Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.36 (hyaluronidase)
4,606 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A zymographic assay for the determination of hyaluronidase activity in cell-free extracts on native polyacrylamide gels has been developed. In this assay an agarose replica of the polyacrylamide gel which contains hyaluronic acid and bovine serum albumin (BSA) was used. After an incubation at 37 degrees C to allow transfer and development of enzymatic activity, the hyaluronic acid and BSA were precipitated in the agarose gel with 2 M acetic acid. Areas of enzymatic activity appeared as clear zones in the agarose replica. The assay was sensitive and was used to demonstrate hyaluronidase activity in cell-free extracts from a number of bacterial and mammalian species.
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PMID:A zymographic assay for detection of hyaluronidase activity on polyacrylamide gels and its application to enzymatic activity found in bacteria. 163 6

The application of flow cytometry to enrich airway epithelial cell subpopulations is described. A complementary epithelial cell preparative technique is also outlined. The ability of the airway epithelium to protect the lung from environmental insults results from a complex interaction among the different cells that form its matrix. The separation of the different epithelial cell types is an essential step in the studies of mechanisms of the controlling factors of cell repair, cell differentiation, and neoplastic transformation. Epithelial cells of the New Zealand white rabbit trachea are prepared using enzymatic digestion and microdissection. Small sections of tracheal wall are dissected into pieces approximately 10 mm2. The mucosa is dissected and placed in 0.15% hyaluronidase for 40 min at 22 degrees C. Mucus is removed, and the mucosa is then placed in 0.1% pronase at 37 degrees C for 30 min. With careful dissection, the epithelium can be dissected from the mucosa in 10-mm2 sheets. Sheets of epithelial cells are placed in 6 ml of an enzymatic solution containing collagenase, 0.2% bovine serum albumin, 0.04% soya bean trypsin inhibitor, 0.06 ml of 1 M Hepes buffer for 3 h at 37 degrees C. The cells are gently pipetted during the 3-h period, yielding a suspension of viable cells. Subpopulations of these different cell types are enriched using an Orthocytofluorograph 50111. A krypton ion laser was used for excitation of cells at 488 nm. Forward-angle and 90 degrees scatter were gated on the histogram. The purification of the ciliated, basal, and secretory cells was 90%, 97%, and 94%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Enrichment of subpopulations of respiratory epithelial cells using flow cytometry. 184 47

Following growth in a chemically defined medium (CDM), five strains of Streptococcus uberis were tested for their ability to survive killing by bovine neutrophils. Strains 0140J, ST10, EF20 and C221 were easily killed, whereas strain C197C was highly resistant. The ability of strain 0140J to resist phagocytosis and killing was increased by supplementation of the growth medium with milk whey, casaminoacids, casein, or, to a lesser extent, bovine serum. Supplementation of the growth medium with yeast extract or bovine serum albumin did not affect the resistance of this strain. Following growth in CDM supplemented with casein, strains ST10 and C221, like strain 0140J, were significantly more resistant to killing by neutrophils. The resistance of strains EF20 and C197C was unaffected by the addition of casein to the medium; strain EF20 remained susceptible and strain C197C highly resistant to killing. The effect of supplementing the growth media with components other than casein was only studied for strain 0140J. Decapsulation of strains C197C, ST10 and 0140J, grown in CDM + casein, with type-X hyaluronidase did not significantly affect their ability to survive in the presence of bovine neutrophils.
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PMID:Killing of Streptococcus uberis by bovine neutrophils following growth in chemically defined media. 204 85

Studies were conducted into behaviours of total and extracellular hyaluronidase activities of bull semen under selected incubation phenomena. Particular attention was given to effects generated by media of high and physiological ion strengths (385 mosm and 305 mosm), in vitro capacitation treatment, and effects of various washing techniques as well as incubation temperatures. Total hyaluronidase activity was clearly lowered in washed ejaculate sperms, as compared to original sperm, with extracellular enzyme activity, however, being unchanged. Hyaluronidase activity was also lower in deep-frozen sperm. Increase in extracellular and total hyaluronidase activity was at its highest under the impact of cattle serum albumin and with a defined temperature-time regime (four hours of comparative incubation in a medium of physiological ion strength at 35 degrees C, 37 degrees C, 39 degrees C, 41 degrees C, and 45 degrees C). Release of hyaluronidase was most clearly inducible after pretreatment in medium of physiological ion strength and by four hours of incubation at 39 degrees C. The preliminary conclusion is that the values so far recorded appeared to reflect some superimposition of physiological and degenerative processes which could not be distinguished from each other under the experimental conditions chosen for the studies.
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PMID:[Hyaluronidase release in bull sperm--influence of media and temperature]. 234 33

