The Notch ligand Jagged1 is required for inner ear sensory development

Within the mammalian inner ear there are six separate sensory regions that subserve the functions of hearing and balance, although how these sensory regions become specified remains unknown. Each sensory region is populated by two cell types, the mechanosensory hair cell and the supporting cell, which are arranged in a mosaic in which each hair cell is surrounded by supporting cells. The proposed mechanism for creating the sensory mosaic is lateral inhibition mediated by the Notch signaling pathway. However, one of the Notch ligands, Jagged1 (Jag1), does not show an expression pattern wholly consistent with a role in lateral inhibition, as it marks the sensory patches from very early in their development—presumably long before cells make their final fate decisions. It has been proposed that Jag1 has a role in specifying sensory versus nonsensory epithelium within the ear [Adam, J., Myat, A., Roux, I. L., Eddison, M., Henrique, D., Ish-Horowicz, D. & Lewis, J. (1998) Development (Cambridge, U.K.) 125, 4645–4654]. Here we provide experimental evidence that Notch signaling may be involved in specifying sensory regions by showing that a dominant mouse mutant headturner (Htu) contains a missense mutation in the Jag1 gene and displays missing posterior and sometimes anterior ampullae, structures that house the sensory cristae. Htu/+ mutants also demonstrate a significant reduction in the numbers of outer hair cells in the organ of Corti. Because lateral inhibition mediated by Notch predicts that disruptions in this pathway would lead to an increase in hair cells, we believe these data indicate an earlier role for Notch within the inner ear.

On the absorbance changes in the photocycle of the photoactive yellow protein: A quantum-chemical analysis

Spectral changes in the photocycle of the photoactive yellow protein (PYP) are investigated by using ab initio multiconfigurational second-order perturbation theory at the available structures experimentally determined. Using the dark ground-state crystal structure [Genick, U. K., Soltis, S. M., Kuhn, P., Canestrelli, I. L. & Getzoff, E. D. (1998) Nature (London) 392, 206–209], the ππ* transition to the lowest excited state is related to the typical blue-light absorption observed at 446 nm. The different nature of the second excited state (nπ*) is consistent with the alternative route detected at 395-nm excitation. The results suggest the low-temperature photoproduct PYPHL as the most plausible candidate for the assignment of the cryogenically trapped early intermediate (Genick et al.). We cannot establish, however, a successful correspondence between the theoretical spectrum for the nanosecond time-resolved x-ray structure [Perman, B., Šrajer, V., Ren, Z., Teng, T., Pradervand, C., et al. (1998) Science 279, 1946–1950] and any of the spectroscopic photoproducts known up to date. It is fully confirmed that the colorless light-activated intermediate recorded by millisecond time-resolved crystallography [Genick, U. K., Borgstahl, G. E. O., Ng, K., Ren, Z., Pradervand, C., et al. (1997) Science 275, 1471–1475] is protonated, nicely matching the spectroscopic features of the photoproduct PYPM. The overall contribution demonstrates that a combined analysis of high-level theoretical results and experimental data can be of great value to perform assignments of detected intermediates in a photocycle.

Ultraviolet-B Radiation Effects on Water Relations, Leaf Development, and Photosynthesis in Droughted Pea Plants1

The effects of ultraviolet-B (UV-B) radiation on water relations, leaf development, and gas-exchange characteristics in pea (Pisum sativum L. cv Meteor) plants subjected to drought were investigated. Plants grown throughout their development under a high irradiance of UV-B radiation (0.63 W m−2) were compared with those grown without UV-B radiation, and after 12 d one-half of the plants were subjected to 24 d of drought that resulted in mild water stress. UV-B radiation resulted in a decrease of adaxial stomatal conductance by approximately 65%, increasing stomatal limitation of CO2 uptake by 10 to 15%. However, there was no loss of mesophyll light-saturated photosynthetic activity. Growth in UV-B radiation resulted in large reductions of leaf area and plant biomass, which were associated with a decline in leaf cell numbers and cell division. UV-B radiation also inhibited epidermal cell expansion of the exposed surface of leaves. There was an interaction between UV-B radiation and drought treatments: UV-B radiation both delayed and reduced the severity of drought stress through reductions in plant water-loss rates, stomatal conductance, and leaf area.

A developmental switch in lymphocyte homing receptor and endothelial vascular addressin expression regulates lymphocyte homing and permits CD4+ CD3- cells to colonize lymph nodes.

IN adult mice, the dominant adhesion molecules involved in homing to lymph nodes are L-selectin homing receptors on lymphocytes and the peripheral lymph node addressins on specialized high endothelial venules. Here we show that, from fetal life through the first 24 hr of life, the dominant adhesion molecules are the mucosal addressin MAdCAM-1 on lymph node high endothelial venules and its counterreceptor, the Peyer's patch homing receptor, integrin alpha 4 beta 7 on circulating cells. Before birth, 40-70% of peripheral blood leukocytes are L-selectin-positive, while only 1-2% expresses alpha 4 beta 7. However, the fetal lymph nodes preferentially attract alpha 4 beta 7-expressing cells, and this can be blocked by fetal administration of anti-MAdCAM-1 antibodies. During fetal and early neonatal life, when only MAdCAM-1 is expressed on high endothelial venules, an unusual subset of CD4 + CD3- cells, exclusively expressing alpha 4 beta 7 as homing receptors, enters the lymph nodes. Beginning 24 hr after birth a developmental switch occurs, and the peripheral node addressins are upregulated on high endothelial venules in peripheral and mesenteric lymph nodes. This switch in addressin expression facilitates tissue-selective lymphocyte migration and mediates a sequential entry of different cell populations into the lymph nodes.

