Proceedings of the National Academy of Sciences

das Neves, Liomar; Duchala, Cynthia S.; Godinho, Fatima; Haxhiu, Musa A.; Colmenares, Clemencia; Macklin, Wendy B.; Campbell, Christine E.; Butz, Kenneth G.; Gronostajski, Richard M.

doi: N/Apmid: 10518556

The phylogenetically conserved nuclear factor I (NFI) family of transcription/replication proteins is essential both for adenoviral DNA replication and for the transcription of many cellular genes. We showed previously that the four murine NFI genes (Nfia, Nfib, Nfic, and Nfix) are expressed in unique but overlapping patterns during mouse development and in adult tissues. Here we show that disruption of the Nfia gene causes perinatal lethality, with >95% of homozygous Nfia −/− animals dying within 2 weeks after birth. Newborn Nfia −/− animals lack a corpus callosum and show ventricular dilation indicating early hydrocephalus. Rare surviving homozygous Nfia −/− mice lack a corpus callosum, show severe communicating hydrocephalus, a full-axial tremor indicative of neurological defects, male-sterility, low female fertility, but near normal life spans. These findings indicate that while the Nfia gene appears nonessential for cell viability and DNA replication in embryonic stem cells and fibroblasts, loss of Nfia function causes severe developmental defects. This finding of an NFI gene required for a developmental process suggests that the four NFI genes may have distinct roles in vertebrate development.

Orimo, Akira; Inoue, Satoshi; Minowa, Osamu; Tominaga, Nobuko; Tomioka, Yasuhiro; Sato, Miyuki; Kuno, Junko; Hiroi, Hisahiko; Shimizu, Yoshihiko; Suzuki, Masami; Noda, Tetsuo; Muramatsu, Masami

doi: N/Apmid: 10518570

The biological roles of estrogen-responsive finger protein (efp) in vivo were evaluated in mice carrying a loss-of-function mutation in efp by gene-targeted mutagenesis. Although efp homozygous mice were viable and fertile in both sexes, the uterus that expressed abundant estrogen receptor α exhibited significant underdevelopment. When the ovariectomized homozygotes were subjected to 17β-estradiol treatment, they showed remarkably attenuated responses to estrogen, as exemplified by decreased interstitial water imbibition and retarded endometrial cell increase, at least, attributable to the lower ratio of G1 to S-phase progression in epithelial cells. These results suggest that efp is essential for the normal estrogen-induced cell proliferation and uterine swelling as one of the direct targets of estrogen receptor α.

Bosma, Gayle C.; Chang, Yung; Karasuyama, Hajime; Bosma, Melvin J.

doi: N/Apmid: 10518557

Progression of pro-B lymphocytes to the pre-B stage depends on the expression of a pre-B cell receptor (pre-BCR), consisting of an Ig μ H chain, Ig surrogate light chain, and associated signal transducing chains. Mice that are unable to express a pre-BCR show an arrest of B cell development at the pro-B stage. Such is the case for severe combined immune deficient (SCID) mice in which μ chains are not made because of a defect in V(D)J recombination. When μ chains are made, as in SCID mice bearing a functional μ transgene, then B cell differentiation can proceed to the pre-B stage. However, as reported here, a μ transgene (M54) that promotes development of SCID pre-B cells in adult bone marrow fails to do so in fetal liver. We suggest that a pre-BCR containing the M54 μ chain cannot signal progression of pro-B cells to the pre-B stage in the fetal liver microenvironment.

Kovács, Ilona; Kozma, Petra; Fehér, Ákos; Benedek, György

doi: N/Apmid: 10518600

Visual development is thought to be completed at an early age. We suggest that the maturation of the visual brain is not homogeneous: functions with greater need for early availability, such as visuomotor control, mature earlier, and the development of other visual functions may extend well into childhood. We found significant improvement in children between 5 and 14 years in visual spatial integration by using a contour-detection task. The data show that long-range spatial interactions—subserving the integration of orientational information across the visual field—span a shorter spatial range in children than in adults. Performance in the task improves in a cue-specific manner with practice, which indicates the participation of fairly low-level perceptual mechanisms. We interpret our findings in terms of a protracted development of ventral visual-stream function in humans.

