Hyaluronan (HA) is a glycosaminoglycan that is abundant in the extracellular matrix of vertebrate cells. Under physiological conditions HA exists in a high-molecular-weight form, whereas HA fragments accumulate at sites of tissue injury and inflammation. Hyaluronidases are a group of enzymes that initiate the breakdown of HA. In humans, six hyaluronidase-like sequences have been identified in two locations, 3p21.3 (HYAL1, HYAL2 and HYAL3) and 7q31.3 (HYAL4, SPAM1 and HYALP1). Deficiency of one of these enzymes, HYAL1, was identified in a patient with Mucopolysaccharidosis IX, a disorder characterized by peri-articular soft masses containing HA-filled lysosomes. Given the broad distribution of HA and the mild phenotype of the patient, it is likely that other hyaluronidases or possibly the exoglycosidases, β-hexosaminidase and β-glucuronidase, are playing a major role in HA degradation. To address the potential role of HYAL3 in HA degradation in health and disease, a Hyal3-deficient mouse model was generated. Hyal3-deficient mice were viable, fertile and appeared to have no gross phenotype. The only difference observed was a subtle change in the cellularity and tissue structure of lungs from aged Hyal3-deficient mice. Further studies focused on analysis of HA homeostasis revealed a significant increase in HA in the airways of Hyal3-deficient lungs. Altered HA homeostasis is observed in rodent models of several lung conditions. In order to further study the role of Hyal3 in lungs, an ovalbumin-challenged inflammation model was generated in Hyal3-deficient mice. A significant increase in lung HA levels and altered distribution of HA in the airways of lungs was detected in ovalbumin-challenged Hyal3-deficient mice. Moreover, lung inflammation and airway resistance were increased in Hyal3-deficient mice after ovalbumin-challenge compared to similarly treated Hyal3-control mice. This suggests HA homeostasis that is altered during Hyal3-deficiency might be directly or indirectly promoting inflammation and airway resistance. Because the reported level of HA accumulation was very low in Hyal1-deficient and Hyal2-deficient mice, and in our studies of Hyal3-deficient mice, we performed preliminary studies to assess a role for an exoglycosidase, β-hexosaminidase, in HA turnover. Our preliminary studies indicate there is no or little HA accumulation in β-hexosaminidase-deficient mouse tissues. To conclude, our study of Hyal3- and β-hexosaminidase-deficient mice suggests that these are not the major enzymes involved in HA degradation

Hyaluronan (HA) is a glycosaminoglycan that is abundant in the extracellular matrix of vertebrate cells. Under physiological conditions HA exists in a high-molecular-weight form, whereas HA fragments accumulate at sites of tissue injury and inflammation. Hyaluronidases are a group of enzymes that initiate the breakdown of HA. In humans, six hyaluronidase-like sequences have been identified in two locations, 3p21.3 (HYAL1, HYAL2 and HYAL3) and 7q31.3 (HYAL4, SPAM1 and HYALP1). Deficiency of one of these enzymes, HYAL1, was identified in a patient with Mucopolysaccharidosis IX, a disorder characterized by peri-articular soft masses containing HA-filled lysosomes. Given the broad distribution of HA and the mild phenotype of the patient, it is likely that other hyaluronidases or possibly the exoglycosidases, β-hexosaminidase and β-glucuronidase, are playing a major role in HA degradation. To address the potential role of HYAL3 in HA degradation in health and disease, a Hyal3-deficient mouse model was generated. Hyal3-deficient mice were viable, fertile and appeared to have no gross phenotype. The only difference observed was a subtle change in the cellularity and tissue structure of lungs from aged Hyal3-deficient mice. Further studies focused on analysis of HA homeostasis revealed a significant increase in HA in the airways of Hyal3-deficient lungs. Altered HA homeostasis is observed in rodent models of several lung conditions. In order to further study the role of Hyal3 in lungs, an ovalbumin-challenged inflammation model was generated in Hyal3-deficient mice. A significant increase in lung HA levels and altered distribution of HA in the airways of lungs was detected in ovalbumin-challenged Hyal3-deficient mice. Moreover, lung inflammation and airway resistance were increased in Hyal3-deficient mice after ovalbumin-challenge compared to similarly treated Hyal3-control mice. This suggests HA homeostasis that is altered during Hyal3-deficiency might be directly or indirectly promoting inflammation and airway resistance. Because the reported level of HA accumulation was very low in Hyal1-deficient and Hyal2-deficient mice, and in our studies of Hyal3-deficient mice, we performed preliminary studies to assess a role for an exoglycosidase, β-hexosaminidase, in HA turnover. Our preliminary studies indicate there is no or little HA accumulation in β-hexosaminidase-deficient mouse tissues. To conclude, our study of Hyal3- and β-hexosaminidase-deficient mice suggests that these are not the major enzymes involved in HA degradation

