DNA binding proteins of rat thigh muscle: purification and characterization of an endonuclease

Two major DNA binding proteins of molecular weights 34,000 and 38,000 have been identified in the 30,000 g supernatant (S-30) fraction of rat thigh muscle extracts. The presence of 38 KD DNA binding protein in the muscle S-30 could be demonstrated only if Triton X-100 treated extracts were used for Afinity chromatography suggesting that this protein may be a membrane associated DNA binding protein. The 38 KD DNA binding protein differed from the 34 KD DNA binding protein also in its chromatographic behaviour in DE-52 columns in which the 38 KD protein was retained, while the 34 KD protein came out in the flow-through in an electrophoretically pure form. The 34 KD DNA binding protein can also be purified by precipitation with MgCl2. Incubation of 0 · 15 M NaCl eluates (containing the 38 KD and/or 34 KD DNA binding protein) in the presence of 100 mM Mg2+ resulted in the specific precipitation of the 34 KD protein. Prolonged incubation (30 days) of the 0 · 15 Ì NaCl eluates containing the two DNA binding proteins at 4°C led to the preferential degradation of the 34 KD DNA binding protein. Nitrocellulose filter binding assays indicated selective binding of purified 34 KD protein to ss DNA. Purified 34 KD DNA binding protein cleaved pBR 322 supercoiled DNA, and electrophoresis of the cleavage products in agarose gels revealed a major DNA band corresponding to the circular form of DNA

Bioflavonoid luteolin prevents sFlt-1 release via HIF-1α inhibition in cultured human placenta

Preeclampsia (PE) is a serious hypertensive complication of pregnancy and is a leading cause of maternal death and major contributor to maternal and perinatal morbidity, including establishment of long-term complications. The continued prevalence of PE stresses the need for identification of novel treatments which can target prohypertensive factors implicated in the disease pathophysiology, such as soluble fms-like tyrosine kinase 1 (sFlt-1). We set out to identify novel compounds to reduce placental sFlt-1 and determine whether this occurs via hypoxia-inducible factor (HIF)-1α inhibition. We utilized a commercially available library of natural compounds to assess their ability to reduce sFlt-1 release from primary human placental cytotrophoblast cells (CTBs). Human placental explants from normotensive (NT) and preeclamptic (PE) pregnancies were treated with varying concentrations of luteolin. Protein and mRNA expression of sFlt-1 and upstream mediators were evaluated using ELISA, western blot, and real-time PCR. Of the natural compounds examined, luteolin showed the most potent inhibition of sFlt-1 release, with >95% reduction compared to vehicle-treated. Luteolin significantly inhibited sFlt-1 in cultured placental explants compared to vehicle-treated in a dose- and time-dependent manner. Additionally, significant decreases in HIF-1α expression were observed in luteolin-treated explants, suggesting a mechanism for sFlt-1 downregulation. The ability of luteolin to inhibit HIF-1α may be mediated through the Akt pathway, as inhibitors to Akt and its upstream regulator phosphatidylinositol-3 kinase (PI3K) resulted in significant HIF-1α reduction. Luteolin reduces anti-angiogenic sFlt-1 through inhibition of HIF-1α, making it a novel candidate for the treatment of PE

FLT1 and transcriptome-wide polyadenylation site (PAS) analysis in preeclampsia

Maternal symptoms of preeclampsia (PE) are primarily driven by excess anti-angiogenic factors originating from the placenta. Chief among these are soluble Flt1 proteins (sFlt1s) produced from alternatively polyadenylated mRNA isoforms. Here we used polyadenylation site sequencing (PAS-Seq) of RNA from normal and PE human placentae to interrogate transcriptome-wide gene expression and alternative polyadenylation signatures associated with early-onset PE (EO-PE; symptom onset 34 weeks) cohorts. While we observed no general shift in alternative polyadenylation associated with PE, the EO-PE and LO-PE cohorts do exhibit gene expression profiles distinct from both each other and from normal placentae. The only two genes upregulated across all transcriptome-wide PE analyses to date (microarray, RNA-Seq and PAS-Seq) are NRIP1 (RIP140), a transcriptional co-regulator linked to metabolic syndromes associated with obesity, and Flt1. Consistent with sFlt1 overproduction being a significant driver of clinical symptoms, placental Flt1 mRNA levels strongly correlate with maternal blood pressure. For Flt1, just three mRNA isoforms account for > 94% of all transcripts, with increased transcription of the entire locus driving Flt1 upregulation in both EO-PE and LO-PE. These three isoforms thus represent potential targets for therapeutic RNA interference (RNAi) in both early and late presentations

