Characterization of genetic‐origin‐dependent monoallelic expression in mouse embryonic stem cells

Monoallelic gene expression occurs in various mammalian cells and can be regulated genetically, epigenetically and/or stochastically. We identified 145 monoallelically expressed genes (MoEGs), including seven known imprinted genes, in mouse embryonic stem cells (ESCs) derived from reciprocal F1 hybrid blastocysts and cultured in 2i/LIF. As all MoEGs except for the imprinted genes were expressed in a genetic‐origin‐dependent manner, we focused on this class of MoEGs for mechanistic studies. We showed that a majority of the genetic‐origin‐dependent MoEGs identified in 2i/LIF ESCs remain monoallelically expressed in serum/LIF ESCs, but become more relaxed or even biallelically expressed upon differentiation. These MoEGs and their regulatory regions were highly enriched for single nucleotide polymorphisms. In addition, some MoEGs were associated with retrotransposon insertions/deletions, consistent with the fact that certain retrotransposons act as regulatory elements in pluripotent stem cells. Interestingly, most MoEGs showed allelic differences in enrichment of histone H3K27me and H3K4me marks, linking allelic epigenetic differences and monoallelic expression. In contrast, there was little or no allelic difference in CpG methylation or H3K9me. Taken together, our study highlights the impact of genetic variation including single nucleotide polymorphisms and retrotransposon insertions/deletions on monoallelic epigenetic marks and expression in ESCs.     

The Minimum Alveolar Concentration of Xenon in the Elderly Is Sex-dependent

Background: The minimum alveolar concentration (MAC) of xenon in the elderly has not been determined. Moreover, because xenon inhibits the activity of the N-methyl-d-aspartate receptors, and because N-methyl-d-aspartate receptor antagonists such as ketamine and MK-801 exert sex-dependent actions, we hypothesized that the MAC of xenon would also be sex-dependent.

Methods: Forty-eight patients of both sexes (24 patients of each sex), who were aged 65 yr or older and were undergoing elective laparotomy, were anesthetized with inhalational induction of xenon. Those who demonstrated marked agitation received supplemental propofol intravenously. After tracheal intubation, the end-tidal concentration of xenon was maintained at 45 (women only), 50, 55, 60, 65, 70, or 75% (men only) for at least 15 min before skin incision. These concentrations were randomly allocated to four patients of each sex. Each patient was monitored for the presence or absence of any purposeful bodily movement for 1 min following skin incision. The MAC of xenon was calculated separately for men and women using logistic regression analysis.

Results: The MAC of xenon was 69.3% (95% CI, 63.0-75.6%) for men and 51.1% (44.6-57.6%) for women. The two 95% confidence intervals did not overlap, indicating a statistically significant difference (P < 0.05).     

Oxidized low density lipoprotein potentiation of Fas-induced apoptosis through lectin-like oxidized-low density lipoprotein receptor-1 in human umbilical vascular endothelial cells.

Under normal conditions, vascular endothelial cells are resistant to Fas-mediated apoptosis, although they express detectable Fas on their cell surface. Because oxidized Low density lipoprotein (Ox-LDL) is thought to promote atherogenesis, the potential role that Ox-LDL may play in Fas-mediated apoptosis was investigated in human umbilical vascular endothelial cells (HUVECs), focusing particularly on the involvement of the lectin-like Ox-LDL receptor-1 (LOX-1). HUVECs were treated with agonistic anti-Fas antibody (CH11) and Ox-LDL and then the degree of apoptosis was determined by cell death ELISA. Ox-LDL concentration-dependently sensitized Fas-mediated apoptosis. Flow cytometry demonstrated that Ox-LDL dose-dependently up-regulated cell surface Fas expression. On the other hand, treating HUVECs with Ox-LDL did not lead to any significant change in the expression of death mediators, including Fas, Fas ligand (FasL), FADD, and FLICE as assessed by multiplex polymerase chain reaction amplification. More importantly, these effects of Ox-LDL on Fas-mediated apoptosis were significantly blocked by a neutralizing LOX-1 monoclonal antibody, which can block LOX-1-mediated cellular uptake of Ox-LDL. Ox-LDL may be an important factor involved in the regulation of Fas-induced apoptosis via Ox-LDL/LOX-1 interaction in vascular endothelial cells. The results may provide insights into the pathogenesis of accelerated atherosclerosis in patients with hyperlipidemia.     