No description available.

No description available.

Staphylococcus aureus encodes for a secreted hyaluronidase, hysA. Hyaluronidases are bacterial enzymes that cleave hyaluronic acid (HA) at the β-1,4 glycosidic bond, yielding unsaturated disaccharides. Initially, little was known about the regulation of this enzyme as well as its roles in S. aureus physiology and pathogenesis. The goal of this dissertation was to determine the regulation of hysA, and to determine the biological and physiological roles of this enzyme. Studies presented in Chapter II focus on determining the regulation of hysA and role of hysA in S. aureus pathogenesis. By screening the Nebraska Transposon Mutant Library (NTML) we identified 8 mutations that significantly altered HysA activity. Further analysis revealed that CodY directly represses hysA by binding to the CodY consensus binding sequence upstream of the hysA translational start site. Additionally, we found that a hysA mutant was attenuated in a neutropenic murine pneumonia model of infection and that there was reduced degradation of HA in the lungs of mice infected with the hysA mutant compared to wildtype by immunofluorescence. Studies presented in Chapter III focus on examining if HA can be incorporated into the S. aureus biofilm matrix and if HysA is involved in biofilm dispersal. We show that HA is incorporated into the biofilm matrix both in vitro and in vivo by confocal microscopy and HA ELISA. Additionally, we found that HA can enhance biofilm formation of the hysA mutant as well as other staphylococcal species in vitro. We show that induction of hysA can prevent biofilm formation in the presence of HA and that exogenous addition of purified HysA can disperse established HA containing biofilms. Finally, we found that a hysA mutant has reduced dissemination in an implant-associated infection model. Together these studies support our hypothesis that HA is incorporated into staphylococcal biofilms and that HysA is involved in dispersing S. aureus from the biofilm.

Indiana University-Purdue University Indianapolis (IUPUI) / This study was undertaken to investigate histologically the effect of a combination of an antibiotic and an anti-inflammatory enzyme when used as a medication in direct pulp therapy. The pulps of 56 teeth in two Macaca Speciosa monkeys, exposed and left open to the oral environment for 24 hours to insure contamination, received direct treatment with one of four experimental medications: (1) vancomycin, starch, and hyaluronidase; (2) vancomycin, starch, and water; (3) starch and water; and (4) starch and hyaluronidase. At 30 days the teeth were removed from one animal and at 90 days•from the other for histologic interpretation. A satisfactory response was observed in 92.9 per cent of the teeth treated with vancomycin, starch, and hyaluronidase; in 71.5 per cent of the teeth treated with vancomycin, starch, and water; and in 42.9 per cent of the teeth treated with both starch and water and starch and hyaluronidase. None of the teeth treated with vancomycin, starch, and water and vancomycin, starch, and hyaluronidase became necrotic,while 35.7 per cent of the teeth treated with starch and water or starch and hyaluronidase became necrotic. Under the conditions of this investigation, vancomycin containing pulp capping agents are effective in controlling infection and in promoting reparative dentin formation in monkeys. The benefit of hyaluronidase when used in combination with vancomycin was questionable.

