Direct interaction of the extracellular matrix protein DANCE with apolipoprotein(a) mediated by the kringle IV-type 2 domain

Lipoprotein(a) [Lp(a)] consists of LDL and apolipoprotein(a), and has been shown to be a major, independent, risk factor for arteriosclerosis and thrombosis in humans. To further elucidate the (patho)physiological function(s) of Lp(a)/apo(a), we searched for new protein ligands, using the yeast two-hybrid system and employing the highly repetitive kringle IV type 2 (KIV-2) sequence from apo(a) as bait. The extracellular matrix protein DANCE [developmental arteries and neural crest epidermal growth factor (EGF)-like] recently implicated in atherogenesis was identified as an interactor. A direct physical interaction between DANCE and apo(a) was confirmed by co-purification of both recombinant proteins derived from culture supernatants of transiently transfected COS-1 cells. Furthermore, binding of human plasma-derived Lp(a) to recombinant DANCE was also observed. Finally, when monoclonal anti-apo(a) and polyclonal anti-DANCE antibodies were applied to tissue slices of atherosclerotic carotid artery, the two proteins were found to be co-localized in endothelial and smooth muscle cells, suggesting that they occur together in the arterial wall. However, as yet, the in vivo relevance and the possible functional role of this newly identified DANCE:Lp(a)/apo(a) interaction remains speculative.     

Novel iodine-124 labeled EGFR inhibitors as potential PET agents for molecular imaging in cancer

The in vivo results with our previously reported irreversible labeled inhibitor [(11)C]-ML03 suggested that more chemically stable inhibitors, labeled with a longer-lived radioisotope, could be better candidates for molecular imaging of epidermal growth factor receptor (EGFR) positive tumors. On the basis of this hypothesis we synthesized three new irreversible tyrosine kinase (TK) inhibitors with various chemical reactivities. The three new inhibitors were successfully labeled on the anilino moiety with [(124)I], starting with the 6-amino-4-[(3-tributylstannylphenyl)amino]-quinazoline (9) precursor. The cell-free results, obtained with these new irreversible inhibitors, indicated that compounds 5 (alpha-chloro-acetamide derivative) and 6 (4-dimethylamino-but-2-enoic amide derivative) possessed high potencies toward the EGFR with an irreversible inhibition effect. Compound 4 (alpha-methoxy-acetamide derivative) was found to be less potent, with only a partially irreversible effect. The high potency of compounds 5 and 6 toward the EGFR establishes their potential as PET agents for molecular imaging of EGFR positive tumors. Their prospect as PET biomarkers is further being investigated.     

Percutaneous balloon mitral valvuloplasty in patients with severe mitral stenosis and low transmitral diastolic pressure gradient

An elevated left atrial pressure and high diastolic pressure gradient (DPG) across the mitral valve are the major hemodynamic abnormalities in mitral stenosis (MS). However, a subgroup of patients with severe MS is characterized by low initial DPG. The authors reviewed the clinical, echocardiographic and hemodynamic data as well as procedural results in 180 patients who underwent percutaneous balloon mitral valvuloplasty (PBMV). An initial mean DPG > 10 mmHg was found in 144 patients (80%) (group A) and mean DPG < or = 10 mmHg in 36 patients (20%) (group B). Patients in group A had higher left ventricular ejection fraction (LVEF) than in group B (61 +/- 5% versus 42 +/- 6%, respectively) and higher cardiac index (2.8 +/- 0.4 versus 2.0 +/- 0.3 l/min/m(2) ). In group B 12 patients (33%) had normal LVEF, whereas 24/36 (67%) had reduced LVEF. All the latter had wall motion abnormalities on ventriculography. Unlike group A, intraprocedural echocardiography was essential for monitoring and evaluating immediate results of PBMV in group B. On follow-up of three years, 75% of group A patients and 55% in group B were in functional class I (p < 0.05). PBMV did not significantly improve symptoms in patients in group B who had preprocedure LVEF < or = 35%.     