An improved micro method for measuring sulfated glycosaminoglycans (S-GAG) in chondrocyte cultures using 1,9-Dimethylmethylene Blue (DMB) has been developed. By increasing the protein concentration in the DMB assay a soluble GAG-DMB complex is prolonged. Without bovine serum albumin (BSA) in the phosphate-buffered saline (PBS) medium, the half time for loss of absorbance was 18 min; with 1% BSA-PBS there was no loss of absorbance over this time period. The limit of detection in a 96 well microtiter plate assay was 2 micrograms/ml; for a cuvette assay it was 1 microgram/ml. Collagen, DNA and RNA did not interfere with this assay. Hyaluronate caused an increase in absorbance at 530 nm that was lost by preincubating with Streptomyces hyaluronidase. The increase in absorbance was due to a turbidity change because there was no color shift from 600 to 530 nm but rather a uniform increase in absorbance between 400 to 700 nm. To validate the assay, the S-GAG was measured in conditioned medium from primary bovine articular chondrocyte monolayer cultures. A protein synthesis inhibitor, cycloheximide, blocked proteoglycan synthesis by greater than 90%. A cytokine, Interleukin-1 alpha, caused a dose-dependent decrease in proteoglycan accumulation. Chondroitinase ABC digestion of the chondrocyte conditioned medium completely prevented reactivity with the DMB. By preincubating samples with specific enzymes, different types of S-GAG can be measured with this assay. This assay can be used to measure changes in proteoglycans synthesized by chondrocytes.
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PMID:An improved method for determining proteoglycans synthesized by chondrocytes in culture. 237 28

The binding of a hyaluronic acid-binding glycoprotein, hyaluronectin (HN), isolated from human brain, to hyaluronic acid (HA) was investigated with the enzyme-linked immunosorbent assay technique using plastic microtest plates coated with a 50 mg/liter solution of HA in 0.1 M bicarbonate. Optimum conditions for HN binding to HA were in 0.2 M NaCl buffered with 0.1 M sodium phosphate at pH 7. An assay for HA in solution was set up exploiting the fact that HN binding could be inhibited by soluble HA. HA was preincubated for 1 h in a test tube with a 30-ng/ml HN solution (v/v) in the buffer containing 0.1% bovine serum albumin. Incubation on HA-coated microtest plate lasted 4 h and maximum sensitivity was achieved when incubation was carried out at 4 degrees C. HN bound to the plate was revealed by means of alkaline phosphatase-conjugated anti-HN antibodies. The test was used to measure HA inhibitory activity after depolymerization by ferrous ions. No difference was found between inhibitory activity or smaller fragments and that of high-molecular-weight HA. The assay was applied to determination of HA in sera. Specificity was demonstrated by Streptomyces hyaluronidase digestion of reactive material in sera. Other glycosaminoglycans did not interfere with the assay. Recovery of HA was good and intra- and interassay variation coefficients were 6 +/- 2.2 and 12%. In 103 blood donor sera, HA was found at 22.4 +/- 16.7 micrograms/liter. HA was elevated in most of the cancer patient sera tested.
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PMID:Immunoenzymoassay of the hyaluronic acid-hyaluronectin interaction: application to the detection of hyaluronic acid in serum of normal subjects and cancer patients. 241 43

A hyaluronidase-sensitive component of human peritoneal fluid from a patient with Wilms' tumor when injected into rabbits has been shown to suppress the formation of humoral precipitating antibodies to certain major classes of proteins present in the fluid. Furthermore, it has been found that hyaluronic acid, when included with certain test antigens (serum albumin, fetuin) or antigen mixtures (tumor isolates or mixtures of albumin, immunoglobulin G and immunoglobulin M), produces a marked distortion or complete blockage of immunoelectrophoresis precipitin arcs, as well as altered gel chromatography elution profiles. These findings that hyaluronic acid can interfere profoundly with both the elicitation of a complete antibody response and the formation of "normal" patterns of antigen-antibody precipitates in laboratory tests supports the possibility that this polysaccharide may play an immuno-regulatory role by masking potential immunogens. Consideration of the mechanisms for these in vivo and in vitro effects suggests that there may be some common basis in an "excluded volume" property of the hyaluronate, but this does not appear sufficient to explain the complexity and selectivity of the observed phenomena.
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PMID:The selective suppression of immunogenicity by hyaluronic acid. 242 4