Sperm Adhesion Molecule 1 (SPAM1) est une protéine spermatique possédant une activité hyaluronidase dans son domaine N-terminal, contribuant à la dispersion des cellules du cumulus entourant l’ovocyte. Elle possède aussi une capacité de liaison à la zone pellucide (ZP) dans son domaine C-terminal. Nos études précédentes chez le taureau ont démontré la présence de deux isoformes potentielles de SPAM1 de ~70 et 80 kDa. Ces mêmes études ont permis d’émettre l’hypothèse que ces deux isoformes de SPAM1 ont des domaines C-terminaux différents, des origines différentes (testicule ou épididyme) et sont localisées dans la région acrosomale ou post-acrosomale du spermatozoïde. Puisque le domaine C-terminal est impliqué dans les interactions spermatozoïdes-ZP, nous voulions caractériser les deux domaines C-terminaux afin de mieux connaître le rôle de SPAM1 dans les interactions entre les gamètes. Deux transcrits de Spam1 ont été trouvés dans les tissus testiculaires et épididymaires. Ces transcrits possèdent une identité dans leur séquence nucléotidique en 3’ de la région codante, mais diffèrent par la présence ou l’absence de 90 nucléotides (exon 3 du gène Spam1). Nous avons identifié PH-20, une homologue potentielle de SPAM1 chez l’espèce bovine. Puisque SPAM1 et PH-20 sont similaires en N-terminal, notre anticorps dirigé contre la portion N-terminale de SPAM1 pourrait, théoriquement, reconnaître PH-20. Pour confirmer ceci, nous avons tenté de produire une protéine recombinante PH-20, mais sans succès. Nous voulions déterminer si SPAM1 fait partie d’un complexe multiprotéique impliqué dans les interactions spermatozoïdes-ZP, tel que rapporté chez l’humain. Nos résultats suggèrent que SPAM1 est associée aux protéines d’ancrage AKAPs, retrouvées majoritairement au niveau de la gaine fibreuse du flagelle des spermatozoïdes. La caractérisation des isoformes de SPAM1, de son homologue PH-20, ainsi que des complexes multiprotéiques dont fait partie SPAM1 sont importantes afin d’approfondir nos connaissances sur le rôle de SPAM1 dans les interactions entre les gamètes. / Sperm Adhesion Molecule 1 (SPAM1) is a sperm protein that has a hyaluronidase activity in its N-terminus, aiding in the dispersal of the cumulus cells surrounding the egg. It also has a zona pellucida (ZP) binding activity in its C-terminus. Our previous studies showed that there are two potential SPAM1 isoforms that have a molecular weight of ~70 and 80 kDa in the bovine species. From these studies, we hypothesized that these two SPAM1 isoforms had different C-terminal domains, different origins (testis or epididymis) and were localised in the acrosomal or post-acrosomal regions of spermatozoa. Seeing as it is the C-terminal domain that is involved in ZP binding, we aimed to characterize the two C-terminal domains in order to better understand SPAM1’s role in gamete interactions. Although the 3’ nucleotide sequences were identical, two Spam1 transcripts varying by the presence or absence of 90 nucleotides (exon 3 of the Spam1 gene) were found in both testicular and epididymal tissues. During our studies, we also identified PH-20, a potential SPAM1 homolog. In order to determine if PH-20 is one of the two potential SPAM1 isoforms that is recognized by our antibody directed against the N-terminal domain, we attempted the production of a PH-20 recombinant protein, without success. We also sought to determine if SPAM1 is part of a multimeric protein complex involved in spermatozoa-ZP interactions, as reported in humans. Our results suggest that SPAM1 is associated with AKAPs, which are anchoring proteins abundantly found in the fibrous sheath of sperm flagella. Characterizing the SPAM1 isoforms, its homolog PH-20, as well as the multimeric protein complexes SPAM1 is part of, are important in order to better understand SPAM1’s role in gamete interactions.

