F3/contactin acts as a functional ligand for Notch during oligodendrocyte maturation

Axon-derived molecules are temporally and spatially required as positive or negative signals to coordinate oligodendrocyte differentiation. Increasing evidence suggests that, in addition to the inhibitory Jagged1/Notch1 signaling cascade, other pathways act via Notch to mediate oligodendrocyte differentiation. The GPI-linked neural cell recognition molecule F3/contactin is clustered during development at the paranodal region, a vital site for axoglial interaction. Here, we show that F3/contactin acts as a functional ligand of Notch. This trans-extracellular interaction triggers gamma-secretase-dependent nuclear translocation of the Notch intracellular domain. F3/Notch signaling promotes oligodendrocyte precursor cell differentiation and upregulates the myelin-related protein MAG in OLN-93 cells. This can be blocked by dominant negative Notch1, Notch2, and two Deltex1 mutants lacking the RING-H2 finger motif, but not by dominant-negative RBP-J or Hes1 antisense oligonucleotides. Expression of constitutively active Notch1 or Notch2 does not upregulate MAG. Thus, F3/contactin specifically initiates a Notch/Deltex1 signaling pathway that promotes oligodendrocyte maturation and myelination.     

Functional correlates of leaf demographic response to gap release in saplings of a shade-tolerant tree,<Emphasis Type="Italic"> Elateriospermum tapos</Emphasis>

For a shade-tolerant SE Asian tropical tree, Elateriospermum tapos (Euphorbiaceae), we studied field-established saplings in gaps and the shaded understory to test the hypothesis that differences in leaf demography and leaf life span under contrasting light regimes should be functionally correlated with architecture, self-shading and nitrogen distribution within the sapling crown. Rates of leaf production and net leaf gain were greater for saplings in gaps than those in the understory. Median leaf life span was approximately 26 months in the gap saplings, while it was estimated to be greater than 38 months in the understory saplings. Consequently, gap saplings had a greater standing leaf number and experienced greater degrees of self-shading than understory saplings. Light availability at individual leaves, estimated by a combination of canopy photos and a three-dimensional architecture model, were negatively correlated with leaf age in gap saplings but not so in understory saplings. Leaf nitrogen content per unit area (N_area) was influenced more by light availability than by leaf age in the gap saplings. In contrast, in understory saplings, N_area was neither correlated with light availability nor with leaf age, and did not decrease significantly before 38 months in leaf age. We conclude that saplings of this shade-tolerant species apparently prolong their leaf life span in the shaded understory through slower rates of leaf production, lower standing number of leaves and lower degrees of self shading than in gap, and that the rate of decline of N_area with leaf age depends on architecture and self-shading regimes that respond to changes in light regimes.     

Comparative study of urinary reproductive hormones in great apes

Urinary estrone conjugates (E₁C), pregnanediol-3-glucuronide (PdG), and follicle-stimulating hormone (FSH) were determined by enzyme immunoassays (EIAs) during the normal menstrual cycle in the orangutan, gorilla, chimpanzee, and bonobo. Furthermore, the data were compared to those levels in the human and long-tailed macaque. The results showed a typical preovulatory E₁C surge and postovulatory increase in PdG in all species. The pattern of E₁C during the menstrual cycle in the great apes more closely resembled the human than do the long-tailed macaque. A major difference of E₁C pattern between these species appeared in the luteal phase. In the great apes and the human, E₁C exhibited two peaks, the first peak detected at approximately mid cycle and the second peak detected during the luteal phase. On the other hand, in the long-tailed macaque, increase of E₁C in the luteal phase was small or nonexistent. The gorilla, chimpanzee, and bonobo exhibited similar PdG trends. The orangutan excreted one tenth less PdG than these species during the luteal phase. The long-tailed macaque also excreted low levels of PdG. The patterns of FSH in orangutan, chimpanzee, bonobo and long-tailed macaque showed a marked mid-cycle rise and an early follicular phase rise, similar to those in the human. Comparing similar taxa, a large difference was found in FSH of gorilla; there were three peaks during the menstrual cycle. Thus, there is considerable species variation in the excretion of these hormones during the menstrual cycle and comparative studies could be approached with a single method. The methods and baseline data presented here provide the basis for a practical approach to evaluation and monitoring of ovarian events in the female great apes. Electronic Publication     

Multiple roles of Rev3, the catalytic subunit of polzeta in maintaining genome stability in vertebrates

