Mucosal galectin genes in all freshwater eels of the genus Anguilla

In this study, we determined the genomic DNA sequences of the mucosal galectin-encoding genes from all 19 species and subspecies of the genus Anguilla. The nucleotide sequences of the galectin genes were c. 2.3–2.5 kb long and the organisation of their four exons and three introns was conserved in all species. An unusual sequence was found in the fourth exon of Anguilla reinhardtii, resulting in a unique deduced amino-acid sequence at the C-terminus. All six amino-acid residues important for β-galactoside binding were conserved in three species, while one residue (R⁷³) was substituted to K⁷³ in the other 16 species–subspecies, including Anguilla marmorata. However, this substitution did not appear to affect the sugar-binding ability of galectins because the galectin of A. marmorata was previously shown to bind to lactose. We also discuss the molecular evolution of galectins among Anguilla spp. and the homologues previously identified in Conger myriaster.     

Vasomotor effects of noradrenaline, acetylcholine, histamine, 5-hydroxytryptamine and bradykinin on snake (Trimeresurus flavoviridis) basilar arteries

We investigated the responsiveness of basilar arterial rings isolated from snakes to noradrenaline (NA), acetylcholine (ACh), histamine (His), 5-hydroxytryptamine (5-HT), mammalian bradykinin (BK) and rattlesnake BK. We also examined whether endothelial cells were involved in the responsiveness to ACh, BK, rattlesnake BK and in their resting vascular tone. NA and 5-HT induced concentration-dependent contractions. The cumulative concentration response curves of NA and 5-HT were shifted to the right in parallel by phentolamine (an alpha antagonist) and methiothepin (a 5-HT(1) and 5-HT(2) antagonist), respectively. However, ketanserin (a 5-HT(2) antagonist) had no effect on the cumulative concentration response curve of 5-HT. His, ACh, BK and rattlesnake BK had no effect on resting vascular tone; however, rattlesnake BK and sodium nitroprusside relaxed arteries precontracted by 5-HT. The rattlesnake BK-induced relaxations were almost abolished by L-nitro arginine (L-NA, a nitric oxide synthase inhibitor). L-NA and indomethacin (a cyclooxygenase inhibitor) had no effect on resting vascular tone or on precontracted arteries. These results suggest that alpha and 5-HT(1) receptor subtypes might be important in arterial contraction. Endothelial cells might play an important role in the responsiveness of snake basilar arteries to rattlesnake BK, but they might not be involved in the responsiveness to ACh, BK and in resting vascular tone.     

PepSplice: cache-efficient search algorithms for comprehensive identification of tandem mass spectra

MOTIVATION: Tandem mass spectrometry allows for high-throughput identification of complex protein samples. Searching tandem mass spectra against sequence databases is the main analysis method nowadays. Since many peptide variations are possible, including them in the search space seems only logical. However, the search space usually grows exponentially with the number of independent variations and may therefore overwhelm computational resources. RESULTS: We provide fast, cache-efficient search algorithms to screen large peptide search spaces including non-tryptic peptides, whole genomes, dozens of posttranslational modifications, unannotated point mutations and even unannotated splice sites. All these search spaces can be screened simultaneously. By optimizing the cache usage, we achieve a calculation speed that closely approaches the limits of the hardware. At the same time, we control the size of the overall search space by limiting the combinations of variations that can co-occur on the same peptide. Using a hypergeometric scoring scheme, we applied these algorithms to a dataset of 1 420 632 spectra. We were able to identify a considerable number of peptide variations within a modest amount of computing time on standard desktop computers.     