The endothelial glycocalyx plays an important role as part of the permeability barrier between the blood and the interstitium. In this study, we used different sized fluorescently labeled dextran molecules to determine the size of the macromolecular exclusion zone in capillaries. The width of the exclusion zone was calculated as one half the difference between the anatomic luminal diameter, as determined by transillumination, and the width of a fluorescent dextran column. During the first hour after systemic injection of labeled dextrans, neither 70 kDa dextran (Dextran 70) nor 500 kDa dextran (Dextran 500) labeled with the anionic fluorescein isothiocyanate (FITC) penetrated the endothelial glycocalyx to the endothelial cell surface. However, the 40 kDa dextran (Dextran 40) labeled with the neutral fluorophore Texas Red was able to penetrate to the endothelial cell surface. Under these control conditions, the width of the exclusion zone for Dextran 500 was 0.55 ± 0.02 mm (n=46); for Dextran 70 it was 0.50 ± 0.01 mm (n=111); and for Dextran 40 it was 0.08 ± 0.01 mm (n=53). One hour after systemically injecting the enzyme hyaluronidase, measurements of the exclusion zone were made using Dextrans 40, 70 and 500. After the enzyme treatment, Dextran 70 appeared to penetrate the glycocalyx layer, whereas Dextran 500 did not. Following hyaluronidase treatment, the width of the exclusion zone for Dextran 500 was 0.56 ± 0.02 mm (n=71); for Dextran 70 it was 0.05 ± 0.01 mm (n=103); and for Dextran 40 it was 0.03 ± 0.01 mm (n=33). These results indicate that the enzyme hyaluronidase was able to degrade the structural integrity of the glycocalyx, since after enzymatic treatment Dextran 70 was able to permeate the glycocalyx layer, while it was unable to prior to this treatment. However, the glycocalyx barrier was not completely compromised following hyaluronidase treatment since Dextran 500 still was not able to permeate the exclusion zone. In conclusion, macromolecules with 5.3 nm or larger radii will more than likely not be able to permeate an intact glycocalyx; in addition, degradation of hyaluronan will increase the permeability of the glycocalyx so that macromolecules with 5.3 nm radii will permeate.

Class of 2009 Abstract / OBJECTIVES: The objective of this study was to characterize the Hyaluronidase (HYA) gene family throughout gestational development of murine hearts to provide greater insight regarding its role in cardiac morphogenesis. METHODS: Microdissection of murine embryos was accomplished to extract embryonic heart tissue. RNA was extracted using the standard Trizol protocol. cDNA templates were created using a standard protocol. Polymerase chain reaction (PCR) was used to verify presence of HYA, isolate a sample for insertion into a cloning plasmid to make a recombinant clone. A TOPO cloning reaction followed by a double DNA digest was accomplished to verify gene sequencing and orientation in the clone. SYBR Green real time RT- PCR was used to quantify gene expression relative to 18S RNA. RESULTS: RT-PCR provided qualitative data indicating HYA1, HYA2, and HYA3 are present at all observed time points (E8.5, E9.5, E10.5, E11.5, E12.5, E13.5, E14.5, E15.5, and E16.5). Real time RT-PCR data results characterizing relative expression for HYA2: E9.0 (Rel. Exp. = 1.00; SD = 0), E10.5 (Rel. Exp. = 1.33; SD = 0.577), E12.5 (Rel. Exp. = 2.00; SD = 0), E13.5 (Rel. Exp. = 2.66; SD = 0.577), E14.5 (Rel. Exp. = 3.00; SD = 0), E15.0 (Rel. Exp. = 2.00; SD = Error). CONCLUSIONS: HYA1, HYA2, and HYA3 are present at al time points observed in embryonic heart tissue. Relative expression of HYA2 progressively increased from E9.0 until E14.5 and then started tapering downward at time point E15.0.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1956. / Typescript. Abstracted in dissertation abstracts, v. 16 (1956) no. 2, p. 210-211. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.