[Show abstract][Hide abstract] ABSTRACT: Endometrial cancer (EC) is the leading malignant tumor occurring in the female genital tract
and some subtypes are highly invasive and metastatic. miRNAs are small non-coding RNAs
that have a broad impact on cancer progression. In particular, miR-194 regulates epithelial to
mesenchymal transition (EMT) by suppressing the expression of BMI-1 in EC. In this
retrospective study, the clinical significance of miR-194 was investigated in archival EC
specimens. We extracted total RNA from thirty-two EC samples and quantified the
expression level of miR-194. We discovered that the expression level of miR-194 was
significantly (P = 0.03) lower in type I EC patients with more advanced stage. In addition,
patients with higher miR-194 levels have better prognosis than those with lower miR-194
levels (P = 0.0067; Cut-off value of miR-194 = 0.3). These results indicate that miR-194 has
potential to serve as prognostic biomarker for EC patients.
[Show abstract][Hide abstract] ABSTRACT: Ca(2+) is implicated in almost every step of the life cycle of viruses, including virus entry into host cells, virus replication, virion assembly, maturation, and release. However, due to the lack of prediction algorithms and rigorous validation methods, only limited cases of viral Ca(2+)-binding sites are reported. Here, we introduce a method to predict continuous EF-hand or EF-hand-like motifs in the viral genomes based on their primary sequences. We then introduce a grafting approach, and the use of luminescence resonance energy transfer and Ca(2+) competition assay for experimental verification of predicted Ca(2+)-binding sites. This protocol will be valuable for the prediction and identification of unknown Ca(2+)-binding sites in virus.
[Show abstract][Hide abstract] ABSTRACT: Human phospholipid scramblase 1 (hPLSCR1) belongs to ATP independent class of phospholipid translocators which possesses a single EF-hand like Ca(2+) -binding motif and also C-terminal helix (CTH). CTH domain of hPLSCR1 was believed to be a putative single transmembrane helix at C-terminus. Recent homology modelling studies by Bateman et al predicted that hydrophobic nature of this helix is due to its packing in the core of the protein domain and proposed that this helix is not a true transmembrane helix. To determine the exact function of CTH of hPLSCR1, we deleted CTH domain and determined (i) whether CTH plays any role beyond membrane anchorage (ii) functional consequence of CTH deletion and (iii) conformation changes associated with it in presence of lipid environment. In vitro reconstitution studies confirm that predicted CTH is indeed required for membrane insertion and scrambling activity. CTH deletion caused 50% decrease in binding affinity of Ca(2+) for ∆CTH-hPLSCR1 (Ka = 115 µM) than hPLSCR1 (Ka = 249 µM). Far UV-CD studies revealed that CTH peptide adopts α-helicity only in presence of SDS micelles and negatively charged vesicles indicating electrostatic interactions are required for insertion of peptide. CTH peptide quenching studies confirm that the predicted CTH inserts into membrane and its ability to interact with membrane depends on the presence of charge interactions. TOXCAT assay revealed that CTH of hPLSCR1 does not oligomerize in the membrane. We conclude that CTH is required for membrane insertion, Ca(2+) co-ordination and also plays an important role in the functional conformation of hPLSCR1. This article is protected by copyright. All rights reserved.
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