Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A recently described method for the preparation of isletlike cell clusters (ICC) from human fetal pancreas has been applied to the fetal pig with the ultimate aim of large-scale production of ICC. Fetuses ranging in age from 51 to 77 days were used, and after a brief collagenase-incubation the pancreatic digest was plated into culture dishes containing medium RPMI 1640 supplemented with either 10% fetal calf serum (FCS) or human serum (HS). HS seemed to increase the number of ICC formed as compared to that obtained with FCS. A total of more than 100,000 ICC were produced from each of 3 litters, ages 67-77 days, after culture in the presence of HS. The DNA content of such ICC was reduced by about 50% as compared to those maintained with FCS supplementation. Immunocytochemical staining revealed insulin- and glucagon-positive cells scattered among a majority of nonstained cells within the cell clusters. ICC maintained in either FCS or HS displayed significant rates of (pro)insulin biosynthesis in vitro and an increased insulin release when exposed to 16.7 mM glucose plus 5 mM theophylline. Four weeks after implantation, ICC grafted under the kidney capsule of nondiabetic nude mice contained frequent insulin- and glucagon-positive cells. In 2 nude mice transplanted with ICC, the functional capacity of the graft was tested by perfusing the graft-bearing kidney. When the perfusion fluid was changed from one containing 2.8 mM glucose to one containing 16.7 mM glucose +/- 5 mM theophylline, the secretion of insulin increased within a few min. It is concluded that the fetal porcine pancreas can be used for large-scale production of ICC, which have a very consistent, but immature functional capacity. Because of their inherent growth and differentiation properties, fetal porcine ICC constitute a potential source of xenogenic islet grafts intended for human diabetics.
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PMID:Large-scale production of fetal porcine pancreatic isletlike cell clusters. An experimental tool for studies of islet cell differentiation and xenotransplantation. 327 71

Cell culture systems allow the examination of cell populations in a functional state. To simulate in vivo conditions as closely as possible freshly established cell strains are superior to permanent cell lines. Different aspects for the establishment of primary cell cultures obtained from various tissues are compared: Disintegration, culture media supplemented with basal additions, special supplements (growth factors, hormones), and attachment factors. The proliferation rates of the attained cell strains were evaluated by determination of cell doubling times. Procedures for how to obtain a relatively high plating efficiency (approx. 70% in our series of 219 attempts) of primary growth in vitro are described: (1) Mechanical disintegration is superior to enzymatic digestion. If mechanical treatment alone did not produce a sufficient number of viable cells, additional digestion with collagenase/dispase revealed a higher number of proliferating primary cultures than with trypsin. (2) Proliferation of cell cultures from normal and tumorous tissues of epithelial origin was superior in Leibovitz L 15 medium (58 of 87 (67%) cases). Cultures from mesenchymal tissues and tumors were found to have shortest cell doubling times in MEM and RPMI 1640 (16 of 23 (70%) cases). The media were supplemented with the basal additions indicated. (3) In approx. 30% of the cases special supplements like growth factors or hormones increased cell replication, although they were almost always not essential for cell growth. (4) Attachment factors only rarely contributed to the initiation of primary monolayer cultures. The application of various culture conditions does not lead to a protocol optimal for all tissues, for all probes of the same type of tumor, or for all tumor specimens of unique differentiation.
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PMID:Establishment of primary cell cultures: experiences with 155 cell strains. 330 16

The human fetal pancreas represents a source of insulin-producing beta-cells with a potential for transplantation to diabetic patients. It has previously been shown that such cells can be viably maintained in tissue culture media containing fetal calf serum (FCS) and that these explants continue to synthesize and release insulin. In this study the effects of human serum (HS) on the growth and function of human fetal pancreatic explants have been compared with those of FCS. For this purpose, pancreatic glands, obtained after prostaglandin-induced abortions, were briefly exposed to collagenase, and the digest was cultured in RPMI-1640 medium plus 10% pooled HS or FCS. The outgrowth of isletlike cell clusters (ICCs) was monitored. In 31 of 58 consecutively explanted glands, development of ICCs was observed. In the presence of FCS the outgrowth of ICC took place on top of a fibroblast monocellular cell layer; HS effected less growth of fibroblasts and increased the formation of ICCs about sevenfold compared with explants from the same glands maintained in FCS. However, in the explant cultures with HS, the cell number per ICC, expressed as DNA content, was reduced by 50%. In both FCS and HS the insulin content of the medium showed great variability and progressively declined from day 2 to day 5. The medium glucagon concentration also decreased but not to the same extent as that of insulin. Immunocytochemical-stained ICCs showed insulin- and glucagon-positive cells scattered among most nonstained, presumably nonendocrine cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tissue culture of human fetal pancreas. Effects of human serum on development and endocrine function of isletlike cell clusters. 331 88

