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

---

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

The hamster histone H3.2 promoter contains an AP-1-like element (referred to as site X) that contains the sequence CGAGTCA. This site differs from the Jun/AP-1 consensus sequence by one base and is also similar to the cyclic AMP response element. Similar AP-1/cyclic AMP response element-like sites have been found in the promoters of other histone H3 genes and are known to bind proteins either in vivo or in vitro. Using site directed mutagenesis, we demonstrate that a 10-base pair region which encompasses site X is a positive control element that is necessary for the S phase dependent increase in H3.2 transcription in cells synchronized by serum stimulation or aphidicolin block. DNase I footprint analysis shows that mutating site X eliminates v-Jun and hamster cellular factor(s) binding. Further in vitro analysis with gel retardation assays reveals that the flanking sequence of this site is necessary for the formation of an H3.2 specific complex that can be distinguished from complexes formed with a collagenase or SV40 AP-1 element. Antibodies specific to the different members of the Jun and Fos family of transcription factors show that, in gel retardation assays, a Jun-like factor is a component of the H3.2 specific complex. However, the H3.2 specific complex exhibits different reactivity toward the Jun and Fos specific antibodies as compared to complexes formed with a collagenase AP-1 element. We hypothesize that a unique protein complex, containing a component related to the AP-1 family of transcription factors, binds to the AP-1-like motif of the hamster H3.2 promoter and may be involved in the S phase dependent regulation of transcription.
...
PMID:Identification of a 10-base pair protein binding site in the promoter of the hamster H3.2 gene required for the S phase dependent increase in transcription and its interaction with a Jun-like nuclear factor. 147 72

The following studies were undertaken to develop a cultured uterine myocyte model which would allow further clarification of the adrenergic signal transduction mechanisms utilized by these myocytes. After mechanical removal of the endometrium, rabbit uterine myocytes were isolated by an overnight enzymatic disaggregation using collagenase and DNase I. The isolated myocytes were maintained in culture in 75-cm2 flasks containing Waymouth's MB 751/1 medium-10% fetal bovine serum along with 10(-8) M estradiol, penicillin, streptomycin, and Fungizone. The phase contrast and electron micrographic appearance of these cells was consistent with that previously reported for smooth muscle myocytes in culture. Immunocytochemical studies utilizing monoclonal anti-alpha-smooth muscle actin antibodies confirmed the presence of smooth muscle actin in these cultured myocytes. Western blot studies similarly confirmed the presence of alpha-smooth muscle actin in rabbit myometrial tissue and the cultured myocytes, both the primary and F1 generation. After prelabeling the myocytes with [3H]inositol, adrenergic stimulation experiments demonstrated alpha-1 receptor mediated stimulation of inositol phosphates. Beta receptor stimulation experiments confirmed cAMP production in these cultured myocytes, and the ability of clonidine, an alpha-2 agonist, to inhibit forskolin stimulated cAMP production confirmed the presence of functional alpha-2 adrenergic receptors in these myocytes. In conclusion, these cultured rabbit uterine myocytes have provided an in vitro model which can be utilized to further clarify the adrenergic receptor signal transduction mechanisms in genital tract smooth muscle.
...
PMID:Alpha-1, alpha-2, and beta adrenergic signal transduction in cultured uterine myocytes. 216 Sep 25

Characterization of cells comprising solid tumors will facilitate the rational design of cancer chemotherapy for individual patients. We have prepared cell suspensions from human melanoma, sarcoma, and lung tumors by thinly slicing the tissue with a microtome and scalpels (mechanical release), followed by treatment with a mixture of collagenase II and DNase I (enzymatic release). This method of disaggregation resulted in two cell suspensions for each tumor specimen, and we characterized these suspensions by assessing their dye exclusion capability, ribonucleoside triphosphate pools, cytological profile and clonogenicity in soft agar. The enzymatic method thus yields cells in addition to those obtainable by a mild mechanical procedure, and these cells are similar in cytological profile and clonogenicity in soft agar to those released mechanically. Furthermore, the enzymatically released population is superior to that released mechanically for purposes requiring large numbers of dye-excluding cells having intact ribonucleotide pools.
...
PMID:Characterization of cells obtained by mechanical and enzymatic means from human melanoma, sarcoma, and lung tumors. 626 Mar 39

