MicroRNAs (miRNAs) in blood plasma are stable under high levels of ribonuclease activity and could function in tissue-to-tissue communication, suggesting that they may have distinctive structural characteristics compared with non-circulating miRNAs. In this study, the expression of miRNAs in horse plasma and their characteristic nucleotide composition were examined and compared with non-plasma miRNAs. Highly expressed plasma miRNA species were not part of the abundant group of miRNAs in non-plasma tissues, except for the eca-let-7 family. eca-miR-486-5p, -92a, and -21 were among the most abundant plasma miRNAs, and their human orthologs also belong to the most abundant group of miRNAs in human plasma. Uracil and guanine were the most common nucleotides of both plasma and non-plasma miRNAs. Cytosine was the least common in plasma and non-plasma miRNAs, although levels were higher in plasma miRNAs. Plasma miRNAs also showed higher expression levels of miRNAs containing adenine and cytosine repeats, compared with non-plasma miRNAs. These observations indicate that miRNAs in the plasma have a unique nucleotide composition.
Matrix metalloproteinase (MMP)-9 degrades type IV collagen in the basement membrane and plays crucial roles in several pathological implications, including tumorigenesis and inflammation. In this study, we analyzed the effect of flavonols on MMP-9 expression in phorbol-12-myristate-13-acetate (PMA)-induced human fibrosarcoma HT-1080 cells. Galangin and kaempferol efficiently decreased MMP-9 secretion, whereas fisetin only weakly decreased its secretion. Galangin and kaempferol did not affect cell viability at concentrations up to 30 M. Luciferase reporter assays showed that galangin and kaempferol decrease transcription of MMP-9 mRNA. Moreover, galangin and kaempferol strongly reduce IB phosphorylation and significantly decrease JNK phosphorylation. These results indicate that galangin and kaempferol suppress PMA-induced MMP-9 expression by blocking activation of NF-B and AP-1. Therefore, these flavonols could be used as chemopreventive agents to lower the risk of diseases involving MMP-9.
Matrix metalloproteinases (MMPs) play an important role in tissue remodeling during normal physiological situations and pathological implications such as tumor invasion and metastasis. MMP inhibitors were screened from extracts of medicinal herbs by an enzymatic assay using the MMP-14 catalytic domain. Among samples tested, a methanol extract of the root of Dalbergia odorifera T. CHEN (Leguminosae) showed the strongest inhibitory activity. The inhibitory component was purified through fractionation methods and identified as fisetin, abundant in many fruits and vegetables. In addition to inhibition of MMP-14, fisetin inhibits MMP-1, MMP-3, MMP-7, and MMP-9, more efficiently than a naturally occurring MMP inhibitor tetracycline. Fisetin dose-dependently inhibits proliferation of fibrosarcoma HT-1080 cells and human umbilical vascular endothelial cells (HUVECs), MMP-14-mediated activation of proMMP-2 in HT-1080 cells, invasiveness of HT-1080 cells, and in vitro tube formation of HUVECs. Therefore, fisetin could be valuable as a chemopreventive agent against cancer and a lead compound for development of therapeutic MMP inhibitors.
Cytochrome P450 1A1 (CYP1A1) is a heme-containing mono-oxygenase involved in metabolism
of environmental contaminants. Two variants of dog CYP1A1 with a single residue difference
were identified and designated Sap1 and Sap2. Compared with Sap1, Sap2 had a Trp50Leu
substitution. The biochemical characteristics of the variants were comparatively analyzed
using heterologous expression in Escherichia coli. The membrane fraction
of E. coli expressing Sap2 exhibited higher CYP holoprotein and heme
contents than the Sap1-containing membranes, although the level of total CYP1A1 protein
(i.e., apoprotein + holoprotein) was comparable between the groups. As normalized to
holo-CYP content, the Sap2-expressing membranes showed lower CYP1A1-specific enzyme
activities, such as 7-ethoxyresorufin O-dealkylation (EROD), than the
Sap1 group. In single substitution variants of residue 50, proteins with hydrophobic
residues having mass similar to Leu exhibited lower EROD activities than those with
hydrophobic residues having larger mass than Leu. In addition, variants with polar or
charged residues having mass similar to Leu showed activities that were comparable to
those of Sap2. Taken together, these findings suggest that the Trp50Leu substitution leads
to an enhancement of holo-CYP1A1 formation, but diminishes the enzyme activity because of
the small size of Leu compared with Trp.
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