Chen, Michellee S.; Tung, Kenneth S. K.; Coonrod, Scott A.; Takahashi, Yuji; Bigler, Dora; Chang, Alice; Yamashita, Yoshio; Kincade, Paul W.; Herr, John C.; White, Judith M.

doi: N/Apmid: 10518536

CD9 is a tetraspan protein that associates with several β1 integrins, including α6β1. Because α6β1 is present on murine eggs and interacts with the sperm-surface glycoprotein ADAM 2 (fertilin β), we first asked whether CD9 is present on murine eggs and whether it functions in sperm–egg binding and fusion. CD9 is present on the plasma membrane of oocytes in the ovary as well as on eggs isolated from the oviduct. The anti-CD9 mAb, JF9, potently inhibits sperm–egg binding and fusion in vitro in a dose-dependent manner. JF9 also disrupts binding of fluorescent beads coated with native fertilin or a recombinant fertilin β disintegrin domain. (Both ligands bind to the egg via α6β1.) Immunohistochemistry showed that CD9 is undetectable in the uterine epithelium, appears basolaterally and as prominent apical patches on the epithelium in the region between the uterus and the oviduct, and then persists apically in the oviduct. The integrin α6A subunit is found in similar apical patches in the region between the uterus and oviduct, but is confined to the basal aspect of the epithelium in the uterus and oviduct. Hence, α6A and CD9 both are expressed on the apical epithelial surface at the uterine–oviduct junction. These findings correlate with the observation that fertilin β “knockout” sperm traverse the uterus but do not progress into the oviduct, contributing to the infertility of fertilin β−/− male mice. Our results suggest that high-avidity binding between fertilin β (ADAM 2) and α6β1 requires cooperation between α6β1 and CD9. Such cooperation may assist sperm passage into the oviduct as well as sperm–egg interactions.

Kaufman, Randal J.

doi: N/Apmid: 10518510

Eukaryotic viruses and their multicellular hosts have coevolved complex interrelationships to permit virus reproduction without destruction of the host. The IFN-induced cellular antiviral response is the first line of defense against viral infection within an animal host. On viral infection, the expression of IFN is induced at the transcriptional level. The IFN is secreted to protect adjacent cells from secondary infection, thereby limiting viral spread. Type I IFNs are composed of the different types of IFN-α that are produced in leukocytes and the IFN-β that is produced in fibroblasts and epithelial cells. Type I IFNs bind to their receptors and activate a signaling cascade that culminates in the transcriptional induction of at least 30 genes (1). Two of these genes encode latent enzymes requiring activation by binding to double-stranded (ds)RNA that is produced as a replication intermediate in the viral life cycle. First, latent 2′-5′-oligoadenylate synthetase is activated by dsRNA to increase synthesis of 2′-5′ oligoadenylates that are required to activate 2′-5′-A-dependent RNase L (2). Activated RNase L nonspecifically degrades single-stranded RNAs and thus limits virus production. Second, the dsRNA-activated protein kinase (PKR), the most well characterized IFN-induced gene product, mediates the antiviral actions of type I IFNs. In studies reported in this issue of PNAS, Lau and coworkers (3) show that reduction in PKR level acts to delay cell death and thereby converts a lytic infection by encephalomyocarditis virus (EMCV) into a persistent infection. These studies as well as those from other laboratories support the idea that viral pathogenesis may be mediated by PKR-induced cell death and suggest that modification of PKR function in vivo may be a feasible approach to influence viral pathogenesis. In addition, the study presents a model system that may be applied to study mechanisms responsible for the establishment of persistent infections for other viruses …

Niles, Jacquin C.; Burney, Samar; Singh, Sukhjeet P.; Wishnok, John S.; Tannenbaum, Steven R.

doi: N/Apmid: 10518518

Of the DNA bases, peroxynitrite (ONOO−) is most reactive toward 2′-deoxyguanosine (dGuo), but even more reactive with 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo), requiring a 1,000-fold excess of dGuo to provide 50% protection against the reaction with 8-oxodGuo. Therefore, it seems reasonable that 8-oxodGuo is a potentially important target in DNA and that the structures of the reaction products with ONOO− should be characterized. Using 3′,5′-di-O-Ac-8-oxodGuo as a model compound, the reaction products with ONOO− have been isolated and identified under simulated physiological reaction conditions (phosphate/bicarbonate buffer at pH 7.2). The major reaction product, II, is unstable and undergoes base-mediated hydrolysis to 2,5-diaminoimidazol-4-one, IIa, and 3-(3,5-di-O-Ac-2-deoxy-β-d-erythro-pentofuranosyl)-5-iminoimidazolidine-2,4-dione, IIb. The latter compound further hydrolyzes to 3-(3,5-di-O-Ac-2-deoxy-β-d-erythro-pentofuranosyl)oxaluric acid, IIc. Other products include 3-(3,5-di-O-Ac-2-deoxy-β-d-erythro-pentofuranosyl)-2,4,6-trioxo-1,3,5triazinane-1-carboxamidine, I, which further hydrolyzes to 1-(3,5-di-O-Ac-2-deoxy-β-d-erythro-pentofuranosyl)cyanuric acid, Ia. 1-(3,5-di-O-Ac-2-deoxy-β-d-erythro-pentofuranosyl)parabanic acid, III, is a minor product that also may contribute to formation of IIc. The major products formed in these reactions are biologically uncharacterized but are similar to modified DNA bases that have been shown to be both premutagenic and blocks to DNA polymerization.