Oval cells and biliary epithelial cells were isolated from livers of rats fed a choline-deficient diet containing 0.1% ethionine and from normal rat livers, respectively. Nonparenchymal cell suspensions prepared from these livers by collagenase perfusion followed by digestion of undissociated tissue with 0.1% collagenase, 0.1% Pronase, and 0.004% DNase I were separated into six fractions by centrifugal elutriation. Cells in each fraction were characterized histochemically for gamma-glutamyl transpeptidase, peroxidase, alkaline phosphatase, and glucose-6-phosphatase activities, and for albumin and alpha-fetoprotein by immunocytochemical methods. Cells from Fraction 5 of the elutriation procedure had various features predicted for oval cells and were selected for further studies. The cell yield in this fraction, from each preneoplastic liver, was 5.7 X 10(7) cells, 93 +/- 2% of which were gamma-glutamyl transpeptidase positive, 6 +/- 1% peroxidase positive, 61% albumin positive, and 29% alpha-fetoprotein positive. Cells in this fraction have a median diameter of 13.1 micron and are diploid and cycling. The majority of these cells has morphological features characteristic of biliary epithelial cells, although some cells display features intermediate between duct cells and hepatocytes. Nucleic acid hybridization using specific probes revealed that these cells contain albumin and alpha-fetoprotein messenger RNAs, while hepatocytes from normal and preneoplastic liver contain only albumin messenger RNA. Biliary cells obtained from normal livers do not contain albumin messenger RNA. The large-scale purification and characterization of cell populations from preneoplastic livers is an important step in elucidating the cellular derivation of liver tumors.
...
PMID:Isolation of oval cells by centrifugal elutriation and comparison with other cell types purified from normal and preneoplastic livers. 669 43

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)
...
PMID:[Cultivation of human cartilage tissue in a 3-dimensional perfusion culture chamber: characterization of collagen synthesis]. 749 39

An enzyme-linked immunosorbent assay (ELISA) was developed that detects PrP(Sc) in crude extracts from brain and spleen tissue of scrapie-affected mice with high sensitivity and specificity. Brain tissue was homogenized in 8% Zwittergent 3-12 and 0.5% Sarkosyl. The homogenate was treated with collagenase and DNase I and then subjected to proteinase K digestion. Precipitates containing PrP(Sc) were obtained by ultracentrifugation. Spleen tissue was homogenized in 4% Triton X-100 and 0.5% Sarkosyl, and the homogenate was treated firstly with collagenase and DNase I, and secondly with proteinase K. PrP(Sc) was then extracted with 6.25% Sarkosyl and precipitated through salting-out with NaCl and by ultracentrifugation. When PrP(Sc) was dissolved in 3-4 M guanidine thiocyanate and adsorbed to microtiter plates, strong and specific reactions to the formation of antigen-antibody complexes could be detected by ELISA. The sensitivity of PrP(Sc)-detection for this ELISA, as measured by serial dilution of scrapie material in tissue homogenates from uninfected animals, was equal or higher than that attained by Western blot. This ELISA is more rapid than Western blot and seems to be more suitable for screening large numbers of animals. It also has potential application for the diagnosis of the transmissible spongiform encephalopathies.
...
PMID:Sensitive enzyme-linked immunosorbent assay for detection of PrP(Sc) in crude tissue extracts from scrapie-affected mice. 907 66

The 92 kDa type IV collagenase (MMP-9), which degrades type IV collagen, has been implicated in tissue remodeling. The purpose of the current study was to determine the role of Jun amino-terminal kinase (JNK)- and extracellular signal-regulated kinase- (ERK)-dependent signaling cascades in the regulation of MMP-9 expression. Towards this end, we first determined the transcriptional requirements for MMP-9 promoter activity in a cell line (UM-SCC-1) which is an avid secretor of this collagenase. Transfection of these cells with a CAT reporter driven by progressive 5' deleted fragments of the MMP-9 promoter indicated the requirement of a region spanning -144 to -73 for optimal promoter activity. DNase I footprinting revealed a protected region of the promoter spanning nucleotides -91 to -68 and containing a consensus AP-1 motif at -79. Mutation of this AP-1 motif practically abolished the activity of the MMP-9 promoter-driven CAT reporter. Mobility shift assays indicated c-Fos and Jun-D bound to this motif and transfection of the cells with a mutated c-Jun, which quenches the function of endogenous Jun and Fos proteins, decreased MMP-9 promoter activity by 80%. UM-SCC-1 cells contained a constitutively activated JNK and the expression of a kinase-deficient JNK1 reduced the activity of a CAT reporter driven either by the MMP-9 promoter or by three tandem AP-1 repeats upstream of a thymidine kinase minimal promoter. Conditioned medium collected from UM-SCC-1 cells transfected with the dominant negative JNK1 expression vector diminished 92 kDa gelatinolysis. Similarly, interfering with MEKK, which lies upstream of JNK1, using a dominant negative expression vector reduced MMP-9 promoter activity over the same concentration range which repressed the AP-1-thymidine kinase CAT reporter construct. UM-SCC-1 cells also contained a constitutively activated ERK1. MMP-9 expression, as determined by CAT assays and by zymography, was reduced by the co-expression of a kinase-deficient ERK1. Interfering with MEK1, which is an upstream activator of ERK1, either with PD 098059, which prevents the activation of MEK1, or with a dominant negative expression construct, reduced 92 kDa gelatinolysis and MMP-9 promoter activity respectively. c-Raf-1 is an upstream activator of MEK1 and a kinase-deficient c-Raf-1 expression construct decreased the activity of a promoter driven by either the MMP-9 promoter or three tandem AP-1 repeats. Conversely, treatment of UM-SCC-1 cells with PMA, which activates c-Raf-1, increased 92 kDa gelatinolysis. These data suggest that MMP-9 expression in UM-SCC-1 cells, is regulated by JNK- and ERK-dependent signaling pathways.
...
PMID:Regulation of 92 kDa type IV collagenase expression by the jun aminoterminal kinase- and the extracellular signal-regulated kinase-dependent signaling cascades. 913 92

