Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.36 (hyaluronidase)
 4,606 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The purpose of this investigation was to provide evidence for the secretion of high molecular weight mucins, CTM-A and CTM-B, in primary culture of canine tracheal epithelial (CTE) cells. The cells were isolated from tracheas of mongrel dogs by pronase treatment. Primary cultures of the epithelial cells were established using ICN collagen inserts in Dulbecco's modified Eagle's/F12 medium supplemented with growth factors and could be maintained for up to 23 days. The evidence for the mucin secretion in culture medium and their localization in the cells was established by a) positive immunocytochemical staining using specific antibodies developed against purified native as well as deglycosylated CTM-A and CTM-B; b) incorporation of labeled amino acids, followed by electrophoresis and autoradiography detection of glycoconjugates purified from the culture medium; c) comparison of the amino acid compositions of mucin purified from canine tracheal pouch secretions and that purified from the culture medium; and d) Western blot analyses using specific polyclonal antibodies directed against deglycosylated CTM-A and CTM-B. Immunoaffinity purified secreted labeled glycoconjugates were resistant to hyaluronidase treatment. The effects of cyclic AMP (1 x 10(-5) M), dibutyryl cyclic AMP (1 x 10(-5) M), 8-bromocyclic AMP (1 x 10(-5) M), and prostaglandin E1 (1 x 10(-6) M) on mucin secretion by CTE cells were also investigated. Secretion of mucins by CTE cells in culture was considerably more enhanced by 8-bromocyclic AMP than that observed for other secretagogues used in this study.
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PMID:Evidence for secretion of high molecular weight mucins by canine tracheal epithelial cells in primary culture: effects of select secretagogues in mucin secretion. 131 Dec 94

We describe a method for establishing the culture of bovine tracheal submucosal gland (BTG) cells, in which we have also examined the influence of a reconstituted basement membrane matrix derived from the Engelbreth-Holm-Swarm tumor (EHS) on the growth and morphological differentiation of these cells. BTG cells have been isolated by tissue enzymatic digestion using trypsin, deoxyribonuclease I, elastase, hyaluronidase and EGTA for 1 hr at 37 degrees C. Afterwards, cells and tissue were collected by centrifugation and were incubated for 15 min with 15% newborn calf serum to inactivate the proteolytic enzymes. Enzymatic digestion using only trypsin, centrifugation and inactivation steps were repeated three times. Using this protocol, we obtained 15 +/- 4 (X 10(6] cells per g of tracheal submucosa with 72 +/- 2% (n = 5) cell viability. On microscopic observation, isolated cells were mainly composed of serous type glandular cells. Cells were cultured in a 1:1 medium of Dulbecco's Modified Eagle's/Ham's F12 supplemented with 10% fetal calf serum and subcultured in either plastic flasks or flasks coated with EHS matrix. On the plastic, the BTG cells exhibited at confluency an epithelioid appearance. They stained positively with the immunofluorescent anticytokeratin antibody and contained PAS-staining granules. By electron microscopy, lactoferrin, a protein marker specific to the serous cells, was demonstrated immunocytochemically in small secretory vesicles. BTG cells cultured on EHS matrix revealed a significantly increased growth in comparison to those cultured on plastic. In post-confluent culture of BTG cells on EHS matrix, we observed numerous dome-like structures formed by differentiated cells which were joined together around luminal spaces.
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PMID:Growth and characterization of isolated bovine tracheal gland cells in culture. Influence of a reconstituted basement membrane matrix. 260 69

In experimental animal models the susceptibility of the mammary gland to neoplastic transformation is related to its degree of development and proliferative activity; this observation led us to determine whether the human breast epithelium also exhibits development-related differences, and whether these differences could be detected in an in vitro system. Normal breast tissue obtained from reduction mammoplasties of 9 patients ranging in age from 18 to 56 years were characterized in both whole mount preparations and organoids obtained after collagenase-hyaluronidase digestion by their degree of development based upon the types of lobules present. Lobules were classified into type 1 (Lob 1), composed of approximately 11 alveolar buds, the less developed; lobules type 2 (Lob 2), of moderate development, composed of approximately 47 ductules each, and lobules type 3 (Lob 3), composed of 80 ductules each, represented the highest level of development. Epithelial organoids obtained after digestion were plated in DMEM:F12 medium supplemented with hydrocortisone, cholera toxin, insulin and 5% horse serum with a calcium concentration of 1.05 mM Ca++. Following attachment, the medium was replaced by medium containing 0.040 mM Ca++. The percentage of attachment of organoids to the flask was greater in cells from Lob 1 (89-99%) and Lob 1+2 (79-100%) than in cells from Lob 3, which had a 53-67% attachment. The total yield of cells after 7 weeks in culture was also greater in cells derived from Lob 1 and Lob 1+2 than in cells from Lob 3. The total yield of cells obtained from primary cultures was not related to the number of organoids plated, but to the degree of development of the gland. The DNA-labeling index (DNA-LI) in intact breast tissue correlated with that in primary cultures; it was greater in Lob 1 and Lob 1+2 than in Lob 3. By flow cytometry, the highest percentage of cells in S-phase was seen in cells with the highest DNA-LI. We concluded that the growth characteristics of mammary epithelial cells in vitro in a low Ca++ medium is modulated by the degree of development and differentiation of the gland.
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PMID:Influence of human breast development on the growth properties of primary cultures. 275 52