The enteric nervous system is a major division of the autonomic nervous system and is responsible for the regulation of gastrointestinal function. The objective of the present study was to develop a simple and effective technique for isolating and culturing neurons of the enteric nervous system that would permit characterization of their development and regulatory peptide content. This was accomplished using a dispersed intestinal cell preparation cultured under conditions designed to support the growth and differentiation of neurons and neuroendocrine cells. Newborn hamster intestine was digested in 0.1% collagenase, mechanically dispersed, and cultured in RPMI 1640 supplemented with 2.5% serum and other additives. Phase and bright-field microscopy demonstrated neuronal cells and fibers after the second day in culture. This was confirmed by immunohistochemistry using antibodies directed against neurofilament and vasoactive intestinal polypeptide. Acetic acid extracts of the culture indicated that during the first 4 days of the culture the content of vasoactive intestinal polypeptide increased, whereas the content of substance P, mammalian bombesin, and neurotensin declined. High-performance liquid chromatography and fast protein liquid chromatography confirmed that the immunoreactive vasoactive intestinal polypeptide coeluted with synthetic and iodinated forms of the peptide. This study describes a technique for primary culture of intestinal tissue that supports the survival of enteric neurons and permits analysis of the development and synthetic and secretory characteristics of the enteric nervous system.
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PMID:Primary culture of the enteric nervous system from neonatal hamster intestine. Selection of vasoactive intestinal polypeptide-containing neurons. 341 Feb 13

Epithelial and stromal cells were isolated from endometrium of Day 1 pseudopregnant rabbits by enzymatic digestion with trypsin or trypsin:collagenase:deoxyribonuclease. Dispersed cells were grown in RPMI 1640 supplemented with 10% whole or steroid-depleted fetal bovine serum (FBS). Epithelial and stromal cells reached confluency after 6 to 7 days in culture and showed specific characteristics. Cells could be differentiated according to morphology, growth patterns, electrophoretic patterns, and response to estrogen or progesterone. Hormonal stimulation of adenylate cyclase activity was measured in broken cell preparations by catalytic transformation of alpha-32P-adenosine triphosphate into 32P-adenosine 3'-5' cyclic monophosphate (cAMP). Adenylate cyclase activity was present in fresh endometrial tissue and in dispersed cells after 7 days in culture. The enzyme activity was significantly higher in stromal than in epithelial cells at all stimulation levels: basal (9.2 +/- 1.0 vs. 2.3 +/- 0.6, p less than 0.001) and guanosine triphosphate (GTP, 300 microM) (25.4 +/- 2.9 vs. 7.0 +/- 1.6, p less than 0.001). Net response to prostaglandin E2 (PGE2, 10 microM) was three times higher (p less than 0.001) in stromal (17 +/- 2) than in epithelial (5.0 +/- 1) cells. These results suggest that PGE2 can stimulate adenylate cyclase in rabbit endometrium and that the enzyme is preferentially localized in the stroma. Our results are in agreement with the hypothesis that cAMP formed in endometrium in response to PGE2 might be involved in the decidual reaction.
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PMID:Cell-specific localization of prostaglandin E2-sensitive adenylate cyclase in rabbit endometrium. 347 35

Intracellular localization of thyroglobulin (TG) using a pre-embedding diffusion technique and an indirect localization sequence has been made in human thyroid obtained from 20 patients with treated Grave's disease. Both antibodies, anti-human TG-rabbit IgG F(ab')2 and anti-rabbit IgG F(ab')2-goat IgG F(ab')2 fragments easily penetrated the cytoplasm of follicular cells which were dissociated by RPMI-1640 solution containing collagenase, dispase, and deoxyribonuclease. With light microscopic observation of semithin sections positive immuno-reaction for TG was demonstrated as fine granular deposits in the cytoplasm of the dissociated cells. In electron microscopic studies, intracellular antigen was well circumscribed within certain cell organelles in all cases with the positive immuno-reaction for TG being observed in perinuclear space, rough endoplasmic reticulum, Golgi complexes, secretory granules, and reabsorbed colloid droplets. Content of positive immuno-reaction product differed somewhat from one case to another and from one follicle to another even in the same case. There was no immunoreaction product in nuclei, mitochondria, lysosomes, and lipofuscin-like granules.
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PMID:An immuno-electron microscopic study on intracellular localization of thyroglobulin (TG) in the thyroid gland in Grave's disease. 389 13

