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:3.1.25.1 (deoxyribonuclease)
1,471 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed an enzymatic technique for isolating human intestinal mucosal lymphoid cells. This method was found to be superior to mechanical methods in regard to cell yield and survival. It is based on treating mucosa with serum-free solutions containing collagenase and deoxyribonuclease, followed by isolating the lymphoid cells through centrifugation steps involving fetal calf serum and ficoll-hypaque. Exposure of peripheral blood lymphocytes to the components of the enzymatic solution did not appreciably alter their uptake of tritiated thymidine in the presence or absence of mitogens. Application of the method to derive lymphoid cells from Crohn's disease, ulcerative colitis, and normal intestinal mucosa has shown that gut mucosal lymphocytes from inflammatory bowel disease (1) exceed the number of those from normal mucosa by a factor of 3 to 5; (2) show different degrees of tritiated thymidine uptake, spontaneously and in response to mitogens, depending upon the time they are harvested during the dissociation process; (3) are better stimulators than responders in the allogeneic mixed lymphocyte reaction; (4) generate suppressor cell activity comparable to that of peripheral blood lymphocytes; (5) cannot, in contrast to peripheral blood lymphocytes, generate antibody-dependent cell mediated cytotoxicity; and (6) produce an average of 5 times more IgM than equal numbers of peripheral blood lymphocytes.
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PMID:Gut mucosal lymphocytes in inflammatory bowel disease: isolation and preliminary functional characterization. 15 97

Lymphocytes infiltrating synovial membranes were characterized in eight patients with proliferative rheumatoid synovitis. Surface immunoglobulins were studied with use of immunofluorescence, and the C3 receptor was detected by adherence of red cells coated with antibody and complement - both are B-cell markers. Spontaneous rosette formation with sheep erythrocytes was used as a T-cell marker. To obtain viable lymphocytes in suspension, the villous synovium of five of these patients was digested with collagenase and deoxyribonuclease. Populations enriched in lymphocytes could be obtained by velocity sedimentation. Whereas only 9 to 35 per cent of lymphocytes bore surface immunoglobulins, the majority (70 to 85 per cent) formed sheep-erythrocyte rosettes. Cells bearing the C3 receptor constituted a distinct minority of synovial lymphocytes in frozen-tissue sections, and were found in follicle-like accumulations. These data indicate that the predominant infiltrating lymphocyte in proliferative rheumatoid synovitis is a T cell.
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PMID:Predominantly T-cell infiltrate in rheumatoid synovial membranes. 16 88

A simple two-step procedure was developed for isolation of lymphocytes from chronically inflamed human synovial membranes. In the first step minced inflamed synovial tissues are disrupted enzymatically by deoxyribonuclease and collagenase. The second step consists of nylon-wool column filtration of the isolated cells. 7 min of preincubation of up to 37.4 X 10(6) cells in a column packed with 600 mg nylon-wool in 6 ml prior to filtration did not result in significant selective losses of either T or B cells, whereas 45 min of preincubation did. Recovery of lymphocytes after nylon-wool column filtration ranged from 68 to 95% (mean 80%) and viability was always higher than 90%. Nylon-wool column filtration increased the proportion of lymphocytes by a mean 73%. The method allows rapid identification of synovial tissue lymphocyte subpopulations as well as characterization of their function.
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PMID:Lymphocyte isolation from chronically inflamed synovial membranes. 30 51

The intraperitoneal administration of [3H]thymidine to adult rats resulted in the rapid appearance of label in the adipocyte fraction of collagenase digests of adipose tissue. Low-speed centrifugation followed by freezing and slicing showed the label to be uniformly distributed in the adipocyte fraction. The presence of label in DNA was confirmed by hydrolysis with deoxyribonuclease and by inhibition of incorporation with hydroxyurea. Organelle fractionation revealed that the label was predominantly in nuclei, and radioautography showed that only a few adipocyte nuclei were labeled. The label in the adipocyte fraction could not be reduced by increased collagenase digestion or by trypsin treatment. Mixing of labeled adipocytes with unlabeled stroma did not result in decrease of label and addition of labeled stroma to unlabeled adipocytes did not cause significant transfer of radioactivity. Addition of [3H]thymidine to the collagenase digestion medium of unlabeled adipose tissue resulted in more incorporation by adipocytes than by stroma, suggesting the presence of a very rapidly proliferating cell type associated more with adipocytes than with stroma. In vivo turnover studies of labeled DNA indicated that there are two components in both adipocytes and stroma, a rapidly labeled component with a half-life of only several days and another with a half-life of several months. These experiments suggest that there is a rapidly proliferating cell type in adipose tissue, closely associated with mature adipocytes, that may be an adipocyte progenitor or may have some other unknown function.
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PMID:Isotopic labeling of DNA in rat adipose tissue: evidence for proliferating cells associated with mature adipocytes. 49 48

