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)

In this study, we undertook to prove the usefulness of cytofluorometric DNA ploidy analysis in distinguishing benign cartilaginous tumors from chondrosarcomas. We analyzed the DNA ploidy of 47 cartilaginous tumors using DNA cytofluorometry, which is more sensitive than flow cytometry. All of these tumors were classified into six groups on the basis of clinical, radiologic, and histologic criteria. The 25 tumors in the No. 1 group showed no histologic signs of malignancy regardless of their clinical signs. The four tumors in the No. 2 group showed histologic signs of malignancy, but had benign clinical signs like small bone origin or Ollier's disease. The No. 3 group (13 tumors), No. 4 group (four tumors), and No. 5 group (three tumors) were conventional grade I, II, and III chondrosarcomas, respectively, and the No. 6 group included three dedifferentiated chondrosarcomas. Tumor cells isolated from fresh tumor materials treated with papain and collagenase were smeared on a glass slide and their nuclear DNA was stained with propidium iodide. The DNA content of each cell was measured by a cytofluorometer as fluorescence intensity. The results of this study showed that all of the tumors in the No. 1 group had a diploid pattern with a significantly lower (P<.001) cell proliferative activity than the grade I chondrosarcomas in the No. 3 group, all of which had a diploid pattern. Cytofluorometric analysis also indicated that grade II and III chondrosarcomas in the No. 4 and 5 groups had a higher frequency of hyperdiploid cells (%HDC), including aneuploid and polyploid cells than grade I chondrosarcomas. Importantly, all of the grade I chondrosarcomas showed a %HDC >8%, whereas all of the tumors in the No. 1 and 2 groups showed a %HDC <8%. Therefore, we believe that a %HDC value of 8% is borderline between biologically benign and malignant states in cartilaginous tumors. Four of five patients with aneuploid chondrosarcoma had tumor recurrence and two of these patients died of metastatic disease, although all of the patients except for one with diploid chondrosarcoma were continuously disease free after surgery. Based on these results, we concluded that the data of DNA ploidy analysis, especially cell proliferative activity expressed as %HDC, is more reliable and clinically more useful than the histologic and clinical signs of malignancy in distinguishing benign cartilaginous tumors from chondrosarcomas and even from low grade chondrosarcomas.
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PMID:Usefulness of cytofluorometric DNA ploidy analysis in distinguishing benign cartilaginous tumors from chondrosarcomas. 1049 94

To explore the method of isolating acutely the smooth muscle cells from pulmonary artery in rats, small pulmonary arteries (700-200 microns, ID) were dissected free of connective tissue and were allowed to digest in a N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid(HEPES)-buffered physiological saline solution (HPSS) containing collagenase, papain and bovine serum albumin. The tissue was then triturated to disperse smooth muscle cells. The isolated cells in suspension were identified and photographed with film on electron microscope (EM). We succeeded in isolating the single smooth muscle cell, which appeared compressed typically. 90% cells in suspension were identified smooth muscle cells on EM. We conclude that the method for isolation of pulmonary arterial smooth muscle cells is simple, stable and effective and is recommanded for use.
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PMID:[Isolation and identification of smooth muscle cells from pulmonary artery in rats]. 1068 82

We have isolated and characterized a novel hemolytic protein from the venom of the Hawaiian box jellyfish (Carybdea alata). Hemolysis of sheep red blood cells was used to quantitate hemolytic potency of crude venom extracted from isolated nematocysts and venom after fractionation and purification procedures. Hemolytic activity of crude venom was reduced or lost after exposure to the proteolytic enzymes trypsin, collagenase and papain. The activity exhibited lectin-like properties in that hemolysis was inhibited by D-lactulose and certain other sugars. Activity was irreversibly lost after dialysis of crude venom against divalent-free, 20mM EDTA buffer; it was optimal in the presence of 10mM Ca2+ or Mg2+. Two chromatographic purification methods, size fractionation on Sephadex G-200 and anion exchange with quaternary ammonium, provided fractions in which hemolytic activity corresponded to the presence of a protein band with an apparent molecular weight of 42kDa by SDS-PAGE. We have designated this protein as CAH1. The N-terminal sequence of CAH1 was determined to be: XAADAXSTDIDD/GIIG.
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PMID:Partial purification and characterization of a hemolysin (CAH1) from Hawaiian box jellyfish (Carybdea alata) venom. 1122 87

Rheumatic diseases are accompanied by a progressive destruction of the cartilage layer of the joints. Despite the frequency of the disease, degradation mechanisms are not yet understood and methods for early diagnosis are not available. Although some information on pathogenesis could be obtained from the analysis of degradation products of cartilage supernatants, the most direct information on degradation processes would come from the native cartilage as such. We have used 1H as well as 13C HR-MAS (high resolution magic angle spinning) NMR spectroscopy to obtain suitable line-widths of NMR resonances of native cartilage. 1D and 2D NMR spectra of native cartilage were compared with those of enzymatically-treated (collagenase and papain) samples. In the 1H NMR spectra of native cartilage, resonances of polysaccharides, lipids and a few amino acids of collagen were detectable, whereas the 13C NMR spectra primarily indicated the presence of chondroitin sulfate. Treatment with papain resulted only in small changes in the 1H NMR spectrum, whereas a clear diminution of all resonances was detectable in the 13C NMR spectra. On the other hand, treatment with collagenase caused the formation of peptides with an amino acid composition typical for collagen (glycine, proline, hydroxyproline and lysine). It is concluded that the HR-MAS NMR spectra of cartilage may be of significance for the investigation of cartilage degradation since they allow the fast evaluation of cartilage composition and only very small amounts of sample are required.
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PMID:1H and 13C HR-MAS NMR investigations on native and enzymatically digested bovine nasal cartilage. 1141 Mar 93