The transplantable hormone-responsive rat mammary adenocarcinoma 13762NF was dissociated with collagenase and hyaluronidase. Cells were cloned directly or lines were established from mass cultures and cells from these lines were cloned. Clones differed in cellular morphology, colony morphology on plastic or in collagen gel, growth rate, growth response to hormones, and hormone receptor levels. Growth response to prolactin, estradiol, progesterone, cortisol, and epidermal growth factor (EGF) was determined by culturing the cells within collagen gel and using a serum-free medium base of DME/F12 (1:1) with insulin, linoleic acid, and BSA. The clones varied in their hormone responses, with all 20 of the clones tested responding to cortisol in combination with EGF. Some clones would respond to EGF, cortisol, or progesterone when used alone. None of the clones tested could be stimulated by prolactin or estradiol. Receptor levels for estradiol, progesterone, glucocorticoids, and EGF were assessed in 3 selected clones differing in their hormone responsiveness. Receptor levels appeared to correlate with hormonal sensitivity. Selected clones transplanted into female F344 rats produced carcinomas with histopathologies similar to the original tumor.
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PMID:Heterogeneity in the hormonal responsiveness of clones derived from the 13762NF rat mammary tumor. 300 10

In reconstructive head and neck surgery, there is a great need for cartilage transplants. Sufficient autologous graft is often not available. Heterologous cartilage is used frequently, although there is danger of transmitting viral infections and resorption rates are high. We have developed a three-dimensional model for the formation of cartilage in vitro. The aim of this study was to characterize the collagen synthesis under these culture conditions. Human chondrocytes were isolated by digesting septal cartilage matrix in the presence of type II collagenase, hyaluronidase, and Dnase II in Ham's F12 medium. The resulting cells were kept in monolayer culture for one week and then suspended in 2% ultra-low-melting agarose (1:1). The cell-agarose conglomerate was encapsulated with a 3% ultra-low-melting agarose solution and placed in a perfusion culture chamber. A permanent flow of fresh medium (Ham's F-12 supplemented with 50 micrograms/ml ascorbic acid and 2% fetal calf serum) was provided by a peristaltic pump which delivered 1 ml/h with on/off intervals of 30 min. Samples were recovered after two weeks. Using electron microscopy abundant collagen fibril formation was shown. The collagen fibrils were identified histologically as cartilage specific type II collagen. No mRNA expression of collagen type X was observed using in situ hybridization. The cells appeared in a round cell shape with round nucleus and only slight variations in form and size. The present results indicate that the chondrocytes maintain their differentiated phenotype and continue to synthesize typical matrix products in this three-dimensional perfusion culture chamber.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Cultivation of human cartilage tissue in a 3-dimensional perfusion culture chamber: characterization of collagen synthesis]. 749 39

In the field of otolaryngology cartilage grafting is commonly performed to reconstruct skeletal defects. Knowledge of chondrocyte growth and differentiation can now be used to engineer cartilage tissue for grafting. The first condition is that chondrocytes maintain their differentiated phenotype besides being able to produce a new cartilage matrix. The target of this study was to develop a three-dimensional culture system for in-vitro formation of vital cartilage transplants. Chondrocytes were isolated by digesting the cartilage matrix with collagenase and hyaluronidase. After embedding in "low-melting" agarose, the chondrocytes were placed into a perfusion culture chamber to provide a constant supply of nutrients to the cultures. The peristaltic pump was operated with on/off intervals of 30 min. Ham's F12 supplemented with 2% FCS and 50 micrograms/ml ascorbic acid was employed as culture medium. Monoclonal antibodies specific to collagens type I and type II were used to characterise cells and matrix synthesis. Synthesis of proteoglycans and collagens was achieved using toluidine blue and azan staining. Under the described culture conditions, the chondrocytes maintained a differentiated phenotype (expression of collagen type II) with synthesis of collagens and proteoglycans. An accumulation of matrix products was achieved pericellularly. After 2-8 weeks the obtained tissue exhibited an excellent histological appearance showing the typical features of cartilage tissue. The results show that the perfusion chamber allows a quick in-vitro fabrication of a piece of pure cartilage tissue for transplantation.
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PMID:[Culture of human cartilage tissue using a perfusion chamber]. 781 42

The major functions of granulosa cells (GCs) include the production of steroids, as well as a myriad of growth factors to interact with the oocyte during its development within the ovarian follicle. Also FSH stimulates GCs to convert androgens (coming from the thecal cells) to estradiol by aromatase. However, after ovulation the GCs produce progesterone that may maintain a potential pregnancy. Experiments with human GCs are mainly focused on the purification of GCs from ovarian follicular fluid followed by FACS analysis or short-term cultivation. The aim of our study was to cultivate GCs for a long period, to characterize their morphology and phenotype. Moreover, we have cultivated GCs under gonadotropin stimulation in order to simulate different pathological mechanisms during folliculogenesis (e.g. ovarian hyperstimulation syndrome). GCs were harvested from women undergoing in vitro fertilization. Complex oocyte-cumulus oophorus was dissociated by hyaluronidase. The best condition for transport of GCs was optimized as short transport in follicular fluid at 37 degrees C. GCs expansion medium consisted of DMEM/F12, 2% FCS, ascorbic acid, dexamethasone, L-glutamine, gentamycine, penicillin, streptomycin and growth factors (EGF, bFGF). GCs transported in follicular fluid and cultivated in 2% FCS containing DMEM/F12 medium supplemented with follicular fluid presented increased adhesion, proliferation, viability and decreased doubling time. Cell viability was 92% and mean cell doubling time was 52 hrs. We have optimized transport and cultivation protocols for long-term cultivation of GCs.
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PMID:The cultivation of human granulosa cells. 1927 84