Experimental autoimmune thyroiditis was induced in out-bred guinea pigs by isoimmunization with thyroid extract in complete Freund's adjuvant. A digestion procedure using collagenase and deoxyribonuclease was used to make viable single-cell suspensions of pooled thyroid glands from groups of animals with advanced degrees of thyroiditis. Thymus-derived or T lymphocytes, identified by their capacity to form E rosettes with rabbit erythrocytes, were found to be the predominant (75%) infiltrating lymphocyte; bone marrow-derived or B cells consitituted most of the remainder. The infiltrates of inbred animals with thyroiditis were demonstrated to contain cells capable of mediating antibody-dependent lymphoid cell-mediated cytotoxicity.
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PMID:Experimental autoimmune thyroiditis in the guinea pig: characterization of infiltrating lymphocyte populations. 79 44

A method is described for the dissociation of mouse ovaries and the isolation of oocytes free of somatic cells by agitating pieces of ovary in collagenase and deoxyribonuclease in a calcium and magnesium free salt solution. This method yielded about 50% of the growing oocytes from immature mice. The utilization of exogenously administered 14C-labelled energy sources by oocytes in various growth stages was determined by measurement of evolved 14CO2. Little or no evolution of 14CO2 was detected from oocytes of any size incubated in 14C-glucose, lactate or succinate. The production of 14CO2 from 14C-pyruvate increased logarithmically when plotted against increasing oocyte volume with a plateau occurring after occytes reached a volume of 65,500 mum3 (50 mum diameter). Thus, the pattern of energy metabolism for oocyte maturation and early egg cleavage, wherein glucose and lactate are not utilized as efficiently as pyruvate, has been established by the earliest stages of oocyte growth.
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PMID:Analysis of mouse oogenesis in vitro. Oocyte isolation and the utilization of exogenous energy sources by growing oocytes. 100 46

Mediators released from injured human skin that initiate the inflammatory response have not been adequately identified. Organ culture of full-thickness skin explants enables us to do so, because injury to the skin can be made in vitro, eliminating the rapid leakage of serum and infiltration of leukocytes that occur in vivo. In our studies, the military vesicant sulfur mustard (SM) (10 microliters of a 0.01 to 1.0% dilution) was topically applied to injure the epidermis of the explant. Then, the explants were cultured in small Petri dishes, usually for 18 h at 36 degrees C, and the organ-culture fluids were assayed for various inflammatory mediators. We found that the culture fluids from SM-exposed and control explants contained similar amounts of angiotensin-converting enzyme, trypsin-like and chymotrypsin-like proteases, acid phosphatase, beta-glucuronidase, beta-galactosidase, lysozyme, deoxyribonuclease, ribonuclease, interleukin 1, and lactic dehydrogenase. However, the culture fluids from SM-exposed explants contained increased amounts of histamine and plasminogen-activating activity, and often prostaglandin E2, when compared to culture fluids from control explants. After 3 to 4 d in culture, full-thickness human skin explants, when exposed to 0.2% SM (but not when exposed to 1.0% SM), sometimes showed separation of the epidermis and increased collagenase activity (i.e., hydroxyproline release). Thus, histamine (from local mast cells), and prostaglandin E2 and plasminogen-activating activity (probably from both mast cells and epidermal cells) are apparently involved in early mediation of the inflammatory response.
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PMID:Mediators, initiating the inflammatory response, released in organ culture by full-thickness human skin explants exposed to the irritant, sulfur mustard. 171 Jun 39

