We found chondroitinase activity in medium conditioned by baculovirus-infected insect cells and identified a novel chondroitinase. Sequence analysis revealed that the enzyme was a truncated form of occlusion-derived virus envelope protein 66 (ODV-E66) of Autographa californica nucleopolyhedrovirus. The enzyme was a novel chondroitin lyase with distinct substrate specificity. The enzyme was active over a wide range of pH (pH 4-9) and temperature (30-60 °C) and was unaffected by divalent metal ions. The ODV-E66 truncated protein digested chondroitin most efficiently followed by chondroitin 6-sulfate. It degraded hyaluronan to a minimal extent but did not degrade dermatan sulfate, heparin, and N-acetylheparosan. Further analysis using chemo-enzymatically synthesized substrates revealed that the enzyme specifically acted on glucuronate residues in non-sulfated and chondroitin 6-sulfate structures but not in chondroitin 4-sulfate structures. These results suggest that this chondroitinase is useful for detailed structural and compositional analysis of chondroitin sulfate, preparation of specific chondroitin oligosaccharides, and study of baculovirus infection mechanism.

Chondroitinase activity can be transduced by a lentiviral vector in vitro and in vivo

The bacterial enzyme chondroitinase ABC (ChABC), which cleaves chondroitin sulfate glycosaminoglycan chains, can degrade inhibitory scar tissue formed following spinal cord injury, thereby promoting axonal growth and regeneration. However, delivering the active enzyme for prolonged periods presents practical limitations. To overcome these problems, we prepared a lentiviral vector (LV) encoding chondroitinase AC (Chase) together with the green fluorescent protein (GFP) reporter (Chase/LV) and demonstrated its expression and enzymatic activity in vitro and in vivo. Neural precursor cells infected with Chase/LV expressed the GFP reporter at levels that increased dramatically with time in culture. Enzymatic activity from the supernatant of the infected cells was demonstrated by dot blot assay using an antibody that recognizes the digested form of CSPG and was compared with the bacterial ChABC enzyme. Chick DRG cultures plated adjacent to the CSPG border and incubated with supernatant from Chase/LV-infected cells showed neurites growing into the CSPG area, a response similar to that after treatment with ChABC. In contrast, in control cultures, the neurites turned to avoid the inhibitory CSPG interface. Degradation of CSPG in these cultures was confirmed by specific CSPG antibodies. A single injection of Chase/LV into the spinal cord resulted in sustained secretion of the enzyme, whose activity was detected for 8 weeks by expression of GFP and evidence of the digested form of CSPG. This study demonstrates the efficacy of the Chase/LV vector and its potential as a therapeutic tool to reduce scar inhibition and promote axonal growth and repair following central nervous system injury.

Identification, characterization, and molecular cloning of a novel hyaluronidase, a member of glycosyl hydrolase family 16, from Penicillium spp.

Hyaluronidase (HAase) activity was detected in the culture supernatants of Penicillium purpurogenum and Penicillium funiculosum. The HAase from Penicillium spp. (HAase-P) was a hyaluronate 4-glycanohydrolase, which catalyzed the endolytic hydrolysis of the β-1,4 glycosidic linkage, as do vertebrate HAases. The gene encoding HAase-P was cloned and expressed in Escherichia coli. According to homology analyses of the deduced amino acid sequences, HAase-P is not classified into any of the known HAase groups, but belongs to glycoside hydrolase family 16, which includes endo-β-1,3(4)-glucanase. Regarding the substrate specificities, no chondroitinase and glucanase activities were detected. Judging from homology analyses and enzymatic properties, HAase-P seems to be a new type of HAase.