Combined exposure to X-irradiation followed by N-ethyl-N-nitrosourea treatment alters the frequency and spectrum of Ikaros point mutations in murine T-cell lymphoma

Ionizing radiation is a well-known carcinogen, but its potency may be influenced by other environmental carcinogens, which is of practical importance in the assessment of risk. Data are scarce, however, on the combined effect of radiation with other environmental carcinogens and the underlying mechanisms involved. We studied the mode and mechanism of the carcinogenic effect of radiation in combination with N-ethyl-N-nitrosourea (ENU) using doses approximately equal to the corresponding thresholds. B6C3F1 mice exposed to fractionated X-irradiation (Kaplan's method) followed by ENU developed T-cell lymphomas in a dose-dependent manner. Radiation doses above an apparent threshold acted synergistically with ENU to promote lymphoma development, whereas radiation doses below that threshold antagonized lymphoma development. Ikaros, which regulates the commitment and differentiation of lymphoid lineage cells, is a critical tumor suppressor gene frequently altered in both human and mouse lymphomas and shows distinct mutation spectra between X-ray- and ENU-induced lymphomas. In the synergistically induced lymphomas, we observed a low frequency of LOH and an inordinate increase of Ikaros base substitutions characteristic of ENU-indcued point mutations, G:C to A:T at non-CpG, A:T to G:C, G:C to T:A and A:T to T:A. This suggests that radiation doses above an apparent threshold activate the ENU mutagenic pathway. This is the first report on the carcinogenic mechanism elicited by combined exposure to carcinogens below and above threshold doses based on the mutation spectrum of the causative gene. These findings constitute a basis for assessing human cancer risk following exposure to multiple carcinogens.     

Hyrtioreticulins A-E, indole alkaloids inhibiting the ubiquitin-activating enzyme, from the marine sponge Hyrtios reticulatus

Hyrtioreticulins A-E (1-5) were isolated from the marine sponge Hyrtios reticulatus, along with a known alkaloid, hyrtioerectine B (6). Structural elucidation on the basis of spectral data showed that 1, 2, and 5 are new tetrahydro-β-carboline alkaloids, while 3 and 4 are new azepinoindole-type alkaloids. Hyrtioreticulins A and B (1 and 2) inhibited ubiquitin-activating enzyme (E1) with IC(50) values of 0.75 and 11μg/mL, respectively, measured by their inhibitory abilities against the formation of an E1-ubiquitin intermediate. So far, only five E1 inhibitors, panapophenanthrine, himeic acid A, largazole, and hyrtioreticulins A and B (1 and 2), have been isolated from natural sources and, among them, 1 is the most potent E1 inhibitor.     

A genetic resource for early-morning flowering trait of wild rice Oryza officinalis to mitigate high temperature-induced spikelet sterility at anthesis

Background and Aims

High temperatures over 32–36 °C at anthesis induce spikelet sterility in rice. The use of a germplasm with an early-morning flowering (EMF) trait has been hypothesized as a way of avoiding this problem. In this study, the effect of the EMF trait on avoiding high temperature-induced sterility at anthesis by flowering at a cooler temperature in the early morning was evaluated.

Methods

The EMF trait was introgressed from wild rice (Oryza officinalis) into the rice cultivar ‘Koshihikari’ (O. sativa). First, spikelets of the EMF line and Koshihikari were subjected to rising temperatures during the daytime in the greenhouse to test for differences in spikelet sterility. Secondly, spikelets of both plants were exposed to 26, 34 and 38 °C at anthesis and to 38 °C beginning at least 1 h after flowering, in the growth chambers at 70 % relative humidity, to test for differences in tolerance to high temperatures.

Key Results

Spikelets of the EMF line started and completed flowering a few hours earlier than Koshihikari. In a greenhouse experiment, spikelets of Koshihikari opened after the air temperature reached 35 °C, but those of the EMF line could open at cooler temperatures. Under these conditions, spikelet sterility significantly increased in Koshihikari, but did not in the EMF line. The number of sterile spikelets increased as their flowering time was delayed in Koshihikari. Furthermore, the chamber experiments revealed that 60 % of the spikelets from both lines were sterile when exposed to 38 °C at anthesis, indicating that tolerance of high temperature was similar in both genotypes.

