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:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Parechymal cells (PC) were prepared by digestion of perfused, lavaged rat lung in a mixture of collagenase and DNase, and harvested on a discontinuous percoll gradient. The process yielded on average 1.0 X 10(8) viable cells/gram tissue. PC were pulsed with soluble antigen, and tested for their capacity to trigger antigen-specific activation of immune T-cells in vitro, or to replace adherent accessory cells necessary for Concanavalin A (Con A)-induced T-cell proliferation. Unfractionated PC exhibited only minor antigen-presenting cell (APC) activity. However, removal of adherent or FcR-positive cells unmasked substantial APC activity. Subsequent experiments indicated that the majority of the APC banded at the top of the percoll gradient (density less than 1.048 g/ml). The same cell preparations substituted for adherent accessory cells in Con A- activation of T cells, suggesting capacity to secrete soluble factors such as interleukin-1 (IL-1) as well to present antigen. The PC preparation also contained T-cells, which were refractory to Con A stimulation unless endogenous adherent cells were first removed. Collectively, these data suggest the presence of non-adherent, FcR-negative, low density accessory cells in the lung parenchyma, capable of both APC activity and soluble factor production. Their T-cell-activation functions appear to be down regulated by endogenous adherent, FcR-positive cells. It is speculated that the accessory cells in these lung preparations may be dendritic cells, the activity of which is subject to inhibition by macrophages.
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PMID:T cell activation by antigen-presenting cells from lung tissue digests: suppression by endogenous macrophages. 387 50

Ovaries of immature, intact rats were dispersed by collagenase-DNase treatment and cultured in serum-free medium (ovarian cell culture). The hormonal responsiveness of the ovarian cell was compared to that exhibited by pure granulosa cells isolated via needle puncturing. Surprisingly, despite the fact that the majority of the cultured cells should have been comprised of granulosa type, no follicle-stimulating hormone-inducible progesterone or 20 alpha-OH-progesterone (20 alpha-OH-P) could be detected by radioimmunoassay, as typically occurs in cultures of pure granulosa cells. Therefore, in order to unravel the cause for the different responsiveness between the granulosa and the ovarian cell, we applied thin-layer chromatography analysis to follow the metabolic fate of added radioactive pregnenolone to intact monolayers in culture. Such TLC analysis revealed that, after priming with follicle-stimulating hormone, added [3H]pregnenolone was converted to progesterone which was rapidly reduced and finally accumulated as 5 alpha-pregnane-3 alpha,20 alpha-diol(pregnanediol). In addition to pregnanediol, a second class of steroid hormones accumulated in the coculture medium, namely androsterone and 5 alpha-pregnane-3 alpha,17 alpha,20 alpha-triol (pregnanetriol). These latter two were specific products of the ovarian coculture, indicating the presence of theca-interstitial cells bearing 17 alpha-hydroxylase and 17,20-lyase activities. Pregnanediol, rather than progesterone, was found to be the progestin precursor for androgen formation. We thus conclude that due to exchange of steroid metabolites between the cocultured cell types, the final steroid products are different by far from the expected contributions of each individually cultured cell type. Moreover, these findings reveal an additional aspect of the "two-cell theory," suggesting a granulosa-thecal concerted metabolism of progestin steroids, rather than exchange of aromatizable androgens.
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PMID:Concerted metabolism of steroid hormones produced by cocultured ovarian cell types. 391 35

In the current study, we found evidence for the existence of binding sites for TRH in synaptic membrane preparations of several regions of the postmortem adult human brain. High levels of specific binding (fmol [3H]Me-TRH/mg protein/2 hr) were found in limbic structures: amygdala (7.1 +/- 0.6, Mean +/- SE), hippocampus (2.8 +/- 0.3), and temporal cortex (2.4 +/- 0.8). Intermediate levels of binding were found in the hypothalamus and nucleus accumbens whereas binding was low to undetectable in frontal and occipital cortex, cerebellum, pons, medulla and corpus striatum. Binding of the radioligand was linear over protein concentrations of 0.05-1.5 mg, and greater than 6 hr of incubation was required to achieve maximal binding. In the amygdala, binding was inhibited in the presence of TRH and Me-TRH but not in the presence of up to 1 microM concentrations of cyclo (His-Pro), TRH-OH, pGlu-His or peptides unrelated to TRH. Pretreatment of amygdala synaptic membranes with detergents, proteases or phospholipases disrupted [3H]Me-TRH binding; pretreatment with DNase or collagenase had no effect on binding. Saturation and association/dissociation analyses of the binding of [3H]Me-TRH to purified amygdala synaptic membranes revealed the presence of a high affinity (KD = 2.0 nM), low capacity (Bmax = 180 +/- 16 fmoles/mg protein) binding site. These results demonstrate that a highly specific membrane associated receptor for TRH is present in the adult human brain. The specific role that this receptor plays in brain function remains to be elucidated.
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PMID:Thyrotropin releasing hormone (TRH) binding sites in the adult human brain: localization and characterization. 609 73