Production of human hyaluronidase in a plant-derived protein expression system: Plant-based transient production of active human hyaluronidase

Four types of human hyaluronidases (rHuHyal-1, -2, -3 and -4) were transiently expressed and purified from Nicotiana benthamiana, and their biochemical characteristics were analyzed. The recombinant HuHyals were expressed via agrobacteria-mediated infiltration and generated and expressed in terms of micrograms per 5 leaves of N. benthamiana. Expressed recombinant HuHyals were purified using a His(6) tagging system and Ni column chromatography, respectively, at pH 8.0, after which the purified rHuHyals were concentrated for additional biochemical analyses. The four types of rHuHyals were allowed to react with hyaluronic acids and chondroitin sulfates. The biochemical properties of rHuHyal-1 fit those of the commercially available Hyal, PH-20, which was extracted from animal testes under acidic conditions (pH 3.5). However, rHuHyal-1 evidenced activity levels 2 to 6-fold greater than the three other rHuHyals (rHuHyal-2, -3 and -4) at pH 3.5. However, only rHuHyal-4 exhibited chondroitinase activity with both 6- S-chondroitin sulfate (chondroitin sulfate C) and 4- S-chondroitin sulfate (chondroitin sulfate A) as standard substrates. The results of zymography demonstrated that recombinant HuHyal 1 was modified by glycosylation, but Escherichia coli Hyal was not. This result demonstrated that plant-based rHuHyal was functionally active and evidenced biochemical characteristics and post-translational protein modifications similar to those of animal testis-derived Hyal.

The C. elegans hyaluronidase: A developmentally significant enzyme with chondroitin-degrading activity at both acidic and neutral pH

Mammalian hyaluronidases degrade hyaluronan and some structurally related glycosaminoglycans. We generated a deletion mutant in the Caenorhabditis elegans orthologue of mammalian hyaluronidase, hya-1. Mutant animals are viable and grossly normal, but exhibit defects in vulval morphogenesis and egg-laying and showed increased staining with alcian blue, consistent with an accumulation of glycosaminoglycan. A hya-1::GFP reporter was expressed in a restricted pattern in somatic tissues of the animal with strongest expression in the intestine, the PLM sensory neurons and the vulva. Total protein extracts from wild-type animals exhibited chondroitin-degrading but not hyaluronan-degrading activity. Chondroitinase activities were observed at both neutral and acidic pH conditions while both neutral and acidic activities were absent in extracts from hya-1 mutant strains. We also evaluated the function of oga-1, which encodes the C. elegans orthologue of MGEA-5, a protein with hyaluronan-degrading activity in vitro. oga-1 is expressed in muscles, vulval cells and the scavenger-like coelomocytes. An oga-1 mutant strain exhibited egg-laying and vulval defects similar to those of hya-1; chondroitinase activity was unaffected in this mutant.

Characterization of Edwardsiella tarda rpoS: effect on serum resistance, chondroitinase activity, biofilm formation, and autoinducer synthetases expression

In this study, rpoS gene was identified from Edwardsiella tarda EIB202 and its functional role was analyzed by using an in-frame deletion mutant rpoS and the complemental strain rpoS (+). Compared with the wild type and rpoS (+), rpoS was impaired in terms of the ability to survive under oxidative stress and nutrient starvation, as well as the resistance to 50% serum of Scophthalmus maximus in 3 h, demonstrating essential roles of RpoS in stress adaptation. The rpoS mutant also displayed markedly increased chondroitinase activity and biofilm formation. Real-time polymerase chain reaction revealed that the expression level of quorum sensing autoinducer synthetase genes luxS and edwI was increased by 3.7- and 2.5-fold in the rpoS mutant strain. Those results suggested that rpoS might be involved in the negative or positive regulation of chondroitinase and biofilm formation, or quorum sensing networks in E. tarda, respectively. Although there were no obvious differences between the wild-type and the rpoS mutant in adherence of epithelioma papulosum cyprini (EPC) cell and in the lethality on fish model, rpoS deletion leads to the drastically reduced capacity for E. tarda to internalize in EPC cells, indicating that RpoS was, while not the main, the factor required for the virulence network of E. tarda.