Single smooth muscle cells (SMCs) isolated from guinea pig ileum using collagenase and papain were cultured on laminin-coated dishes in MEM containing fetal calf serum. Temporal changes in intracellular calcium ion concentration in response to carbachol and to ATP were investigated using fluo-3/AM and fluorescence microscopy. It was observed that carbachol caused an increased intracellular calcium ion in freshly isolated single SMCs but a reduced or negative response of cultured SMCs before confluence. On the other hand, ATP was observed to cause an increase in the calcium ion content of SMCs throughout the culture. SDS-PAGE and Western blot analyses revealed changes in the expression of contractile proteins as follows. l-Caldesmon and non-muscle type myosin heavy chain (NMHC) (considered to be marker molecules for dedifferentiation in smooth muscle cells) and non-muscle type tropomyosin were not observed in freshly isolated single SMCs. l-Caldesmon and NMHC appeared in the cultured SMCs within 2 days and the tropomyosin isoform was observed 6 days following seeding. Simultaneously, smooth muscle type myosin heavy chain (SMHC) decreased strikingly and the 41 kDa tropomyosin monomer was lost. The content of alpha-actin decreased gradually to a minimum on day 6 when non-muscle type tropomyosin appeared, and the cells began to proliferate rapidly. These results suggest that the loss of contractility in cultured smooth muscle cells is more closely related to changes in contractile protein profiles than to receptor-mediated signal transduction and that in addition to NMHC and l-caldesmon, non-muscle type tropomyosin may be useful as a marker molecule for de-differentiation of smooth muscle cells.
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PMID:Changes in Ca2+ signaling and contractile protein isoforms in smooth muscle cells from guinea pig ileum during culture. 1159 84

We previously reported a simple method of acutely preparing dissociated smooth muscle cells from urinary bladder tissue, but the feasibility of this method has not been well ascertained. In the present study, we assessed whether this method is applicable for measuring muscarinic receptor function in intestinal smooth muscle cells. Single smooth muscle cells were prepared from the longitudinal muscle tissue of guinea pig colon by the enzymatic dissociation with papain and hyaluronidase, followed by collagenase digestion. Muscarinic responses in the isolated smooth muscle cells were measured by intracellular Ca(2+) mobilization and extracellular acidification through Fura-2 fluorometry and Cytosensor microphysiometry, respectively. A single, viable population of colon longitudinal smooth muscle cells (approximately 6 x 10(6) cells/animal) was obtained. In these cells, carbachol (muscarinic agonist) induced Ca(2+) mobilization and extracellular acidification over the concentration range similar to that previously reported to produce contraction of the intact colon muscle strips. Atropine (nonselective muscarinic antagonist) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, M(3)-selective antagonist) inhibited the Ca(2+) mobilization with potencies approximately 3 log units greater than that for methoctramine (M(2)-selective antagonist). For extracellular acidification, the potency differences between these antagonists was approximately 2 log units. In addition, the carbachol-induced extracellular acidification was inhibited by 5-[N-ethyl-N-isopropyl]-amiloride, a selective inhibitor of the Na(+)/H(+) exchanger. These findings indicate that in isolated colonic smooth muscle cells, M(3) receptors are predominantly involved in Ca(2+) mobilization, while a mixed population of M(2) and M(3) receptors seems to contribute to extracellular acidification. Our results further suggest the role of the Na(+)/H(+) exchanger in muscarinic-mediated extracellular acidification. Consequently, our method produces viable isolated colonic smooth muscle cells that display physiologically appropriate responses to muscarinic receptor activation, and the method may be applicable for several types of nonvascular smooth muscle tissues.
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PMID:A method for measurement of muscarinic receptor-mediated responses in dissociated single colon longitudinal smooth muscle cells. 1175 83