The histological effects of cryocoagulation on the myocardium and coronary arteries

The effects of epicardial and endocardial cryolesions were histologically studied in 29 dogs. To produce epicardial lesions, hypothermic exposure was applied at −60°C for 3 minutes, over or adjacent to the left anterior descending coronary artery. To produce endocardial lesions, exposure was applied at −60°C for 2 minutes, using the inflow occlusion technique over the ventricular septum. The dogs were killed 30 minutes to 6 months later. The cryolesions were sharply demarcated from the surrounding tissues and showed similar healing processes. The lesions showed no tendency to form aneurysms or rupture, although moderate intimal thickening of the coronary artery subjacent to the probe was observed. Our results indicate that cryocoagulation may greatly contribute to the surgical treatment of cardiac arrhythmias.     

The Study of Clinical Usefulness of Basic FGF

Fibroblast‐collagen matrix contraction has been used as a model system to study how cells organize connective tissue. Previous work showed that lysophosphatidic acid (LPA)‐stimulated collagen matrix contraction is independent of Rho kinase whereas platelet‐derived growth factor (PDGF)‐stimulated contraction is Rho kinase dependent. The current studies were carried out to learn more about the molecular motors responsible for LPA‐ and PDGF‐stimulated fibroblast‐collagen matrix contraction. Fibroblasts whose MLC kinase was knocked down using siRNA were used to measure matrix contraction in the presence of LPA or PDGF with or without the Rho kinase inhibitor added. The extent of contraction of MLC kinase‐silenced cells was not detectably different from control cells. Other experiments were carried out to test the effects of LPA and PDGF on MLC phosphorylation with and without Rho kinase inhibitor. After 15 min of growth factor stimulation, levels of diphosphorylated MLC were highest in cells in LPA‐containing medium and lowest in PDGF containing medium. Prior addition of Rho kinase inhibitor markedly reduced phosphorylation in every case. These observations suggested that stimulation of collagen matrix contraction required neither growth factor stimulation of MLC phosphorylation nor MLC kinase.     

The detection of coronary ostial lesions by intraoperative transesophageal echocardiography: Report of a case

Transesophageal echocardiography (TEE) is a valuable diagnostic tool for providing clear images of the proximal coronary arteries. We describe herein the case of an elderly man in whom dissection and an atherosclerotic plaque in the proximal coronary arteries were demonstrated by TEE during combined coronary artery bypass grafting and aortic valve replacement. Thus, retrograde cardioplegia was employed, whereby trauma to the coronary ostia was avoided.     

Characterization of intracellular Ca2+ transient by the hybrid logistic function in aequorin-injected rabbit and mouse papillary muscles

Myocardial intracellular calcium (Ca2+) transients (CaTs) regulate tension generation and relaxation. Isometric tension curves are often analyzed using exponential equations; however, we previously demonstrated that hybrid logistic (HL) functions, which describe the difference between two S-shaped logistic functions, provide more accurate representations. In the present study, we investigated the potential application of HL functions for analyzing CaTs directly. CaTs were measured using the calcium-sensitive bioluminescent protein, aequorin, in 7 isolated rabbit right ventricular and 15 isolated mouse left ventricular papillary muscles. CaT data were fit by the least-squares method using HL and polynomial exponential (PE) function equations. The mean correlation coefficient (r) values of HL and PE fits were 0.9934 vs. 0.9523 in rabbit and 0.9980 vs. 0.9407 in mouse, respectively. The Z transformation of r value and the adjusted coefficient of determination (r squares) were higher, and the residual mean squares and Akaike information criterion values, which estimate goodness of fit between functions with different numbers of parameters, were lower for the HL curves than for the PE curves in both rabbit and mouse. There were significant correlations between the calculated values from the best-fit HL function curve and the primary CaT data. Thus the HL function curves more accurately described the amplitudes and time courses of CaTs in both rabbit and mouse papillary muscles. We speculate that the first logistic component curve reflects the concentration and time course of Ca2+ inflow into the cytoplasmic space, and that the second logistic component curve reflects the concentrations and time courses of Ca2+ removal from the cytoplasmic space as well as Ca2+ binding to troponin. This approach might provide a more robust model for studying CaTs and cardiac cycle regulation.