Evaluation of seprase activity

Seprase is a serine protease that is integral to the plasma membrane and is overexpressed by invasive tumor cells (Piñeiro-Sánchez et al., J Biol Chem 1997; 272: 7595–601; Monsky et al., Cancer Res 1994; 54: 5702–10). Seprase activity is most often assessed by zymography, which is not a quantitative assay. This study establishes a relatively simple and quantitative method for determining seprase activity. The degradation of a 3H-gelatin substrate is measured in the presence of 5 mM EDTA which inhibits matrix metalloproteinases but not seprase. The quantitative character of the assay was demonstrated using partially purified seprase from chicken embryos, a preparation that lacks detectable matrix metalloproteinase activity. In this assay, release of 3H-gelatin fragments is linear over time for 1.5 g/assay seprase concentration as well as for preparations concentrated or diluted by five fold (7.5 g/assay and 0.3 g/assay respectively). Additional experiments were performed to validate the quantification of seprase activity using the radiographic assay by comparing the results to zymography. Exposure to 22 or 37°C results in maximal seprase activity while exposure to 80 or 100°C completely abolishes seprase activity in both zymography and the radiographic assay. Exposure to 60°C abolished seprase activity as judged by zymography, but about 50% gelatinase activity was observed using the 3H-gelatin substrate. Immunopreciptiation with seprase-specific antibody specifically removed seprase and lowered the seprase activity remaining in the extracts as judged by both assays. Investigation of the seprase that was partially purified from human breast cancer tissue revealed that its specific activity (cpm gelatin fragments released/ {mg protein×h}) is five times greater than that of seprase purified from chicken embryos. This assay will be useful for determining the seprase activity in extracts of tumor tissues and cells as well as for identifying inhibitors of seprase.     

Infiltrative capacity of T leukemia cell lines: A distinct functional property coupled to expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1)

Infiltrative capacity was found to distinguish separate T leukemia cell lines. Of seven T-cell lines four exhibited capacity to infiltrate Matrigel. Analysis of infiltration was performed at the single-cell level throughout the Matrigel using a depth meter. Further, we examined differences in migration capacity and metalloproteinase production between infiltrating and non-infiltrating T-cell lines. The capacity to infiltrate was not directly correlated to the capacity to adhere to the Matrigel or to migrate on/to extracellular matrix components. It is concluded that infiltration capacity does not simply reflect capacity to migrate but represents a distinct functional property. The production of metalloproteinases and their inhibitors by the separate T-cell lines was analyzed using rt PCR, biosynthetic labelling, zymography, immunoprecipitation and ELISA. All T-cell lines with capacity to infiltrate produced matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) while non-infiltrating cell lines did not express MMP-9. Expression of MMP-1, 2, 3, 10, 14 and 17 showed no correlation to capacity to infiltrate. Analysis of infiltration in the presence of a metalloprotease inhibitor showed an increased number of cells within the gel. This enhancement of infiltration suggests that the function of MMPs and/or their inhibitors in lymphocyte infiltration is more complex than previously thought. This revised version was published online in July 2006 with corrections to the Cover Date.     

大鼠肝纤维化模型中基质金属蛋白酶-2及其抑制物的表达

目的了解基质金属蛋白酶－２（ＭＭＰ－２）及其组织型抑制剂（ＴＩＭＰ－２）在纤维化过程中的变化,分析它们在始动阶段的作用。方法用免疫组化和酶图法,在异种血清诱导的大鼠肝纤维化模型不同阶段,作肝细胞内ＭＭＰ－２、ＴＩＭＰ－２、酶降解底物Ⅳ型胶原（ＣｏｌⅣ）以及结蛋白（Ｄｍ）定位和半定量研究。结果实验鼠于４周末形成细胞间隔,８周末发展为肝纤维化,含细胞－纤维间隔或纤维间隔,１２、１６周仍为纤维间隔。酶图法表明４周末ＭＭＰ－２活性升高２．５倍左右,８周末仍高于对照鼠。免疫组化显示４周末ＭＭＰ－２阳性细胞数最多,连续切片观察ＭＭＰ－２与Ｄｍ阳性细胞（活化的肝星状细胞）几乎重叠。８周末阳性细胞数有所减少,１２周末明显减少。半定量变化趋势与酶图法一致。ＴＩＭＰ－２于８周末表达开始升高,１２周末达最高。２周末ＣｏｌⅣ表达开始上升,４周末达高峰,以后缓慢下降。结论肝纤维化早期ＭＭＰ－２活性增高,继而ＴＩＭＰ－２分泌过多,ＭＭＰ－２活性受抑,ＥＣＭ降解受阻,纤维化进一步发展。     