EMT-6/UW tumours were treated in vivo with X-rays, cyclophosphamide, or bleomycin. Cell survival was assayed in vitro following tumour disaggregation with trypsin or an enzyme cocktail (EC) consisting of pronase, collagenase and DNase which gives a 10-20 x higher cell yield. Surviving fraction was lower after cyclophosphamide treatment for cells isolated with EC than for cells prepared with trypsin. The opposite result was obtained with bleomycin; trypsin-isolated cells appeared more sensitive. In attempting to determine the basis for this discrepancy, it was found that both dissociation methods isolate a non-representative cell sample with fewer cells in DNA synthesis (12-13%) than in the original tumour (approximately 22%). The specific nature of the interaction between the injury caused by drug and enzyme remains to be elucidated.
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PMID:Response of an in vivo-in vitro tumour to X-rays and cytotoxic drugs: effect of tumour disaggregation method on cell survival. 615 76

Cell suspensions were prepared by either mechanical or enzymatic disaggregation methods from biopsy specimens from 54 patients with various tumors. The biologic activities of cells derived from the two suspensions were then examined. Biopsy specimens of solid tumors were minced, and one-half of each specimen was further processed by being teased with needles, whereas the other half was exposed to a combination of collagenase, hyaluronidase, and DNase. The enzymatic disaggregation method yielded fewer cells per gram of tissue than the mechanical method. However, the percentage of dye-excluding cells was increased by the enzymatic procedure in 93% of the cases. Cells obtained by enzymatic means also had higher cloning efficiencies than those obtained by mincing. The histologic types of cells present in the initial cell suspensions were the same for cells obtained by the enzymatic or mechanical disaggregation methods. The number of colonies obtained was linearly related to the number of cells plated in both cases. The tritiated thymidine suicide indices (estimates of the percentage of cells in the S-phase of the cell cycle) were the same for the two cell populations obtained by the two methods. The results indicate that cells obtained from solid tumors by enzymatic dissociation methods did not differ significantly from cells obtained by the more conventional mechanical techniques. However, cell viabilities and cloning efficiencies were significantly improved by the enzymatic technique.
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PMID:Effect of enzymatic disaggregation on proliferation of human tumor cells in soft agar. 628 27

Production of monodispersed cell suspensions from primary human breast tumors is difficult due to the predominant stromal composition of most breast tumors. Our studies were designed to optimize dispersion of breast tumors of known stromal content and histopathology. In a first series of experiments three enzymatic protocols were compared to disperse minced tissue: (A) treatment with collagenase (2 mg/ml) in the presence of 5% serum for 24 hours; (B) treatment with collagenase (6 mg/ml) and DNase (0.002%) in 10% serum for 3 hours; (C) treatment with collagenase (2 mg/ml) for 3 hours followed by pronase (0.075%) for 1 hour. Protocol A produced better cell yields than B or C for all tumors tested. The monodispersed cells were suspended in a 0.3% semi-solid agar with alpha modified Eagles medium (alpha MEM), 10% serum, and selected hormones, then layered over similarly enriched 0.5% semi-solid agar. The cells prepared by protocol A had a higher plating efficiency and larger average colony size than B or C. In a second series of experiments, protocol A was repeated and compared to two additional procedures: (D) treatment with collagenase (2 mg/ml) and hyaluronidase (1 mg/ml) in the presence of 5% serum for 24 hours; and (E) mechanical disaggregation. Protocol D exhibited a small but significant negative difference from A, while E was the least efficient in producing viable monodispersed cells from the tumors. All enzymatically monodispersed cells produced clonal growth in our agar system. However, mechanically dispersed cells gave growth in only 4 of 7 tumors. Protocol A, in addition to yielding the highest number of viable cells per gram of tissue, gave the highest plating efficiency of all protocols tested.
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PMID:A comparison of methods for the production of monodispersed cell suspensions from human primary breast carcinomas. 630 35