In neutrophils preincubated with non-inflamed synovial fluid, opsonized zymosan-induced O2- production was increased by 232.7 +/- 19.1% and degranulation induced by zymosan or phorbol myristate acetate was increased by 152.8 +/- 21.8% and 217.4 +/- 26.3 respectively. Unstimulated neutrophils were not directly activated by the fluid, nor did it affect their chemotactic response. The activity was not abolished by heat (56 degrees C, 30 min, or 100 degrees C, 3 min) or by treatment with trypsin or hyaluronidase. The activity was not present in the serum albumin-bound lipid extract of the synovial fluid. Thus, this activity may represent a heat- and trypsin-resistant factor which is neither a complement component nor hyaluronic acid. It is proposed that this synovial fluid factor plays a role in increasing the neutrophil killing potential during the inflammatory process.
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PMID:The effect of non-inflamed synovial fluid on neutrophil function in vitro. 299 26

Prepuberal (P) gilts were induced to ovulate with pregnant mare serum gonadotropin followed 72 h later by human chorionic gonadotropin (hCG). Three P gilts and three mature (M) gilts each were ovariectomized on d 10, 14, 18, 22 and 26 (d 0 = day of hCG for P gilts and onset of estrus for M gilts). Gilts ovariectomized on d 14, 18, 22 and 26 were hysterectomized on d 6 to ensure maintenance of the corpora lutea (CL). Two to five grams of minced luteal tissue were dispersed using collagenase and hyaluronidase in HEPES buffered salt solution supplemented with glucose and bovine serum albumin. Dispersed cells were rinsed in Dulbecco's Modified Eagle Medium (DMEM), counted (ratio of large to total number of luteal cells determined) and then incubated for 1 h in DMEM. With aliquots standardized to 2.5 X 10(4) viable, large cells (greater than 25 micron diameter) were incubated in 1 ml DMEM for 2 h in the presence of either 10, 50, 100 or 1,000 ng luteinizing hormone (LH); .1, 1, 10 or 100 ng hCG; 10, 100 or 1,000 ng norepinephrine (NE) or either .75, or 1.5 mM dibutyrl cyclic adenosine monophosphate (dbcAMP). Progesterone (P4) in the medium was quantified by radioimmunoassay. Basal P4 production (no P4 stimulator added to the medium) on d 10, 14, 18, 22 and 26 for P gilts was 246 +/- 9, 66 +/- 4, 64 +/- 6, 41 +/- 3 and 69 +/- 6 ng/ml medium, respectively, and for M gilts was 281 +/- 12, 128 +/- 8, 53 +/- 4, 82 +/- 6, 101 +/- 5 ng/ml medium, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Comparison of induced corpora lutea from prepuberal gilts and spontaneous corpora lutea from mature gilts: in vitro progesterone production. 303 Sep 95

Previous studies have shown that hamster sperm release a significant amount of hyaluronidase before and independently of the normal acrosome reaction. In this study, we have used improved methods for in vitro incubation to investigate the time course of the release of hyaluronidase and hexosaminidase from hamster sperm. When hamster sperm are incubated in medium which allows capacitation, 34 to 47% of the total mechanically extractable hyaluronidase and 34 to 51% of beta-N-acetylhexosaminidase are released into solution prior to and independently of the normal acrosome reaction (ARx). An additional 40 to 50% of the hyaluronidase and 34 to 51% of the hexosaminidase are released at the time of the normal ARx. Control experiments indicate that the early release is not due to the presence of dead sperm in culture and that the normal ARx is required for the second release. Increasing amounts of TCA-precipitated bovine serum albumin in the culture medium stimulated the early (1 hr) release of both enzymes. The data are consistent with the ideas that a significant amount of both enzymes is released from the sperm surface by 1 hr of incubation and that about the same amount of each enzyme is released during the normal ARx. Hyaluronidase and hexosaminidase release at the time of the acrosome reaction was measured for the first time using hamster sperm. The biphasic release of these enzymes may indicate that they have a dual function in fertilization and may help explain how sperm can penetrate the cumulus and corona radiata without undergoing an acrosome reaction.
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PMID:Release of hyaluronidase and beta-N-acetylhexosaminidase during in vitro incubation of hamster sperm. 315 74


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