Chondroitin C lyase [4.2.2.] is unable to cleave fructosylated sequences inside the partially fructosylated Escherichia coli K4 polymer

Chondroitin C lyase was demonstrated to be unable to act on fructosylated sequences inside a partially fructosylated polysaccharide having the chondroitin backbone structure, the Escherichia coli K4 polymer, using different analytical approaches. Chondroitin C lyase produced various unsaturated oligosaccharides by acting on an approximately 27%-fructosylated K4 polymer. The online HPLC-ESI-MS approach showed the disaccharide nature of the main species produced by chondroitinase C as DeltaHexA-GalNAc. Furthermore, the non-digested sequences inside the K4 polymer were demonstrated to be oligosaccharides bearing a fructose for each glucuronic acid unit. In fact, unsaturated fully fructosylated oligomers, from tetrasaccharide to decasaccharide (DeltaHexA(Fru)-GalNAc-[GlcA(Fru)-GalNAc](n) with n between 1 and 4), at decreasing percentages, were produced by the enzyme. These results clearly indicate that chondroitinase C cleaved the innermost glucuronic acid-N-acetylgalactosamine linkage without affecting the 1,4 glycosidic linkage between fructosylated glucuronic acid and N-acetylgalactosamine residues, confirming that the 3-O-fructosylation of the GlcA residue renders the polysaccharide resistant to the enzyme action. This novel specific activity of chondroitinase C was also useful for the production of discrete microgram amounts of fully fructosylated oligomers, from 4- to 10-mers, from E. coli K4 for possible further studies and applications.

Sperm surface arylsulfatase A can disperse the cumulus matrix of cumulus oocyte complexes

Cumulus cell layers of expanded cumulus oocyte complexes (COCs) are interlinked with networks of hyaluronic acid, chondroitin sulfate B proteoglycans and link proteins, and they can be dispersed by sperm surface hyaluronidases. In this report, we showed that arylsulfatase A (AS-A), existing on the sperm head surface, also had this dispersion action. Purified AS-A free of protease, hyaluronidase and chondroitinase activities could disperse the cumulus matrix of expanded COCs. However, this COC dispersion action was not associated with AS-A desulfation activity, assayed by using p-nitrocatecholsulfate (artificial substrate). COCs incubated for 1 h with sperm pretreated with anti-AS-A IgG in the presence of apigenin (a hyaluronidase inhibitor) did not exhibit matrix dispersion, whereas several cumulus layers were already dispersed in COCs incubated with sperm pretreated with preimmune IgG. Furthermore, sperm from AS-A null mice showed a significant delay in COC dispersion, compared with wild-type sperm. Within 1 h of sperm-COC co-incubation, the size of COCs incubated with AS-A null sperm was 65% of the original dimension, whereas that of COCs inseminated with wild-type sperm was only 17%. A further delay in COC dispersion by AS-A(-/-) mouse sperm was observed when apigenin was present in the co-incubation. We also showed for the first time that AS-A had a specific affinity for chondroitin sulfate B, a component of cumulus matrix proteoglycan networks; this might provide a mechanism of cumulus matrix destabilization induced by sperm surface AS-A.

Spreading factors of Mycoplasma alligatoris, a flesh-eating mycoplasma.

Mycoplasma alligatoris causes lethal invasive disease of alligators and caimans. A homolog of the nagH gene, encoding a hyaluronidase secreted by Clostridium perfringens, and a C. perfringens hyaluronidase nagI or nagK pseudogene were discovered in the M. alligatoris genome. The nagH gene was detected by PCR in the closest relative of M. alligatoris, Mycoplasma crocodyli, but not in 40 other species representing the Mycoplasma hominis, Mycoplasma pneumoniae, and Spiroplasma phylogenetic clusters. The hyaluronidase activity in the cellular fraction of M. alligatoris and M. crocodyli SP4 broth cultures was equivalent to 10(-16) U of Streptomyces hyalurolyticus hyaluronidase CFU(-1). Negligible activity was present in the cell-free supernatant fraction. No chondroitinase activity was detected. There is also a novel homolog of the nanI gene, which encodes a sialidase secreted by C. perfringens, in the M. alligatoris genome. The signature YRIP and SXDXGXTW motifs and catalytic residues of the clostridial sialidase are conserved in the mycoplasmal gene, but the leader sequence necessary for its secretion by C. perfringens is absent. The gene was not detected by PCR in any other mycoplasma. Potent cell-associated sialidase activity was present in M. alligatoris colonies on agar but not in the cell-free supernatants of broth cultures or in M. crocodyli. The presence of hyaluronidase and sialidase in M. alligatoris is consistent with the rapid invasiveness and necrotizing effects of this organism, and the lack of sialidase in M. crocodyli is consistent with its comparatively attenuated virulence. This genetic and biochemical evidence suggests that the spreading factors hyaluronidase and sialidase, a combination unprecedented in mycoplasmas, are the basis of the virulence of M. alligatoris.

