Serine Protease Zymography: Low-Cost, Rapid, and Highly Sensitive RAMA Casein Zymography.

To detect serine protease activity by zymography, casein and CBB stain have been used as a substrate and a detection procedure, respectively. Casein zymography has been using substrate concentration at 1 mg/mL and employing conventional CBB stain. Although ordinary casein zymography provides reproducible results, it has several disadvantages including time-consuming and relative low sensitivity. Improved casein zymography, RAMA casein zymography, is rapid and highly sensitive. RAMA casein zymography completes the detection process within 1 h after incubation and increases the sensitivity at least by tenfold. In addition to serine protease, the method also detects metalloprotease 7 (MMP7, Matrilysin) with high sensitivity.

[The application of matrix metalloproteinase-3 and 7 joints ultrasonic score in assessment of disease activity in patients with rheumatoid arthritis].

OBJECTIVE: To evaluate the significance of serum matrix metalloproteinase-3 (MMP-3) and joint ultrasonography in assessing the activity of rheumatoid arthritis (RA) by comparing MMP-3 level and the ultrasonic 7 joints (US7) score in RA patients. METHODS: Serum MMP-3 level and US7 score were measured in 133 RA patients by immune turbidity and Doppler ultrasound. Synchronous 53 healthy subjects were recruited as controls. Clinical data were collected. Erythrocyte sedimentation rate (ESR), serum level of anti-cyclic citrullinated peptide (CCP) antibody, health assessment questionnaire (HAQ) and disease activity score 28 (DAS28) were measured. The level of disease activity is interpreted as remission(DAS28<2.6), low(DAS 28≥2.6-<3.2), moderate(DAS 28≥3.2-<5.1), high(DAS28≥5.1). The discriminating validity of MMP-3 and US7 score in disease was evaluated using receiver operating characteristic (ROC) curve analysis with DAS28 as the reference standard. RESULTS: Compared with that in healthy controls [35.20(25.90, 48.90) µg/L] and remission patients[33.40(22.60, 678.40) µg/L], the MMP-3 level in moderate [105.1(61.70, 172.70) µg/L] and high [363.1(161.50, 475.90) µg/L] groups increased dramatically. US7 score in patients with high disease activity was significantly higher than that in other groups. The level of MMP-3 was significantly correlated with DAS28, HAQ, US7 score, yet did not have correlation with anti-CCP antibody. Serum level of MMP-3 was positively correlated with US7 score(r=0.566, P<0.001). In evaluating the disease activity, US7 score combined with MMP-3 (AUC 0.863 2) was not superior to MMP-3 alone (AUC 0.854 3), but significantly better than single US7 score (AUC 0.764 3, P<0.05). CONCLUSIONS: MMP-3 is an effective and simple index in evaluating RA disease activity. The combination of MMP-3 and US7 score does not further improve the efficacy to evaluate disease activity than MMP-3 alone in patients with RA.

Differential expression of extracellular matrix constituents and cell adhesion molecules between malignant pleural mesothelioma and mesothelial hyperplasia.

INTRODUCTION: Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm associated with asbestos exposure. Currently, the molecular mechanisms that induce MPM development are still unknown. The purpose of this study was to identify new molecular biomarkers for mesothelial carcinogenesis. METHODS: We analyzed a panel of 84 genes involved in extracellular matrix remodeling and cell adhesion by polymerase chain reaction (PCR) array in 15 samples of epithelioid mesothelioma and 10 samples of reactive mesothelial hyperplasia (MH; 3 of 25 samples were inadequate for mRNA analysis). To validate the differentially expressed genes identified by PCR array, we analyzed 27 more samples by immunohistochemistry, in addition to the 25 samples already studied. RESULTS: Twenty-five genes were differentially expressed in MPM and MH by PCR array. Of these we studied matrix metalloproteinase 7 (MMP7), MMP14, CD44, and integrin, alpha3 expression by immunohistochemistry in 26 epithelioid MPM and 26 MH samples from the entire series of 52 cases. We observed higher MMP14 and integrin, alpha3 expression in MPM samples compared with MH samples (p = 0.000002 and p = 0.000002, respectively). Conversely, CD44 expression was low in most (57.7%) mesothelioma samples but only in 11.5% of the MH samples (p = 0.0013). As regards MMP7, we did not observe differential expression between MH and MPM samples. CONCLUSIONS: We have extensively studied genes involved in cell adhesion and extracellular matrix remodeling in MPM and MH samples, gaining new insight into the pathophysiology of mesothelioma. Moreover, our data suggest that these factors could be potential biomarkers for MPM.