Compartmental changes in expression of c-Fos and FosB proteins in intact and dopamine-depleted striatum after chronic apomorphine treatment

Chronic administration of dopaminergic agonists to rats with unilateral 6-OH-dopamine (6-OHDA) lesions of nigrostriatal pathway produces behavioral sensitization to subsequent agonist challenges and may serve as a model for DOPA-induced dyskinesias. In order to understand striatal mechanisms behind this long-term behavioral change we examined striatal c-Fos and FosB immunoreactivity induced by apomorphine challenge (5 mg/kg, s.c.) after 3 days of withdrawal following a 2-week administration (5 mg/kg, b.i.d., s.c.) both in intact and 6-OHDA-lesioned animals. In intact rats, c-Fos induction by acute apomorphine exposure showed a striosomal pattern, whereas FosB immunopositivity was diffusely distributed. Following chronic administration, FosB induction turned to a clear striosome dominant pattern similar to c-Fos expression. In denervated striatum, expression of both proteins was profoundly augmented in a homogeneous pattern after a single dose of apomorphine. A distinct striosomal patterning appeared after chronic apomorphine administration in ventromedial part of the denervated striatum with a down-regulation in the matrix and relative enhancement in striosomes. These results suggest that compartmental reorganization of striatal neuronal activity may play a role in long-term behavioral changes induced by chronic dopaminergic treatments both under normal and dopamine-depleted conditions.     

Expression of extracellular matrix degrading enzymes during migration of xenografted brain cells

Proteolytic enzymes, postulated to create an avenue for cell migration by digestion of host extracellular matrix molecules, have been implicated in neoplastic glial cell migration. A similar process is likely to occur in the developing brain. Fetal rabbit brain fragments transplanted into the striatum of the neonatal Shiverer mouse give rise to cells which migrate from the graft site and differentiate into astrocytes and oligodendrocytes. Proteinase expression by transplanted brain cells was studied using immunohistochemistry and in situ hybridization. Immature donor cells expressed the mRNAs for matrix metalloproteinases (MMP) 1 (collagenase) and 3 (stromelysin). Northern blot analysis of rabbit brain showed that MMP-1 in particular is expressed in the immature rabbit cerebrum and down-regulated during maturation. Immature donor cells exhibited immunoreactivity for urokinase plasminogen activator. However, immunoreactivity was also present in maturing neurons. Donor and host astroglia in the vicinity of grafts were immunoreactive for MMP-2 and tissue-type plasminogen activator. This expression may represent a reactive phenomenon, not specifically related to cell migration, by mature astrocytes. Based upon our findings, MMP-1 appears to be a candidate for involvement in migration of immature brain cells in the cerebrum.     

Matrix Metalloproteinase Inhibitors: Applications in Oncology

Matrix metalloproteinases (MMP) are a group of zinc dependentenzymes which include the interstitial collagenases, stromelysins,gelatinases and membrane-type metalloproteinases. They are involvedin the remodelling and turnover of the extracellular matrixproteins. They play a role in wound healing and the pathogenesis ofarthritis. In malignancies they play a role in tumor invasion,metastasis and angiogenesis. A number of synthetic matrixmetalloproteinase inhibitors (MMPIs) have been developed forclinical use. In preclinical tumor models they have shown promisingactivity in achievinginhibition of MMPs and reducing tumor growth and metastatic spread.Some have also shown additive or synergistic effects with cytotoxicagents. Phase I and II studies in human subjects have defined themain side effects of these agents as beingmusculoskeletal pains or arthralgias. As they are cytostatic agentsrather than cytotoxic in activity conventional measurements ofradiological response for assessment are not applicable in trials.Biological activity has been demonstrated in certain cancers by theeffects on levels of tumor markers as surrogate markers of tumorresponse and also by a fibrotic stromal reaction seen in tumortissue. Newer agents have been developed withselective inhibition of certain MMPs in an attempt to reduce theside effects. A number of phase III human clinical trialsevaluating MMPs are being carried out at present but onlyone has been formally reported so far. This study suggested thatmarimastat had no survival advantage when compared to chemotherapywith gemcitabine in advanced pancreatic carcinoma. Current trialsare assessing efficacy of MMPIs in maintenance of remission afterother modalities of therapy or in combination with cytotoxicagents. MMPs have also been demonstrated to play an important rolein the articular cartilage destruction seen in both rheumatoidarthritis and osteoarthritis. The use of MMPIs in both exvivoand in vivomodels have shown promising resultsand trials are in process to assess their potential role in thecontrol of articular destruction. The true therapeutic role ofMMPIs await the results of these randomized studies.     