Isolation and characterization of DNA-binding proteins of Yoshida ascites tumour cells

DNA-binding proteins from the cytosol and nuclei of Yoshida ascites sarcoma cells were isolated by affinity chromatography on native and denatured DNA-cellulose columns. The proteins that were retained in the columns were eluted by increasing concentrations of NaCl and analysed by electrophoresis in polyacrylamide gels. Two proteins with approximate molecular weights (MW) of 38000 (38K) and 34000 (34K) D showed high affinity for denatured DNA and eluted in large quantities from denatured DNA-cellulose columns. In contrast, only small amounts of 38000 and 34000 D proteins were found to be associated with native DNA and the native DNA-associated 38K and 34K proteins showed higher rate of phosphorylation than the corresponding denatured DNA-binding proteins. The 38000 and 34000 D DNA-binding proteins were present both in nuclei and cytoplasm. These two proteins together comprised over 70% of the total DNA-binding proteins present in the 0.4 M NaCl eluate of the cytoplasmic extracts. DNA-binding proteins of MW 38000 and 34000 D were also present in a methylcholanthrene-induced fibrosarcoma, although in smaller amounts

Relationship between hypoxia and downstream pathogenic pathways in preeclampsia

Defects in angiogenesis and mitochondrial function in the placenta contribute to the pathogenesis of preeclampsia; however upstream regulators of these pathways are not known. It has been argued that placental hypoxia secondary to abnormal spiral artery remodeling may play a causal role in the angiogenic and mitochondrial abnormalities noted in preeclampsia. The aim of this study was to evaluate the relationship between hypoxia-inducible factor-1α (HIF-1α) ¸ a surrogate of hypoxia, and soluble fms-tyrosine kinase 1 (sFlt1), a circulating anti-angiogenic factor, and microRNA 210 (miR-210), a microRNA that regulates mitochondrial function, in human placentas from preeclamptic and non-hypertensive pregnancies. We first confirmed a 2.5-fold upregulation of HIF-1α protein in placentas from preeclampsia when compared to non-hypertensive controls. Consistent with prior studies, we also observed a 10-fold upregulated sFlt1 mRNA and 2-fold upregulated miR-210 in preeclamptic tissue. Interestingly, while sFlt1 mRNA correlated with miR-210 in preeclampsia (R2 = 0.77, p = 0.0004), there were no significant correlations between these molecules and HIF1α expression. We conclude that non-hypoxia pathways may be involved in the abnormal angiogenic and metabolic alterations noted in preeclampsia

Human endometrial stromal cell plasticity: Reversible sFlt1 expression negatively coincides with decidualization

Preeclampsia (PE) is a major complication of pregnancy in which the placenta is known to have shallow implantation into the uterine decidua. Studies have implicated soluble fms-like tyrosine kinase-1 (sFlt1), a soluble vascular endothelial growth factor (VEGF) receptor protein, in the pathogenesis of PE. sFlt1 has the ability to bind to and neutralize the angiogenic functions of VEGF and placental growth factor (PlGF). The presence of sFlt1 and its action in the endometrium is yet to be determined. We hypothesize that endometrial stromal cells (ESC) at the maternal–fetal interface may play a role in sFlt-1 regulation during pregnancy. In this study, we seek to understand the dynamic regulation of sFlt1 production in primary human ESC as a result of hormone stimulation and withdrawal. To mimic a biphasic menstrual cycle, ESC were treated with cAMP to induce endometrial decidualization that occurs during the luteal secretory phase, followed by cAMP withdrawal reflecting the follicular proliferative phase. Here, we present data to show that (1) ESC produce detectable amounts of sFlt1, (2) sFlt1 expression is turned off during decidualization at both the protein and RNA level (3) ESC decidualization and resulting sFlt1 expression are reversible phenomenon, and (4) Decidualization markers prolactin (PRL) and VEGF expressions in ESC are negatively correlated with sFlt1. These findings may have important implications in diseases such as PE that involve abnormal decidualization, implantation and angiogenesis at the maternal-fetal interface