We investigated the role of collagen in the magnetization transfer (MT) effect in contrast to other macromolecules. By means of phantoms made of collagen, chondroitin sulfate (CS) and albumin, MR parameters have been optimized in order to reduce the acquisition time and improve the sensitivity, as well as to minimize the contributions from CS and albumin to the MT induced signal attenuation. The same method was used to study cartilage ex vivo (bovine articular and nasal cartilage plugs) and in vivo (goat knee femoral chondyle). In phantom samples, the MT signal attenuation depended on the collagen concentration while contributions from the other macromolecules were found to be minimal. In average, analysis of MT images revealed a approximately 25%, approximately 35% and approximately 30% signal attenuation in 10% w/v type I collagen gels, cartilage plugs, and cartilage from the weight-bearing areas of the goat knee, respectively. Biochemical data revealed that treatment of cartilage plugs with bacterial collagenase led to collagen depletion and correspondingly to a decrease of the MT response. In contrast, trypsin-induced proteoglycan loss in cartilage plugs did not alter the MT effect. A significant correlation was observed between the collagen content in these plugs and their respective MT ratios and the rate constant k for the exchange process bound versus free water. Finally, data obtained from in vivo MT measurement of the goat knee demonstrated that intra-articular injection of papain might not only cause degradation of proteoglycans but also a change in collagen integrity in a dose-dependent manner. We conclude that in vivo measurement of MT ratios gives quantitative and qualitative information on the collagen status and may be applied for the routine evaluation of normal and abnormal articular cartilage.
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PMID:Quantitative and qualitative assessment of articular cartilage in the goat knee with magnetization transfer imaging. 1180 55

The primary specificity of papain-like cysteine proteases (family C1, clan CA) is determined by S2-P2 interactions. Despite the high amino acid sequence identities and structural similarities between cathepsins K and L, only cathepsin K is capable of cleaving interstitial collagens in their triple helical domains. To investigate this specificity, we have engineered the S2 pocket of human cathepsin K into a cathepsin L-like subsite. Using combinatorial fluorogenic substrate libraries, the P1-P4 substrate specificity of the cathepsin K variant, Tyr67Leu/Leu205Ala, was determined and compared with those of cathepsins K and L. The introduction of the double mutation into the S2 subsite of cathepsin K rendered the unique S2 binding preference of the protease for proline and leucine residues into a cathepsin L-like preference for bulky aromatic residues. Homology modeling and docking calculations supported the experimental findings. The cathepsin L-like S2 specificity of the mutant protein and the integrity of its catalytic site were confirmed by kinetic analysis of synthetic di- and tripeptide substrates as well as pH stability and pH activity profile studies. The loss of the ability to accept proline in the S2 binding pocket by the mutant protease completely abolished the collagenolytic activity of cathepsin K whereas its overall gelatinolytic activity remained unaffected. These results indicate that Tyr67 and Leu205 play a key role in the binding of proline residues in the S2 pocket of cathepsin K and are required for its unique collagenase activity.
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PMID:Selective inhibition of the collagenolytic activity of human cathepsin K by altering its S2 subsite specificity. 1208 94

A serine protease, subtilisin YaB, produced by alkalophilic Bacillus YaB, shows promises as a potent meat tenderizer, because its substrate specificity is for small amino acids, which are found at high levels in meat connective tissue proteins. Substrate specificity engineering of the substrate binding pockets was used to generate more suitable meat-tenderizing mutants, G124A, G124V, G159A, and G159S, derived from recombinant wild subtilisin YaB and expressed in Bacillus subtilis DB104. The characteristics of these recombinant enzymes were studied to evaluate their usefulness as improved meat tenderizers. The proteolytic activities of recombinant subtilisin YaB, engineered subtilisin YaBs, and commercially available papain, bromelain, collagenase, and elastase were compared using elastin, collagen, casein, and myofibrillar proteins as substrates. Hydrolysis of beef proteins was evaluated using the myofibrillar fragmentation index and collagen solubility. The results demonstrated that recombinant mutant G159A was the most improved meat tenderizer and can be used in the meat pH range of 5.5-6.0 and the temperature range of 10-50 degrees C. Contrary to the result obtained from artificial substrate, mutant enzymes engineered on G124 residues did not exhibit better tenderizing ability when elastin, collagen, or meat was used as substrate, suggesting the necessity of evaluation by real substrate before protein-engineered enzymes are applied commercially.
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PMID:Application potency of engineered G159 mutants on P1 substrate pocket of subtilisin YaB as improved meat tenderizers. 1235 2

CD55 is a complement regulatory protein expressed by cells to protect them from bystander killing by complement. CD55 is over-expressed 2-100-fold on tumour cells and is deposited in large amounts within tumour matrix. Vascular endothelial growth factor (VEGF) produced by tumours to stimulate angiogenesis, also up-regulates endothelial cell surface expression of CD55 and stimulates the release of matrix degrading metalloproteinases. This study investigated the effects of VEGF on CD55 deposition into matrix and the release of CD55 by metalloproteinases. In contrast to inflammatory cytokines, CD55 was up-regulated by VEGF at the cell surface and within the extracellular matrix (ECM). Interestingly, human umbilical vein endothelial cells (HUVEC) exposed to VEGF released similar amounts of CD55 into the ECM as a tumour cell line expressing 50-fold higher level of CD55 on its cell surface. Furthermore, in contrast to earlier studies, both tumour and HUVEC-derived CD55 was functionally active. However, in contrast to papain that degrades CD55, and collagenase that fails to release CD55, MMP-7 released intact CD55 from ECM. This suggests that it may have a further role to play in protecting cells during inflammation and invasion.
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PMID:The role of CD55 in protecting the tumour environment from complement attack. 1244 4


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