Regulation of toxin production in the gram-positive anaerobe Clostridium perfringens occurs at the level of transcription and involves a two-component signal transduction system. The sensor histidine kinase is encoded by the virS gene, while its cognate response regulator is encoded by the virR gene. We have constructed a VirR expression plasmid in Escherichia coli and purified the resultant His-tagged VirR protein. Gel mobility shift assays demonstrated that VirR binds to the region upstream of the pfoA gene, which encodes perfringolysin O, but not to regions located upstream of the VirR-regulated plc, colA, and pfoR genes, which encode alpha-toxin, collagenase, and a putative pfoA regulator, respectively. The VirR binding site was shown by DNase I footprinting to be a 52-bp core sequence situated immediately upstream of the pfoA promoter. When this region was deleted, VirR was no longer able to bind to the pfoA promoter. The binding site was further localized to two imperfect direct repeats (CCCAGTTNTNCAC) by site-directed mutagenesis. Binding and protection analysis of these mutants indicated that VirR had the ability to bind independently to the two repeated sequences. Based on these observations it is postulated that the VirR positively regulates the synthesis of perfringolysin O by binding directly to a region located immediately upstream of the pfoA promoter and activating transcription.
...
PMID:The VirR response regulator from Clostridium perfringens binds independently to two imperfect direct repeats located upstream of the pfoA promoter. 1061 63

The effects of hyaluronic acid (HA) derivative on the proliferation and metabolism of human chondrocytes were examined. Cells were obtained from cartilage from metatarsal phalangeal joints of 20 adult humans (aged 22-63) and from femoral knee condyles of 10 subjects (aged 22-77). Chondrocytes isolated by collagenase/Dnase digestion were cultured with addition of different doses of HA for 4 weeks. Morphological studies demonstrated that HA enhanced the adhesion of cells to substrate; HA-treated chondrocytes proliferated better than chondrocytes cultured in HA-free medium. This study shows that HA improves in vitro substrate adhesion ability and proliferative activity of human cartilage cells and that the response to the treatment varies on an individual basis.
...
PMID:Effect of hyaluronic acid on human chondrocyte cell lines from articular cartilage. 1146 44

This study was aimed at the feasibility of using ATP-bioluminescence assay for tumor in vitro chemosensitivity testing. With the use of this assay, the authors determined dose-response curve in mouse fibroma cell line L929 treated with chemotherapeutic agents, and investigated the different in vitro responses of 6 ovarian carcinomas (5 from fresh tumor tissues, 1 from ascites) treated with etopside, cis-plating, 5-fluorouracil and adriamycin. The results showed that the coefficients of variation for triplicate assays ranged from 1.2% to 15.8% which means high reproducibility of the assay. The single cell suspension (including < 30 cells clusters) could be separated from tissue fragments by means of enzyme cocktail (collagenase, Dnase, pronase). The viable cells were over 90%. This study demonstrates that ATP-bioluminescence assay is a sensitive, reliable and efficient method for tumor chemosensitivity testing. In this connection, the correlation between in vitro drug sensitivity and in vivo patient response is worth further studying.
...
PMID:[An evaluation of adenosine triphosphate bioluminescence assay for tumor in vitro chemosensitivity testing]. 1254 24


1 2 Next >>

---