We studied which endothelin (ET) receptor subtypes mediate ET-1-induced vasocontraction in the human pulmonary artery (PA) compared with the rabbit PA. ET-1 produced potent contraction in both human and rabbit isolated PAs. In human PA, ET-1-induced contraction was competitively antagonized by BQ-123 (an ETA receptor antagonist) with a pA2 value of 7.68. In rabbit PA, however, even a high concentration of BQ-123 (1 microM) did not affect the contraction. BQ-3020 (an ETB receptor agonist) produced potent contraction in rabbit PA but not in human PA. Binding assays of the membrane preparations showed that human and rabbit PAs contained ETA and ETB receptors in ratios of 93:7 and 23:77, respectively. These results suggest interspecies differences in the ET receptor subtypes that mediate ET-1-induced vasocontraction; ETA receptors are dominant in the human PA, whereas ETB receptors are dominant in the rabbit PA. Furthermore, the predominance of ETA receptors in human PA was supported by autoradiographical studies. If ET-1 acts as a physiological and/or pathophysiological vasocontractor in the human pulmonary circulation, an ETA receptor antagonist would function as a pulmonary vasodilator in humans.

A new octadentate cavitand forms a stable dimeric molecular capsule via metal-coordination, creating a large and elaborate three-dimensional cavity in which large aromatic guests are accommodated to form supramolecular complexes.

2'-Deoxyoxanosine (dOxo) is a novel DNA lesion produced by the reaction of 2'-deoxyguanosine (dGuo) with nitrous acid and nitric oxide [Suzuki, T., Yamaoka, R., Nishi, M., Ide, H., and Makino, K. (1996) J. Am. Chem. Soc. 118, 2515-2516]. In this work, 2'-deoxyoxanosine 5'-triphosphate (dOTP) was prepared by nitrous acid treatment of 2'-deoxyguanosine 5'-triphosphate (dGTP), and its incorporation into DNA by DNA polymerases was investigated to elucidate the substrate and mutagenic properties of dOTP. Primed M13mp18 DNA was replicated by Escherichia coli DNA polymerase I Klenow fragment (Pol I Kf) in the presence of three normal dNTPs and dOTP or 2'-deoxyxanthosine 5'-triphosphate (dXTP), another major product of reaction of dGTP with nitrous acid and nitric oxide. dOTP substituted for dGTP and to a lesser extent for dATP, while dXTP substituted slightly for dGTP but not for dATP. Neither dOTP nor dXTP substituted for dCTP and dTTP. The similar results were obtained for the incorporation by T7 DNA polymerase deficient in 3'-5' exonuclease [T7(exo-)]. To quantify the substitution efficiency, kinetic parameters for incorporation of dOTP and dXTP opposite template C or T by Pol I Kf (exo-) were determined and compared with those for dGTP using oligodeoxynucleotide templates. Incorporation efficiencies (f = Vmax/Km) of dOTP (f = 0.28% min-1 microM-1) and dXTP (f = 0.10% min-1 microM-1) opposite template C were much lower than that of dGTP (f = 1506% min-1 microM-1). Frequencies of mutagenic incorporation of dOTP opposite template T were dependent on the nearest neighbor base pairs, and 1.6-3.9-fold higher than those for dGTP with the nearest neighbors containing G.C pairs. dXTP was not incorporated opposite template T with all four nearest neighbors. These data suggest that formation of dOTP, but not dXTP, from dGTP with nitrous acid or nitric oxide in the intracellular nucleotide pool would result in the elevation of the mutation frequency.

No abstract

5-Formyluracil (fU) is a major oxidative thymine lesion generated by ionizing radiation and reactive oxygen species. In the present study, we have assessed the influence of fU on DNA replication to elucidate its genotoxic potential. Oligonucleotide templates containing fU at defined sites were replicated in vitro by Escherichia coli DNA polymerase I Klenow fragment deficient in 3-5-exonuclease. Gel electrophoretic analysis of the reaction products showed that fU constituted very weak replication blocks to DNA synthesis, suggesting a weak to negligible cytotoxic effect of this lesion. However, primer extension assays with a single dNTP revealed that fU directed incorporation of not only correct dAMP but also incorrect dGMP, although much less efficiently. No incorporation of dCMP and dTMP was observed. When fU was substituted for T in templates, the incorporation efficiency of dAMP (f A ‫؍‬ V max /K m ) decreased to 1 ⁄4 to 1 ⁄2, depending on the nearest neighbor base pair, and that of dGMP (f G ) increased 1.1-5.6-fold. Thus, the increase in the replication error frequency (f G /f A for fU versus T) was 3.1-14.3-fold. The misincorporation rate of dGMP opposite fU (pK a ‫؍‬ 8.6) but not T (pK a ‫؍‬ 10.0) increased with pH (7.2-8.6) of the reaction mixture, indicating the participation of the ionized (or enolate) form of fU in the mispairing with G. The resulting mismatched fU:G primer terminus was more efficiently extended than the T:G terminus (8.2-11.3-fold). These results show that when T is oxidized to fU in DNA, fU promotes both misincorporation of dGMP at this site and subsequent elongation of the mismatched primer, hence potentially mutagenic.Faithful replication of DNA is essential for maintaining genetic integrity of living organisms. High fidelity of DNA replication is achieved by two cellular functions that involve discrimination of correct versus incorrect nucleotides by DNA polymerases (1, 2) and postreplication mismatch repair (3). The overall error frequency of DNA replication is one in 10 8 to 10 10 base pairs when they function properly. Fidelity of DNA replication also relies on the structural integrity of DNA itself that serves as a template for the newly synthesized strand. A number of endogenous and exogenous agents have been identified to induce structural deterioration of DNA (4). Among them, reactive oxygen species generate a very complicated spectrum of DNA damage (5, 6). These lesions are mostly restored by the base excision repair pathway both in prokaryotic and eukaryotic cells, but if left unrepaired, they arrest DNA synthesis or direct misincorporation of nucleotides during DNA replication, hence exerting deleterious effects on cells (7,8). Replication blocks and nucleotide misincorporation have been related to lethality and mutation of cells, respectively, until recently. However, this concept is now challenged by the discovery of numerous error-prone and error-free DNA polymerases that can bypass the blocking lesions (9). Although the past several years have witnessed the discovery...