Translesion DNA synthesis (TLS) and homologous DNA recombination (HR) are two major postreplicational repair (PRR) pathways. The REV3 gene of Saccharomyces cerevisiae encodes the catalytic subunit of DNA polymerase zeta, which is involved in mutagenic TLS. To investigate the role of REV3 in vertebrates, we disruped the gene in chicken DT40 cells. REV3(-/-) cells are sensitive to various DNA-damaging agents, including UV, methyl methanesulphonate (MMS), cisplatin and ionizing radiation (IR), consistent with its role in TLS. Interestingly, REV3(-/-) cells showed reduced gene targeting efficiencies and significant increase in the level of chromosomal breaks in the subsequent M phase after IR in the G(2) phase, suggesting the involvement of Rev3 in HR-mediated double-strand break repair. REV3(-/-) cells showed significant increase in sister chromatid exchange events and chromosomal breaks even in the absence of exogenous genotoxic stress. Furthermore, double mutants of REV3 and RAD54, genes involved in HR, are synthetic lethal. In conclusion, Rev3 plays critical roles in PRR, which accounts for survival on naturally occurring endogenous as well as induced damages during replication.     

Characterization of Sleeping Beauty transposition and its application to genetic screening in mice

The use of mutant mice plays a pivotal role in determining the function of genes, and the recently reported germ line transposition of the Sleeping Beauty (SB) transposon would provide a novel system to facilitate this approach. In this study, we characterized SB transposition in the mouse germ line and assessed its potential for generating mutant mice. Transposition sites not only were clustered within 3 Mb near the donor site but also were widely distributed outside this cluster, indicating that the SB transposon can be utilized for both region-specific and genome-wide mutagenesis. The complexity of transposition sites in the germ line was high enough for large-scale generation of mutant mice. Based on these initial results, we conducted germ line mutagenesis by using a gene trap scheme, and the use of a green fluorescent protein reporter made it possible to select for mutant mice rapidly and noninvasively. Interestingly, mice with mutations in the same gene, each with a different insertion site, were obtained by local transposition events, demonstrating the feasibility of the SB transposon system for region-specific mutagenesis. Our results indicate that the SB transposon system has unique features that complement other mutagenesis approaches.     

Study of chiral recognition of bilayered phosphatidylcholine vesicles using a helicene probe: characteristic function of cholesterol

Incorporated into bilayered chiral phosphatidylcholine (PC) vesicles, 2-hydroxymethyl[5]thiaheterohelicene (5HM) having a labile helix that functioned as a chirality probe, exhibited induced CD absorptions. The Cotton effects of these absorptions demonstrated opposite signs according to the difference in chirality of PC applied, indicating the chiral recognition of the vesicles. The vesicles formed by PCs with unsaturation or acyl chains shorter than dipalmitoyl-PC (DPPC) exhibited a slightly stronger CD absorptions of 5HM, presumably because of an increase in the constraint by the vesicles. The phenomenon that an increase in fluidity results in a decrease in the CD intensity was examined by CD measurements at various temperatures. The vesicles formed with egg lecithin and bovine heart lecithin also induced CD absorptions of 5HM similar to those of (L)PC vesicles. The influence of cholesterol (Cho) and four kinds of analogs with different structures at the 3- or 5-position of a Cho molecule on the CD intensities was investigated. Following addition of Cho, the CD intensities of 5HM decreased slightly in the (L)DPPC vesicles and increased moderately in the (D)DPPC vesicles. On the other hand, by addition of Cho analogs the CD intensities of 5HM were nearly unchanged in (L)DPPC vesicles and weakened moderately in the (D)DPPC vesicles.     

Angiotensin I-converting enzyme inhibitory peptides isolated from tofuyo fermented soybean food

Angiotensin I-converting enzyme (ACE) inhibitory activity was observed in a tofuyo (fermented soybean food) extract with an IC(50) value of 1.77 mg/ml. Two ACE inhibitors were isolated to homogeneity from the extract by adsorption and gel filtration column chromatography, and by reverse-phase high-performance liquid chromatography (HPLC). The purified substances reacted with 2,4,6-trinitrobenzensulfonic acid sodium salt. The amino acid sequences of these inhibitors determined by Edman degradation were Ile-Phe-Leu (IC(50), 44.8 microM) and Trp-Leu (IC(50), 29.9 microM). The Ile-Phe-Leu sequence is found in the alpha- and beta-subunits of beta-conglycinin, while the Trp-Leu sequence is in the B-, B1A- and BX-subunits of glycinin from soybean. Both of the peptides are non-competitive inhibitors. The inhibitory activity of Trp-Leu was completely preserved after a treatment with pepsin, chymotrypsin or trypsin. Even after successive digestion by these gastrointestinal proteases, the activity remained at 29% of the original value.     

Putative cis-elements in the promoter region of the carrot phenylalanine ammonia-lyase gene induced during anthocyanin synthesis

Deletion mutants of the carrot phenylalanine ammonia-lyase gene promoter were used to survey cis-elements for their effect on expression of promoter activity by transient expression. Two putative cis-elements were required to give full activity, but a third might be the most important in regulation of the promoter by 2,4-dichlorophenoxyacetic acid. Electronic Publication