Impact of oestradiol and progesterone on the glycosaminoglycans and their depolymerizing enzymes of the rat mammary gland.

The influence of oestradiol and progesterone either singly or in combination with each other on the levels of hyaluronic acid, heparan sulphate, chondroitin sulphate, and on the activity of hyaluronidase and chondroitinase were investigated in the mammary gland of ovary-intact and in ovariectomized rats, administered oestradiol and/or progesterone. Administration of oestradiol to ovary-intact rats elevated the levels of hyaluronic acid and decreased the levels of heparan sulphate while progesterone, when administered alone, could elevate only chondroitin sulphate when compared with controls. The steroids when administered in combination, however, increased the levels of all glycosaminoglycans studied. Ovariectomized animals showed a decrease in heparan sulphate alone as compared with controls while administration of oestradiol to these rats elevated the levels of both heparan sulphate and chondroitin sulphate as compared with ovariectomized rats. Also the administration of progesterone either singly or in combination increased the levels of heparan sulphate and also decreased the levels of hyaluronic acid with no impact on the levels of chondoritin sulphate. In ovary-intact animals administration of oestradiol alone had no effect on hyaluronidase activity. Progesterone either singly or in combination with oestradiol reduced the activity of hyaluronidase, whereas it had no influence on the activity of chondroitinase. The activities of both the enzymes were decreased in ovariectomized animals and administration of oestradiol and/or progesterone to the above groups resulted in an increase. This study demonstrates that oestradiol anzd progesterone play an important role in modulating glycosaminoglycans and their depolymerizing enzymes, thereby influencing the activities of the mammary epithelium.

Use of a Mini-Transposon to Study Chondroitinase Activity Associated withEdwardsiella ictaluri

Abstract Edwardsiella ictaluri, the etiological agent of enteric septicemia of catfish (ESC), is the leading cause of bacterial disease in commercially raised channel catfish Ictalurus punctatus. Little work has been conducted at a genotypic level to determine potential virulence characteristics, but the production of chondroitin sulfatase is a suspected virulence factor. Using transpositional mutagenesis, we created stable E. ictaluri mutants that are deficient in chondroitinase activity. Channel catfish were challenged by injection with E. ictaluri transposon mutant MI15. None of the catfish challenged with the mutant died or showed signs of ESC. These fish were held for 2 weeks and then challenged by injection with the known virulent parent strain of E. ictaluri. The challenged naive control fish showed clinical signs of and a mortality rate consistent with ESC, whereas catfish that had been injected with MI15 prior to challenge with the parent strain were resistant to disease. This work represents a p...

An extracellular enzyme with hyaluronidase and chondroitinase activities from some oral anaerobic spirochaetes.

Treponema denticola, Treponema vincentii and Treponema socranskii produce an enzyme that hydrolyses hyaluronic acid (HA) and chondroitin sulphate (CS). The secreted enzyme is specifically inhibited by gold sodium thiomalate and anti-bee-venom antibodies. The use of saturated substrate (HA or CS) transblots allowed the visualization of active enzyme directly from culture supernatants and is a useful tool in clarification of complex polysaccharide-degrading enzyme specificities. The affinity-purified extracellular enzyme of T. denticola contains a single molecular species with a molecular mass of 59 kDa. Since it hydrolyses both HA and CS, it can more appropriately be termed a hyaluronoglucosaminidase (HGase). The HGase has been localized at the cell surface by electron microscopy and may play an active role in the degradation of connective tissue ground substance in the initiation and progression of periodontal disease.