An in vitro model of human ovarian follicles would greatly benefit the study of female reproduction. Ovarian development requires the combination of germ cells and several types of somatic cells. Among these, granulosa cells play a key role in follicle formation and support for oogenesis. Whereas efficient protocols exist for generating human primordial germ cell-like cells (hPGCLCs) from human induced pluripotent stem cells (hiPSCs), a method of generating granulosa cells has been elusive. Here, we report that simultaneous overexpression of two transcription factors (TFs) can direct the differentiation of hiPSCs to granulosa-like cells. We elucidate the regulatory effects of several granulosa-related TFs and establish that overexpression of NR5A1 and either RUNX1 or RUNX2 is sufficient to generate granulosa-like cells. Our granulosa-like cells have transcriptomes similar to human fetal ovarian cells and recapitulate key ovarian phenotypes including follicle formation and steroidogenesis. When aggregated with hPGCLCs, our cells form ovary-like organoids (ovaroids) and support hPGCLC development from the premigratory to the gonadal stage as measured by induction of DAZL expression. This model system will provide unique opportunities for studying human ovarian biology and may enable the development of therapies for female reproductive health.

The synthesis and spectroscopic characterization of a cavitand-based coordination capsule 14 BF4 of nanometer dimensions is described. Encapsulation studies of large aromatic guests as well as aliphatic guests were performed by using 1H NMR spectroscopy in [D1]chloroform. In addition to the computational analysis of the shape and geometry of the capsule, an experimental approach to estimate the interior size of the cavity is discussed. The cavity provides a highly rigid binding space in which molecules with lengths of approximately 14 A can be selectively accommodated. The rigid cavity distinguished slight structural differences in the flexible alkyl-chain guests as well as the rigid aromatic guests. The detailed thermodynamic studies revealed that not only CH-pi interactions between the methyl groups on the guest termini and the aromatic cavity walls, but also desolvation of the inner cavity play a key role in the guest encapsulation. The cavity preferentially selected the hydrogen-bonded heterodimers of a mixture of two or three carboxylic acids 18-20. The chiral capsule encapsulated a chiral guest to show diastereoselection.

5-Formyluracil (fU), a major methyl oxidation product of thymine, forms correct (fU:A) and incorrect (fU:G) base pairs during DNA replication. In the accompanying paper (Masaoka, A., Terato, H., Kobayashi, M., Honsho, A., Ohyama, Y., and Ide, H. (1999) J. Biol. Chem. 274, 25136 -25143), it has been shown that fU correctly paired with A is recognized by AlkA protein (Escherichia coli 3-methyladenine DNA glycosylase II). In the present work, mispairing frequency of fU with G and cellular repair protein that specifically recognized fU:G mispairs were studied using defined oligonucleotide substrates. Mispairing frequency of fU was determined by incorporation of 2-deoxyribonucleoside 5-triphosphate of fU opposite template G using DNA polymerase I Klenow fragment deficient in 3-5 exonuclease. Mispairing frequency of fU was dependent on the nearest neighbor base pair in the primer terminus and 2-12 times higher than that of thymine at pH 7.8 and 2.6 -6.7 times higher at pH 9.0 with an exception of the nearest neighbor T(template):A(primer). AlkA catalyzed the excision of fU placed opposite G, as well as A, and the excision efficiencies of fU for fU:G and fU:A pairs were comparable. In addition, MutS protein involved in methyl-directed mismatch repair also recognized fU:G mispairs and bound them with an efficiency comparable to T:G mispairs, but it did not recognize fU:A pairs. Prior complex formation between MutS and a heteroduplex containing an fU:G mispair inhibited the activity of AlkA to fU. These results suggest that fU present in DNA can be restored by two independent repair pathways, i.e. the base excision repair pathway initiated by AlkA and the methyl-directed mismatch repair pathway initiated by MutS. Biological relevance of the present results is discussed in light of DNA replication and repair in cells.Damage to DNA base moieties alters the base pairing properties, hence generating mutation after DNA replication. Deamination of cytosine and adenine, for example, results in uracil and hypoxanthine, respectively, and changes their coding properties in an explicit manner because the deamination products can fully (uracil) or partly (hypoxanthine) adopt hydrogen bonding schemes of thymine and guanine, respectively.Abasic sites also result in mutation by an explicit mechanism due to the total loss of base pairing information at the sites. In contrast, some of DNA base lesions that seemingly retain intact coding regions have mutagenic potential. 7,8-Dihydro-8-oxoguanine is one of the best studied examples for this type of lesions. In this lesion, the functional groups in the pyrimidine unit responsible for canonical hydrogen bond formation remain intact, but oxidation of the imidazole unit (C-8) tends to shift the anti-syn equilibrium of the base conformation so that 7,8-dihydro-8-oxoguanine in syn conformation forms a mispair with adenine during DNA replication (1-3). Similarly, we have previously shown that 5-formyluracil (fU) 1 retaining an apparently intact coding region forms a mispair with guanine, as well ...