The present study evaluates the development and function of human fetal B-cells in vitro with a view to using such cells in future attempts for transplantation of human fetal pancreas to diabetic patients. A method previously described in our laboratory for preparing islets in vitro from the fetal rat pancreas has been applied and modified for use with human fetal pancreas. Pancreatic glands of different gestational ages were obtained from 37 consecutive prostaglandin-induced abortions. After a mild collagenase treatment, the partially disintegrated tissue was maintained in culture for 7 days in tissue culture medium RPMI 1640 plus 20% fetal calf serum to permit cell attachment and out-growth of endocrine cells. In 17 of the 37 consecutively cultured fetal pancreatic glands, islet-like cell clusters were formed. The 20 remaining glands were lost because of either bacterial contamination or lack of viability already before dissection had occurred. Sections of the newly formed cell clusters revealed well-preserved pancreatic cells showing frequent mitotic figures. The tissue exhibited a high rate of (pro)insulin biosynthesis and a modest insulin response to secretory stimuli, suggesting that the mechanism of glucose regulation by the fetal B-cells is not yet fully developed. Electron micrographs showed a large number of granule-containing cells, some of which were identified as B-cells. In nine cases, harvested cell clusters were implanted beneath the kidney capsule of nude mice. When these animals were killed after 2 mo, seven mice showed a considerable growth of the grafts with numerous islet-like structures containing insulin- and glucagon-positive cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tissue culture of human fetal pancreas. Development and function of B-cells in vitro and transplantation of explants to nude mice. 393 Mar 24

Various conditions for tissue culture of collagenase-isolated mouse pancreatic islets were studied for their effects on the glucagon production of the cultured specimens. Culture media containing heat-treated bovine calf serum degraded [125I]glucagon to a much less extent than those supplemented with untreated serum. Addition of aprotinin to the heat-treated serum gave a further reduction of the [125I]glucagon degradation in the culture medium. A similar supplementation of Medium 199, used for culture of isolated islets, resulted in the most extensive glucagon accumulation in the culture medium. Islets cultured free-floating or attached to the bottom of the culture dishes contained similar amounts of glucagon. However, the free-floating islets released less glucagon when tested in short-term experiments performed at the end of the 1 wk culture period. A comparison between different culture media showed that islets cultured in RPMI-1640 had the highest glucagon content and released most glucagon to the culture medium. Moreover, these islets responded most actively to an acute arginine challenge at the end of the culture period. The present data suggest that the optimal conditions for culture of isolated islets aimed at studies of glucagon production may be obtained by using a culture medium consisting of RPMI-1640 supplemented with both a proteinase inhibitor and heat-inactivated serum.
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PMID:Glucagon production by cultured pancreatic islets: effects of different culture conditions and media. 616 39

Mouse monoclonal antibodies to various human epidermal and basement membrane components were formed by immunizing Balb/c mice with ME-180, a line of human cervical carcinoma cells. The spleen cells from hyperimmunized mice were fused with a nonsecreting mouse myeloma cell line using polyethylene glycol. The resulting hybrids were selected by growth in media containing 20% fetal calf serum, hypoxanthine, thymidine, and methotrexate in RPMI-1640 in 24-well Linbro plates. Wells producing antibodies of interest were grown and eventually cloned over an HGPRT- rat fibroblast feeder layer. These cultures were expanded and recloned. Two cloned antibodies of interest are DUX 5.2 and DUX 1.1.3. DUX 5.2 is the mouse IgG1 subclass and reacts with the membranes of ME-180 cells and the human skin epidermal basement membrane zone as shown by direct immunofluorescent microscopy. Ultrastructural localization using electron microscopic immunoperoxidase techniques showed localization of the DUX 5.2 antigen to be beneath the lamina densa; the reaction product may include the anchoring fibrils. Although DUX 5.2 reacts with the normal human basement membrane zone and the basement membrane zone in several diseases, there is no reactivity in the normal, never-blistered skin of patients with dystrophic epidermolysis bullosa (DEB). This suggests that the increased collagenase in the disease may be destroying antigenicity of the antigen recognized by DUX 5.2 or that the antigen may not be present in DEB. This antibody will thus allow early neonatal and prenatal diagnosis in DEB and allow isolation of the structural moiety which is deficient in DEB. DUX 1.1 is an IgM mouse immunoglobulin specific for the cytoplasm of human basal cells. Its reactivity with upper epidermis is significantly less than that seen in the basal layer. All cells of the basal layer stain uniformly. The slight amount of staining in upper cells probably represents dilution of antigen which is not synthesized beyond the basal layer. Basal cells of hair follicles and sweat glands are stained to some degree.
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PMID:Monoclonal antibodies to normal and abnormal epithelial antigens. 619 49

Human pancreatic islets were isolated by collagenase treatment of pancreatic tissue obtained from 27 individuals aged 12 to 69 years. The islets were maintained free floating in tissue culture medium RPMI 1640 supplemented with calf or human serum. In two cases the insulin production was followed up to nearly two years. The insulin production rate of the individual islet preparations varied between 0.2 and 8 ng per islet per day. No significant correlation with donor age or sex was found. The glucose concentration in the medium influenced the insulin release in a dose dependent manner. The acute response of the cultured islets to glucose was evaluated both by batch incubation and by perifusion. Both in the acute and the chronic experiments maximal insulin release was found at 10 mM glucose. In conclusion, these experiments indicate that viable islets of Langerhans can be obtained from adult human pancreatic tissue and that their beta-cell function can be maintained for up to two years. The variation in insulin production rate could not be ascribed to age or sex and may reflect both physiological and methodological factors.
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PMID:Beta-cell function in isolated human pancreatic islets in long-term tissue culture. 626 82


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