Leitinger, Norbert; Tyner, Timothy R.; Oslund, Laura; Rizza, Cristina; Subbanagounder, Ganesamoorthy; Lee, Hans; Shih, Peggy T.; Mackman, Nigel; Tigyi, Gabor; Territo, Mary C.; Berliner, Judith A.; Vora, Devendra K.

doi: N/Apmid: 10518567

We previously have demonstrated that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), a component of minimally modified low density lipoprotein (MM-LDL), activates endothelial cells to bind monocytes. 1-Palmitoyl-2- (5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC) and 1- palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), which are present in OxPAPC, MM-LDL, and atherosclerotic lesions, were shown to have a major role in the activation of endothelial cells. We now demonstrate that these two highly similar molecules have dramatically different effects on leukocyte endothelial interactions. POVPC is a potent regulator of monocyte-specific endothelial interactions. Treatment of endothelial cells with POVPC increased monocyte binding by inducing the surface expression of the connecting segment 1 domain of fibronectin; no increase in neutrophil binding was observed. In addition, POVPC strongly inhibited lipopolysaccharide-mediated induction of neutrophil binding and expression of E-selectin protein and mRNA. This inhibition was mediated by a protein kinase A-dependent pathway, resulting in down-regulation of NF-κB-dependent transcription. In contrast, PGPC induced both monocyte and neutrophil binding and expression of E-selectin and vascular cell adhesion molecule 1. We present evidence to suggest that the two phospholipids act by different novel receptors present in Xenopus laevis oocytes and that POVPC, but not PGPC, stimulates a cAMP-mediated pathway. At concentrations equal to that present in MM-LDL, the effect of POVPC dominates and inhibits PGPC-induced neutrophil binding and E-selectin expression in endothelial cells. In summary, our data provide evidence that both POVPC and PGPC are important regulators of leukocyte-endothelial interactions and that POVPC may play a dominant role in a number of chronic inflammatory processes where oxidized phospholipids are known to be present.

Korkotian, E.; Segal, M.

doi: N/Apmid: 10518577

The ability to monitor ongoing changes in the shape of dendritic spines has important implications for the understanding of the functional correlates of the great variety of shapes and sizes of dendritic spines in central neurons. We have monitored and three-dimensionally reconstructed dendritic spines in cultured hippocampal neurons over several hours of observation in a confocal laser scanning microscope. In the absence of extrinsic stimulation, the dimensions of dendritic spines of 3-week-old cultured neurons did not change to any significant degree over 3–4 hr in the culture dish, unlike the case with younger cultures. Releasing calcium from stores with pulse application of caffeine causes a transient rise of Ca2+i in dendrites and spines, monitored with the calcium dye Oregon-green. Application of caffeine to a dendrite imaged with calcein caused a fast and significant increase in the size of existing dendritic spines and could lead to formation of new ones. This effect is mediated by calcium released from the ryanodine-sensitive stores, as application of caffeine in the presence of ryanodine blocked this effect on the morphology of dendritic spines. Thus, release of calcium from stores is sufficient to produce significant changes in the shape of dendritic spines of cultured hippocampal neurons.

Cooper, Gillian; Burroughs, Nigel J.; Rand, David A.; Rubinsztein, David C.; Amos, William

doi: N/Apmid: 10518551

We describe a Markov Chain Monte Carlo analysis of five human Y- chromosome microsatellite polymorphisms based on samples from five diverse populations. Our analysis provides strong evidence for mutational bias favoring increase in length at all loci. Estimates of population coalescent times and population size from our two largest samples, one African and one European, suggest that the African population is older but smaller and that the English East Anglian population has undergone significant expansion, being larger but younger. We conclude that Markov Chain Monte Carlo analysis of microsatellite haplotypes can uncover information not apparent when the microsatellites are considered independently. Incorporation of population size as a variable should allow us to estimate the timing and magnitude of major historical population trends.