Modifications in chromatin, including DNA methylation and histone modification, are known to be important epigenetic determinants of gene transcription. DNA methylation levels fluctuate markedly in early mouse development. In preimplantation development, the mouse embryo undergoes active and passive genomic demethylation (3,15). This is restored at the time of implantation by the combined action of de novo and maintenance DNA methyltransferases (Dnmts). Studies of DNA methyltransferase 1-deficient (Dnmt1 Ϫ/Ϫ ) and Dnmt3a/ Dnmt3b-deficient (Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ]) mouse embryos have demonstrated that restoring DNA methylation is essential for development (13,19). Dnmt1 Ϫ/Ϫ and Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] embryos exhibit an early-lethal phenotype. At day 9.5 postcoitus, the embryos appear to have gastrulated but exhibit marked growth delay, having failed to turn or develop somites. In the presence of Dnmt1 deficiency, development is thought to fail because of cell death. Dnmt1-deficient embryoid bodies (EBs) aberrantly express Xist, down-regulate X-linked genes, and apoptose when induced to differentiate (20). Late-passage hypomethylated Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] embryonic stem (ES) cells are unable to form teratomas in vivo, but the cause of their differentiation failure has not been studied (4).Early embryonic development is characterized by high levels of Dnmt3a and Dnmt3b expression. These enzymes clearly have roles in initiating remethylation of the genome following preimplantation demethylation, but it is not known whether continued de novo methyltransferase activity is required for development once global remethylation has taken place. This was our reason for studying the differentiation of Dnmt[3a Ϫ/Ϫ , 3b Ϫ/Ϫ ] ES cells in vitro. These mutant ES cells were derived from fully methylated wild-type ES cells and would have been predicted to have retained most of their methylation because of the continued presence of the maintenance methyltransferase Dnmt1. In fact, while early-passage Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] ES cells are well methylated, DNA methylation levels fall progressively in culture (4). However, the rate of loss and the precise levels of methylation remaining have not been quantified. We have used a quantitative assay of DNA methylation to examine the effects of progressively decreasing genomic methylation levels on differentiation in vitro. Our studies reveal a clear but unexpected difference between the behaviors of hypomethylated Dnmt1 Ϫ/Ϫ and Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] ES cells in in vitro assays of differentiation. At very low levels of DNA methylation, Dnmt[3a Ϫ/Ϫ ,3b Ϫ/Ϫ ] ES cells demonstrate an inability to initiate differentiation upon leukemia inhibitory factor (LIF) withdrawal, remaining viable and retaining markers characteristic of undifferentiated ES cells. MATERIALS AND METHODSES cell culture. ES cells were maintained on gelatin in a Glasgow modification of Eagle medium (Invitrogen) supplemented with 10% fetal calf serum, 100 M 2-mercaptoethanol, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 ...

Plasma extravasation from postcapillary venules is one of the earliest steps of inflammation. Substance P (SP) and bradykinin (BK) mediate extravasation and cause hypotension. The cell-surface enzyme neutral endopeptidase (NEP) inactivates both peptides. Thus, absence of NEP may predispose development of inflammation and hypotension. We examined these possibilities in mice in which the NEP gene was deleted by homologous recombination. There was widespread basal plasma extravasation in postcapillary venular endothelia in NEP-/- mice, which was reversed by recombinant NEP and antagonists of SP (NK1) and BK (B2) receptors. Mean arterial blood pressure was 20% lower in NEP-/- animals, but this was unaffected by reintroduction of recombinant NEP and the kinin receptor antagonists. The hypotension was also independent of nitric oxide (NO), because NEP-/- mice treated with a NO synthase inhibitor remained hypotensive relative to the wild type. Thus, NEP has important roles in regulating basal microvascular permeability by degrading SP and BK, and may regulate blood pressure set point through a mechanism that is independent of SP, BK and NO. The use of NEP antagonists as candidate drugs in cardiovascular disease is suggested by the blood pressure data reported herein.

There is accumulating evidence that cells exposed to low and often environmentally relevant doses of ionizing radiation survive the initial insult, but transmit genomic instability to their progeny. The underlying mechanism of radiation-induced genomic instability is unknown. We present bio-chemical evidence consistent with the hypothesis that enhanced and persistent oxy-radical activity may be responsible.

The widely held view that transfused bone marrow cells will not proliferate in normal mice, not exposed to irradiation or other forms of bone marrow ablation, was reinvestigated. Forty million bone marrow cells from male donors were given to female recipients on each of 5 consecutive days, 5 to 10 times the number customarily used in the past. When the recipients were examined 2-13 weeks after the last transfusion, donor cells were found to average 16-25% of total marrow cells. Similar percentages ofdonor cells were found when variants ofthe enzyme phosphoglycerate kinase determined electrophoretically were used for identification of donor and recipient cells. Evidence is presented that the proportion of donor cells is compatible with a linear dependence on the number of cells transfused over the range tested-i.e., million bone marrow cells injected intravenously. Special proliferative sites thus do not appear to be required.There is a long-standing presumption that intravenously transfused marrow cells proliferate only to a minimal extent, even in congeneic mice (1-3). However, Saxe et aL (4) recently reported the replacement of 10% of marrow cells by donor cells after transfusion of 100 million marrow cells in 5 daily aliquots of 20 million given intravenously, though not after intraperitoneal injection.We have confirmed the substantial proliferation of donor marrow cells after multiple large transfusions. The results appear of particular interest because they make unnecessary the assumption of special proliferative sites for stem cells [colonyforming units, spleen (CFU-S)] thought to be filled in normal marrow and thus precluding seeding of transfused CFU-S (1). MATERIALS AND METHODSMost of the experiments were with CBA mice. To trace the origin ofproliferating bone marrow cells after transfusion, bone marrows of female recipients of cells from male donors were analyzed cytologically for the presence of the Y chromosome as described (3, 5). Suspensions of bone marrow were prepared in phosphate-buffered saline and counted in a Coulter Counter, and the desired number of cells was given intravenously in 0.5 ml of phosphate-buffered saline.Further experiments were performed using donors and recipients carrying A and B alleles of the X chromosome locus PGK-1 of phosphoglycerate kinase (EC 2.7.2.3); the products of these alleles differ in electrophoretic mobility (6). The PGK-1A variant, on a predominantly C3H genetic background, was kindly donated by John West (Oxford University) and was backcrossed onto the CBA/Ca (PGK-1B) genetic background for nine generations. After electrophoretic separation of the two variant enzymes, densitometry provided the percentages of donor and host cells in the recipients' bone marrow or other organs. Full details of the method, modified from that of Buecher et aL (7), will be published elsewhere. Briefly, cell suspensions were prepared in RPMI-1640 medium, centrifuged at 150 X g, and exposed to a 10-sec distilled water shock to lyse the erythrocytes (8). After one washing ...

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