Background: Preeclampsia (PE) is an obstetric disorder with significant morbidities for both the mother and fetus possibly caused by a failure of the placental trophoblast invasion. However, its pathophysiology largely remains unclear. Here, we performed DNA methylation profiling to determine whether differential patterns of DNA methylation correlate with PE and severe features of PE. Materials and methods: We extracted DNA from placental tissues of 13 normal, five PE, and eight PE pregnant women with severe features. Genome-wide DNA methylation analysis was performed using the Illumina HumanMethylation 850K BeadChip. New functional annotations of differentially methylated CpGs (DMCs) in PE were predicted using bioinformatics tools. Results: Significant differences were evident for 398 DMCs, including 243 DMCs in PE and 155 DMCs in PE with severe features, compared with normal placental tissues. Of these, 12 hypermethylated DMCs and three hypomethylated DMCs were observed in both PE groups, thus were independent from severe features. Three hundred seventy-nine DMCs were identified by the presence or absence of severe features. Two hundred genes containing these DMCs were associated with developmental processes and cell morphogenesis. These genes were significantly associated with various PE complications such as disease susceptibility, viral infections, immune system diseases, endocrine disturbance, seizures, hematologic diseases, and thyroid diseases. Conclusions: This is the first study to investigate the genome-scale DNA methylation profiles of PE placentas according to severe features. The epigenetic variation in the placentas probably resulted in altered developmental processes and immune dysregulation, contributing to PE. This study provides basic information to refine the clinical and pathological mechanisms of the severe features in placenta-mediated PE.

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Cystatins are endogenous inhibitors of mammalian lysosomal cysteine proteinases, such as cathepsins B, L, H, and S. Cystatin C belongs to the type 2 cystatin family. In this study, the 751-bp cystatin C cDNA (PoCystatin C) of olive flounder (Paralichthys olivaceus) was cloned by screening from the olive flounder cDNA library. The mRNA expression of the PoCystatin C gene was examined in various tissues from normal and lipopolysaccharide (LPS)-stimulated olive flounder by RT-PCR and was compared with inflammatory cytokines IL-1β, IL-6, and IL-8. PoCystatin C transcripts ubiquitously existed in all normal and LPS-stimulated tissues that were tested. The recombinant PoCystatin C protein was expressed in Escherichia coli BL21(DE3) in pCold™ TF DNA expression vector as a 70-kDa fusion protein. The protease inhibitory activities of recombinant PoCystatin C toward papain cysteine protease, piscine cathepsins (L, S, K, F, and X), and bovine cathepsin B were measured with the synthetic fluorogenic peptide substrates. PoCystatin C tightly inhibited papain cysteine protease, whereas cathepsins L, S, K, F, X, and B were inhibited with lower affinities. Our results indicate that the P. olivaceus cystatin C is a homolog of mammalian cystatin C due to its sequence, structure, tissue expression, and biochemical activity.

Cathepsin X, also known as cathepsin Z, is referred to as a "lysosomal proteolytic enzyme" and a member of the peptidase C1 family, which is involved in various biological processes such as immune response, cell adhesion, and proliferation. In the present study, the cDNA of starfish (Asterina pectinifera), which is known to cause serious damage to commercial shellfish mariculture, cathepsin X (ApCtX) was isolated through the combination of homology molecular cloning and rapid amplification of cDNA ends (RACE) methods for the application to find a way to reduce/control starfish densities. The full-length of ApCtX gene was determined to consist of the 2,240 bp nucleotide sequence, which encoded for a preproprotein of 296 amino acids with a molecular mass of about 32.7 kDa. The tissue type expression of ApCtX was determined in various tissues of A. pectinifera and was shown most abundantly in the liver. The cDNA encoding pro-mature enzyme of ApCtX was expressed in Escherichia coli BL21 (DE3) using the pGEX-4T-1 expression vector. Its activity was quantified by cleaving the synthetic peptide Z-Phe-Arg-AMC. The optimal pH for the protease activity was 6.5. The enzymatic activity of proApCtX was reduced by antipain, NEM, EDTA, EGTA, and 1,10-phenanthroline, and the proApCtX enzyme was significantly inhibited by CuSO₄, HgCl₂, CoCl₂, and SDS whereas Triton X-100 and Brij 35 might have potentially acted as an activator. Here, we demonstrated for the first time that the structural features and enzymatic characteristics of Echinoderms cathepsin X are similar to those of the other mammalian and piscine cathepsin X except its pH optimum, and the results of tissue-specific expression might explain their importance in food digestion by hepatic cecain starfish.