Limited up-regulation of DNA methyltransferase in human colon cancer reflecting increased cell proliferation.

Epigenetic alterations in the genome of tumor cells have attracted considerable attention since the discovery of widespread alterations in DNA methylation of colorectal cancers over 10 years ago. However, the mechanism of these changes has remained obscure. el-Deiry and coworkers [el-Deiry, W. S., Nelkin, B. D., Celano, P., Yen, R. C., Falco, J. P., Hamilton, S. R. & Baylin, S. B. (1991) Proc. Natl. Acad. Sci. USA 88, 3470-3474], using a quantitative reverse transcription-PCR assay, reported 15-fold increased expression of DNA methyltransferase (MTase) in colon cancer, compared with matched normal colon mucosa, and a 200-fold increase in MTase mRNA levels compared with mucosa of unaffected patients. These authors suggested that increases in MTase mRNA levels play a direct pathogenetic role in colon carcinogenesis. To test this hypothesis, we developed a sensitive quantitative RNase protection assay of MTase, linear over three orders of magnitude. Using this assay on 12 colorectal carcinomas and matched normal mucosal specimens, we observed a 1.8- to 2.5-fold increase in MTase mRNA levels in colon carcinoma compared with levels in normal mucosa from the same patients. There was no significant difference between the normal mucosa of affected and unaffected patients. Furthermore, when the assay was normalized to histone H4 expression, a measure of S-phase-specific expression, the moderate increase in tumor MTase mRNA levels was no longer observed. These data are in contrast to the previously reported results, and they indicate that changes in MTase mRNA levels in colon cancer are nonspecific and compatible with other markers of cell proliferation.

A designed beta-hairpin peptide in crystals.

Beta-hairpin structures have been crystallographically characterized only in very short acyclic peptides, in contrast to helices. The structure of the designed beta-hairpin, t-butoxycarbonyl-Leu-Val-Val-D-Pro-Gly-Leu-Val-Val-OMe in crystals is described. The two independent molecules of the octapeptide fold into almost ideal beta-hairpin conformations with the central D-Pro-Gly segment adopting a Type II' beta-turn conformation. The definitive characterization of a beta-hairpin has implications for de novo peptide and protein design, particularly for the development of three- and four-stranded beta-sheets.

The specificity of the transforming growth factor beta receptor kinases determined by a spatially addressable peptide library.

Type I and II receptors for the transforming growth factor beta (TGF-beta) are transmembrane serine/threonine kinases that are essential for TGF-beta signaling. However, little is known about their in vivo substrates or signal transduction pathways. To determine the substrate specificity of these kinases, we developed combinatorial peptide libraries synthesized on a hydrophilic matrix that is easily accessible to proteins in aqueous solutions. When we subjected these libraries to phosphorylation by the cAMP-dependent protein kinase, we obtained the optimal peptide sequence RRXS (I/L/V), in perfect agreement with the substrate sequence deduced from mutagenesis and crystal structure analyses. By using the same libraries, we showed that the optimal substrate peptide for both the type I and II TGF-beta receptors was KKKKKK(S/T)XXX. Since the two kinases are thought to play different roles in intracellular signal transduction, it was a surprise to find that they have almost identical substrate specificity. Our method is direct, sensitive, and simple and provides information about the kinase specificity for all the amino acid residues at each position.

Transgenic mice carrying the diphtheria toxin A chain gene under the control of the granzyme A promoter: expected depletion of cytotoxic cells and unexpected depletion of CD8 T cells.

We have generated transgenic mice bearing the diphtheria toxin A chain (DTA) gene under the control of granzyme A (GrA) promoter sequences (GrA-DTA). GrA is expressed in activated cytotoxic cells but not in their immediate progenitors. These GrA-DTA mice are deficient in cytotoxic functions, indicating that most cytotoxic cells express GrA in vivo. Surprisingly, one founder strain containing a multicopy GrA-DTA insert show a marked and selective deficiency in CD8+ cells in peripheral lymphoid organs. This depletion was not observed in thymus, where the distribution of CD4+ and CD8+ cells is normal. Moreover, the emigration of T cells from thymus is normal, indicating that the depletion occurs in the periphery. GrA-DTA mice should be useful as models to dissect the role of cytotoxic cells in immune responses and as recipients of normal and neoplastic hematopoietic cells. The selective depletion of CD8+ cells in one founder strain could have implications for postthymic T-cell development.

The purification and characterization of fetal liver hematopoietic stem cells.

Thy-1loSca-1+Lin-Mac-1+CD4- cells have been isolated from the livers of C57BL-Thy-1.1 fetuses. This population appears to be an essentially pure population of hematopoietic stem cells (HSC), in that injection of only six cells into lethally irradiated adult recipients yields a limit dilution frequency of donor cell-reconstituted mice. Sixty-seven to 77% of clones in this population exhibit long-term multilineage progenitor activity. This population appears to include all long-term multilineage reconstituting progenitors in the fetal liver. A high proportion of cells are in cycle, and the absolute number of cells in this population doubles daily in the fetal liver until 14.5 days postcoitum. At 15.5 days postcoitum, the frequency of this population falls dramatically. Long-term reconstituting HSC clones from the fetal liver give rise to higher levels of reconstitution in lethally irradiated mice than long-term reconstituting HSC from the bone marrow. The precise phenotypic and functional characteristics of HSC vary according to tissue and time during ontogeny.