Impact of matrix metalloproteinases on inhibition of mineralization by fetuin.

BACKGROUND AND OBJECTIVE: Human subjects affected by inflammatory diseases, such as periodontitis, may be at increased risk for the development of cardiovascular diseases and calcification of atheromas; however, the potential mechanisms have not been defined. Alpha-2-Heremans Schmid glycoprotein (fetuin A) is an abundant serum glycoprotein of ~49 kDa that inhibits ectopic arterial calcification. We examined whether matrix metalloproteinases (MMPs), which are increased in inflammatory diseases, including periodontitis, bind and degrade fetuin and alter its ability to inhibit calcification in vitro. MATERIAL AND METHODS: Binding and cleavage of fetuin by MMPs were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-silico analyses and mass spectrometry. The effects of intact and MMP-degraded human fetuin on mineralization were measured in a cell-free assay. RESULTS: From in-silico analyses and literature review, we found that only MMP-3 (stromelysin) and MMP-7 (matrilysin) were predicted to cleave human fetuin at levels that were physiologically relevant. In-vitro assays showed that MMP-7, and, to a lesser extent, MMP-3, degraded human fetuin in a time- and dose-dependent manner. Fetuin peptides generated by MMP-7 cleavage were identified and sequenced by mass spectrometry; novel cleavage sites were found. Hydroxyapatite mineralization in vitro was strongly inhibited by fetuin (> 1 µm), as was MMP-3-cleaved fetuin, while MMP-7-cleaved fetuin was threefold less effective in blocking mineralization. CONCLUSION: MMP-7 and, to a lesser extent, MMP-3, affect the ability of fetuin to inhibit the formation of hydroxyapatite in vitro. These data suggest that the MMPs increased in inflammatory diseases, such as periodontitis, could affect regulation of mineralization and potentially enhance the risk of calcified atheroma formation.

Antiviral activities of biflavonoids.

Biflavonoids such as amentoflavone (1), agathisflavone (2), robustaflavone (3), hinokiflavone (4), volkensiflavone (5), rhusflavanone (7), succedaneflavanone (9), all isolated from Rhus succedanea and Garcinia multiflora, as well as their methyl ethers and acetates, volkensiflavone hexamethyl ether (6), rhusflavanone hexaacetate (8), and succedaneflavanone hexaacetate (10) were evaluated for their antiviral activities. The inhibitory activities against a number of viruses including respiratory viruses (influenza A, influenza B, respiratory syncytial, parainfluenza type 3, adenovirus type 5, and measles) and herpes viruses (HSV-1, HSV-2, HCMV, and VZV) were investigated. The results indicated that robustaflavone exhibited strong inhibitory effects against influenza A and influenza B viruses with EC50 values of 2.0 micrograms/ml and 0.2 microgram/ml, respectively, and selectivity index values (SI) of 16 and 454, respectively. Amentoflavone and agathisflavone also demonstrated significant activity against influenza A and B viruses. Amentoflavone and robustaflavone exhibited moderate anti-HSV-1 anti-HSV-2 activities with EC50 values of 17.9 micrograms/ml (HSV-1) and 48.0 micrograms/ml (HSV-2) and SI values of > 5.6 (HSV-1) and > 2.1 (HSV-2) for amentoflavone; EC50 values of 8.6 micrograms/ml (HSV-1) and 8.5 micrograms/ml (HSV-2), and SI values of > 11.6 (HSV-1) and > 11.8 (HSV-2) for robustaflavone. Rhusflavanone demonstrated inhibitory activities against influenza B, measles, and HSV-2 viruses with SI values of 9.3, 8 and > 6.4, respectively. Succedaneaflavanone exhibited inhibitory activities against influenza B virus and VZV with SI values of 15 and < 3.0, respectively.

[Uterine rupture in sheep]. / Uterusruptuur bij het schaap.

A number of cases of uterine rupture in sheep occurring in the field are reported. Ruptures of the uterus in sheep which could not be sutured were not found to carry a bad prognosis in every case.