After collagenase treatment of perfused rat liver, isolated washed hepatocytes (parenchymal cells) and washed nonparenchymal cells contained 13-53% and 5-9%, respectively, of the total vitamin A in rat liver. After Pronase E and DNase digestion of liver, nonparenchymal cells contained 1-6% of the total liver vitamin A content. By density-gradient centrifugation, hepatocytes were divided into six fractions, which contained fewer lipid globules per cell and less vitamin A per cell as the cell density increased. In our procedures, lipocytes could not be isolated after either collagenase or pronase E and DNase digestion of liver. Of the total liver vitamin A, 40-80% was found in very low density, vitamin A-containing globules, which have a median diameter of 1.7 microns (range 0.4-4.6 microns), show intense yellow-green fluorescence under UV illumination and contain greater than 95% of their vitamin A in ester form. The distribution of vitamin A among cells and vitamin A globules in rats dosed with vitamin A was the same as in undosed rats. The possible interaction of lipocytes and different classes of hepatocytes in the storage and mobilization of vitamin A is discussed.
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PMID:The storage form of vitamin A in rat liver cells. 631 81

Certain tissues, such as the spleen, are rich sources of mononuclear phagocytes (MP); however, separating the phagocytes from tissues and removing the contaminating cells have been difficult. We report here a method for the extraction and purification of human splenic MP that employs gentle homogenization of splenic fragments with a Tenbroeck tissue homogenizer, controlled digestion with purified collagenase to free MP from splenic stroma, incubation with DNase to dissociate cell clumps and purification by countercurrent centrifugal elutriation (CCE). With homogenization and enzymatic digestion most of the splenic nonspecific-esterase-positive cells are freed into suspension as determined by morphometric analysis of 2 micron sections from plastic embedded spleen stained for alpha-naphthyl butyrate esterase (ANB). Overall cell recovery after homogenization and enzyme treatment is 56 +/- 7%; no selective cell loss occurs as determined by differential cell counts at each purification step. CCE of up to 3 X 10(9) treated spleen cells results in recovery of 63 +/- 6% of the elutriated cells and separates nearly 50% of the recovered MP into enriched fractions. These MP are morphologically intact as determined by light and electron microscopy and are actively phagocytic. Highly purified (96%) autologous splenic lymphocytes are a useful by-product of this separation technique.
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PMID:Isolation of human splenic macrophages and lymphocytes by countercurrent centrifugal elutriation. 636 Nov 49

The reactivity of rabbit anti-HLA-DR antigen antibodies with cells in normal and rheumatoid synovial tissue was investigated by indirect immunofluorescence on frozen sections of tissue. The antibodies reacted with a significant proportion of the synovial lining cells of both normal and rheumatoid synovial tissue, with endothelial cells, and with a number of, most probably, migratory cells. After dispersion of cells from rheumatoid synovial tissue by digestion with collagenase and DNase, adherent cells of both a macrophage-like and a dendritic appearance reacted with the anti-HLA-DR antigen antibodies. The adherent cells were also found to be potent stimulators in the allogeneic MLR. In addition, it was found that a high percentage of T lymphocytes from both peripheral blood and synovial tissue of rheumatoid patients bound anti-HLA-DR antibodies. The present data suggest a role for synovial lining cells in HLA-D-locus-dependent events of importance in the pathogenesis of rheumatoid arthritis and other joint diseases and point to the need for further investigations on T lymphocytes derived from the site of inflammation in the study of rheumatoid arthritis.
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PMID:Appearance of anti-HLA-DR-reactive cells in normal and rheumatoid synovial tissue. 645 80

Clonogenic assays have attracted most attention in the field of chemosensitivity testing procedures. For many practical reasons and theoretical considerations detailed in this paper the human tumour stem cell assay (HTSCA) has proven most suitable for this purpose. The slightly modified method originally described by Hamburger and Salmon was used for testing tumour specimens of patients with breast cancer. 77 samples from 65 different patients were sent to our laboratory. 51% of the specimens were suitable for testing. 56% of all tested samples were biopsies and 44% were effusions. To enhance cell yield and viability solid tumours were disaggregated enzymatically using an enzyme cocktail consisting of collagenase 0.15% and DNase 0.015%. The median viability of the solid samples was 55%, that of the effusions was 91%. 44% of the culture samples showed colony growth (greater than 5 colonies per control plate) and 28% sufficient colony growth (greater than 20 colonies per control plate). The average number of colonies per control plate was 65, the average cloning efficiency was 0.0111%. Half of the samples with sufficient colony growth (5/11) was adequate for drug prediction. Positive correlation of sensitivity was observed in 2/2 and correct resistance prediction in 3/3.
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PMID:[In vitro chemosensitivity testing with the human tumor stem cell assay (HTSCA) in breast cancer]. 647 79


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