Cell growth modulates nitric oxide synthase expression in fetal pulmonary artery endothelial cells

Nitric oxide (NO), produced by endothelial (e) nitric oxide synthase (NOS), is a critical mediator of vascular function and growth in the developing lung. Pulmonary eNOS expression is diminished in conditions associated with altered pulmonary vascular development, suggesting that eNOS may be modulated by changes in pulmonary artery endothelial cell (PAEC) growth. We determined the effects of cell growth on eNOS expression in cultured ovine fetal PAEC studied at varying levels of confluence. NOS enzymatic activity was sixfold greater in quiescent PAEC at 100% confluence compared with more rapidly replicating cells at 50% confluence. To determine if there is a reciprocal effect of NO on PAEC growth, studies of NOS inhibition or the provision of exogenous NO from spermine NONOate were performed. Neither intervention had a discernable effect on PAEC growth. The influence of cell growth on NOS activity was unique to pulmonary endothelium, because varying confluence did not alter NOS activity in fetal systemic endothelial cells. The effects of cell growth induced by serum stimulation were also evaluated, and NOS enzymatic activity was threefold greater in quiescent, serum-deprived cells compared with that in serum-stimulated cells. The increase in NOS activity observed at full confluence was accompanied by parallel increases in eNOS protein and mRNA expression. These findings indicate that eNOS gene expression in fetal PAEC is upregulated during cell quiescence and downregulated during rapid cell growth. Furthermore, the interaction between cell growth and NO in the PAEC is unidirectional.     

Rapid Detection of Senescent Mesenchymal Stromal Cells by a Fluorescent Probe

Despite intense interest in human mesenchymal stromal cells (MSCs), monitoring of the progressive occurrence of senescence has been hindered by the lack of efficient detection tools. Here, the discovery of a novel MSC senescence‐specific fluorescent probe (CyBC9) identified by a high‐throughput screen is reported. Compared with the prototypical senescence‐associated β‐galactosidase (SA‐β‐gal) staining, the CyBC9 assay is rapid (2 h) and nontoxic and can thus be applied to live cells. It is shown that CyBC9 is able to stain early and late senescent populations both in monolayer‐ and in microcarrier‐based cultures. Finally, to investigate the mechanism of CyBC9, colocalization assays are performed and it is found that CyBC9 is accumulated in the mitochondria of senescent MSCs presumably due to the loss of membrane potential. Taken together, it is expected that CyBC9 will be a useful tool to ameliorate cell therapy through rapid and early screening of senescent phenotypes in clinically relevant MSCs.     

An optimized model for hCG stimulation of human mural granulosa cell culture

Ovarian follicular development and ovulation in mammals is a highly-regulated process. Most of the current knowledge of ovarian processes was obtained from the studies of non-human models. Molecular studies on human ovarian processes suffer from lack of material and appropriate research tools. Mural granulosa cells (MGCs) culture is a major tool for studying the effect of different substances but a major problem for using these primary MGCs is their unresponsiveness to hCG stimulation at the time of oocyte retrieval. It is acceptable that MGCs regain responsiveness during days in culture but when the best time is and how to accelerate the regenerative process are unknown.The aim of the current study was to establish an optimized protocol which will provide a practical and efficient tool to examine the effect of LH/hCG on different downstream targets in luteinized MGCs.hCG effects were examined according to days in culture and hCG stimulation time. As read-out, we analyzed the gene expression of known hCG targets, protein production, and progesterone secretion.Our results show that with a daily medium exchange, the strongest effect was achieved already 4 days after seeding. On day 4, hCG stimulation triggers two major patterns of gene expression. Early induced genes were highly expressed 6–8?h after hCG, while 24?h of hCG stimulation was needed for late induced genes.Based on our results, we suggest daily medium exchange for 4 days before adding hCG and examine its effect 6 and 24?h later.     

Targeting Enterococcus faecalis Biofilms with Phage Therapy

Phage therapy has been proven to be more effective, in some cases, than conventional antibiotics, especially regarding multidrug-resistant biofilm infections. The objective here was to isolate an anti-Enterococcus faecalis bacteriophage and to evaluate its efficacy against planktonic and biofilm cultures. E. faecalis is an important pathogen found in many infections, including endocarditis and persistent infections associated with root canal treatment failure. The difficulty in E. faecalis treatment has been attributed to the lack of anti-infective strategies to eradicate its biofilm and to the frequent emergence of multidrug-resistant strains. To this end, an anti-E. faecalis and E. faecium phage, termed EFDG1, was isolated from sewage effluents. The phage was visualized by electron microscopy. EFDG1 coding sequences and phylogeny were determined by whole genome sequencing (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"KP339049","term_id":"761545084","term_text":"KP339049"}}KP339049), revealing it belongs to the Spounavirinae subfamily of the Myoviridae phages, which includes promising candidates for therapy against Gram-positive pathogens. This analysis also showed that the EFDG1 genome does not contain apparent harmful genes. EFDG1 antibacterial efficacy was evaluated in vitro against planktonic and biofilm cultures, showing effective lytic activity against various E. faecalis and E. faecium isolates, regardless of their antibiotic resistance profile. In addition, EFDG1 efficiently prevented ex vivo E. faecalis root canal infection. These findings suggest that phage therapy using EFDG1 might be efficacious to prevent E. faecalis infection after root canal treatment.