Longitudinal muscle strips adhered with myenteric plexus were subjected to enzyme digestion under controlled conditions in a Krebs-bicarbonate buffer solution containing a mixture of collagenase, deoxyribonuclease, protease, choline chloride, and bovine serum albumin for 30 min at 37 degrees C. Myenteric ganglia, singly or in multiple aggregates, were harvested with micropipette and labeled with [3H]choline for [3H]acetylcholine (ACh) release studies. When examined by light or electron (transmission or scanning) microscopy, the ganglia exhibited their normal structural characteristics with axon bundles, dendrites, cell bodies, and vesiculated processes. Depolarization with elevated potassium or veratrine hydrochloride significantly elevated the efflux of [3H] ACh. Perfusion with tachykinins (substance P and substance K), vasoactive intestinal peptide, forskolin, or serotonin also significantly increased the release of [3H]ACh. This study demonstrated that enzyme-dissociated myenteric ganglia, notably free of muscle or connective tissue components, were structurally well preserved and were amenable to functional studies targeted specifically for the enteric plexus neurons.
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PMID:Characterization of acetylcholine release from enzyme-dissociated myenteric ganglia. 253 38

In the present study, a culture system of human placental cells was established to examine the role of estrogen and androgen in progesterone (P4) formation. Normal human placentae were obtained at term, and cells were dispersed in Hank's Balanced Salt Solution (5 ml/g tissue) containing 0.1% collagenase, 0.1% hyaluronidase, 0.01% deoxyribonuclease, and 1% fetal bovine serum for 2 h at 37 C. Dispersed placental cells (10(6) cells/ml) were placed in medium 199 with modified Earle's salts (pH 7.4) containing 10% fetal bovine serum, 12.5 mM HEPES buffer, 26 mM NaHCO3, and 40 micrograms/ml Gentamycin-SO4 and incubated for 72 h at 37 C and 5% CO2 in air to allow cell attachment. Medium was then changed (time zero), and P4 formation was studied thereafter. Culture of placental cells for 96 h resulted in linear increases in P4 and estradiol (E2) formation, indicating the maintenance of cell viability and steroidogenic function. Mean +/- SE P4 formation at 48 h was 246 +/- 16 pg/micrograms DNA. To assess the role of estrogen on P4 formation, placental cells were incubated for a period of 48 h with various amounts (10(-7)-10(-4)M) of the antiestrogen ethamoxytriphetol (MER-25), the aromatase inhibitor 4-hydroxyandrostenedione (4-OHA), and/or E2. Both MER-25 and 4-OHA resulted in a dose-dependent decline (P less than 0.01) in P4 formation (greater than 80% decline at 10(-4)M MER-25 or 4-OHA). The marked reduction in P4 formation caused by 4-OHA alone was reversed by concomitant addition of E2; however, E2 alone had no effect. To assess the role of androgens on P4 formation, cells were incubated for 48 h with increasing amounts (10(-7)-10(-4)M) of androstenedione, dehydroepiandrosterone (DHA), or dihydrotestosterone. Although the formation of E2 was enhanced by DHA, formation of P4 was not affected by the aromatizable androgens DHA or androstenedione or the nonaromatizable dihydrotestosterone. The decline in P4 formation by human placental cells in culture elicited by MER-25 or 4-OHA supports the hypothesis of a regulatory role for estrogen in placental P4 formation during human pregnancy. The lack of effect of exogenous estrogen suggests that the action of estrogen on P4 formation may be permissive.
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PMID:Regulation of progesterone formation by human placental cells in culture. 294 94

An enzymatic method is described for disaggregation of viable tumor cells from human solid tumors. The enzymatic cocktail consists of 0.1% collagenase, 0.01% hyaluronidase, and 0.002% deoxyribonuclease. After mechanical mincing of the tumor tissue, tumor specimens are dissociated by incubation in the enzymatic cocktail for 12-18 hours at room temperature. In 17 cases of sarcoma, the mean yield was 5 X 10(6) viable cells per gram tumor tissue. Yield was 1 X 10(7) viable cells per gram tumor tissue in 23 cases of gastrointestinal carcinoma. The viabilities of tumor cell suspensions ranged from 50 to 98%, except for low viabilities in four specimens that were grossly composed almost entirely of necrotic tissue. The dissociation procedure is simple and the viable cell yield is sufficient for applications in studies of human cancer immunobiology.
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PMID:An enzymatic method for the consistent production of monodispersed viable cell suspensions from human solid tumors. 298 62


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