Purification of alkaline phosphatase from extracellular vesicles of fracture callus cartilage

Summary Extracellular matrix vesicles from fracture callus cartilage were isolated by differential centrifugation and resolved by equilibrium centrifugation on a discontinuous sucrose gradient into two bands. The phosphohydrolytic activity towards p-nitrophenyl phosphate, tetrasodium pyrophosphate and adenosine triphosphate was distributed similarly after differential and equilibrium centrifugation suggesting the association of this activity with the matrix vesicles. The two bands isolated by equilibrium centrifugation of the partially purified vesicular preparation demonstrated high levels of alkaline phosphatase activity. Observed with an electron microscope, the 1.07–1.14 g/cm³ band from the gradient was enriched in electron luscent matrix vesicles while the 1.27 g/cm³ band contained electron dense matrix vesicles. Enzymatic analysis of the 1.27 g/cm³ band indicated a slight contamination due to the presence of mitochondria and lysosomes while the 1.07–1.14 g/cm³ band gave no enzymatic indication of subcellular contamination. A phosphohydrolytic enzyme active towards p-nitrophenyl phosphate, tetrasodium pyrophosphate and adenosine triphosphate was purified from the 1.07–1.14 g/cm³ fraction by DEAE-cellulose column chromatography. Electron micrographs of callus cartilage sections demonstrated densification of the plasma membrane and matrix vesicles following substrate incubation with-glycerophosphate or tetrasodium pyrophosphate. The histochemical and biochemical data indicate that a phosphatase, with multiple substrate specificity, is a component of fracture callus cartilage matrix vesicles.     

Rigid immobilization alters matrix organization in the injured rat medial collateral ligament.

The effects of mobilization on matrix reorganization and density after ligament injury were studied in rat medial collateral ligaments using scanning electron microscopy (SEM). Both medial collateral ligaments of 14 Sprague-Dawley rats were sharply incised transversely at their midpoint. A 1.14-mm threaded Kirschner wire was driven through the tibia and into the femur of the right leg (through the knee) to immobilize that knee at 90 degrees of flexion. Four additional rats were used as controls. The right medial collateral ligament of the control rats was exposed in the same manner as the experimental rats and the wound closed without damaging the ligament. Rats were sacrificed on the 7th and 14th days postinjury and the ligaments evaluated by SEM. The electron micrographs from this study demonstrated that early on, the tissue at the injury site is disorganized on a gross scale with large bundles of poorly organized matrix. Large "defects" were present between bundles in the substance of the ligament and appeared as holes in the ligament around the injury site. As healing progressed, the matrix in the mobilized specimens appeared to bridge the injury site more rapidly and completely with fewer "defects" and thus higher density than the immobilized specimens.     

抗纤软肝冲剂对肝星状细胞胶原降解相关基因表达的影响

目的 从细胞分子水平研究抗纤软肝冲剂对肝星状细胞(HSC)胶原降解的影响。方法 抗纤软肝冲剂药物血清温育传一代HSC，以生理盐水和秋水仙碱为对照。ELISA法测定细胞上清I型胶原，并测定细胞层总蛋白以校正胶原含量；半定量RT—PCR法检测间质胶原酶(MMN)和金属蛋白酶组织抑制因子-1(TIMP-1）的mRNA表达。结果 抗纤软肝冲剂组细胞上清中的I型胶原含量明显低于其他两组(P＜0．05或P＜0．01)；明显促进细胞MMN的mRNA表达(P＜0．05或P＜0.01)，显著抑制细胞TIMP-1的mRNA表达(P＜0．05或P＜0．01)。结论 抗纤软肝冲剂能促进MMN基因表达，抑制TIMP-1基因表达，从而促进胶原降解，这可能是该方抗肝纤维化的细胞分子学机制之一。     

AHPA衍生物的设计、合成及抗癌活性研究

目的 以基质金屑蛋白酶AP—N为靶点，寻找新的抗癌活性化合物。方法 以3—氨基—2—羟基--4—苯基丁酸—亮氨酸(AHPA-Leu)为先导化合物，借助计算机分子结构优化处理进行合理药物设计，合成了8个结构全新的AHPA衍生物，并对目标化合物进行了体内、体外初步药效学评价。结果与结论 初步药效学评价表明，所合成的化合物能增强小鼠的免疫功能，抑制S180肿瘤生长，其中化合物Ⅵ活性最强。