Conclusions

Reduced sterility in the EMF line subjected to rising temperatures at anthesis in the greenhouse was attributed to an earlier flowering time compared with Koshihikari. The EMF trait of wild rice is effective in mitigating anticipated yield loss due to global warming by escaping high-temperature stress at anthesis during the daytime.     

Wnt3a Promotes Hippocampal Neurogenesis by Shortening Cell Cycle Duration of Neural Progenitor Cells

The effects of Wnt signaling on neural progenitor cells have been controversial. Activation of the canonical Wnt signaling pathway either promotes neural progenitor cell proliferation or accelerates their differentiation into postmitotic neurons. This study demonstrates that activation of the Wnt signaling pathway by itself induces neural progenitor cell proliferation but does not directly affect neuronal differentiation processes. To investigate whether Wnt signaling promotes expansion and/or differentiation of neural progenitor cells in the developing hippocampus, we prepared primary mouse hippocampal progenitors and treated them with Wnt3a in a chemically defined culture medium. Wnt3a increased the total number of cells, including the numbers of Ki67⁺ proliferating cells and Tuj1⁺ differentiated neurons. This result verified that Wnt3a promoted neural progenitor cell proliferation. Meanwhile, Wnt3a did not appear to actively enhance the neuronal differentiation process itself, because (1) the ratio of Tuj1⁺ cells to the total cells, and (2) the ratio of BrdU⁺ Tuj1⁺ cells to the total BrdU⁺ cells, were both comparable between cultures with or without Wnt3a. Indeed, Wnt3a caused no significant change in either cell survival or the proportion of symmetric and asymmetric cell divisions that directly affected neuron production. We finally demonstrated that the Wnt3a treatment simply shortened cell cycle duration of neural progenitor cells by 2.9 h. The accelerated cell cycle progression without affecting the ratio of symmetric/asymmetric cell divisions explains how Wnt signaling per se leads to the expansion of both proliferative cell population and differentiated neuronal cell population.     

Possible role of nicaraven in neuroprotective effect on hippocampal slice culture

Nicaraven is an agent that is especially beneficial in vasospasm or brain damage caused by subarachnoid hemorrhage. It ameliorates neurological deficits of patients and protects the central nervous system from ischemia. We investigated the neuroprotective effect of nicaraven against oxygen-glucose deprivation (OGD) induced or N-methyl-D-aspartic acid (NMDA) induced hippocampal neuronal cell death in organotypic brain slice cultures. The effect of nicaraven on hippocampal neuronal injury was evaluated by inhibition of uptake of propidium iodide (PI) into dead cells. The results demonstrated that nicaraven protected neuronal cells from both OGD- and NMDA-induced cell death. While nicaraven has a strong hydroxyl radical scavenging effect, another radical scavenger, N-acetyl-L-cysteine (NAC), inhibited cell death only caused by OGD. In contrast, the poly(ADP-ribose) synthetase (PARS) inhibitors 3-aminobenzamide (3-AB) and theophylline protected cells from both OGD- and NMDA-induced cell death. Since nicaraven has an inhibitory effect in PARS, as well as a radical scavenging effect, these results suggest that inhibition of hippocampal cell death caused by NMDA may be attributable to PARS inhibition by nicaraven.     

Competition between the Rad50 complex and the Ku heterodimer reveals a role for Exo1 in processing double-strand breaks but not telomeres

The Mre11-Rad50-Nbs1(Xrs2) complex and the Ku70-Ku80 heterodimer are thought to compete with each other for binding to DNA ends. To investigate the mechanism underlying this competition, we analyzed both DNA damage sensitivity and telomere overhangs in Schizosaccharomyces pombe rad50-d, rad50-d pku70-d, rad50-d exo1-d, and pku70-d rad50-d exo1-d cells. We found that rad50 exo1 double mutants are more methyl methanesulfonate (MMS) sensitive than the respective single mutants. The MMS sensitivity of rad50-d cells was suppressed by concomitant deletion of pku70+. However, the MMS sensitivity of the rad50 exo1 double mutant was not suppressed by the deletion of pku70+. The G-rich overhang at telomere ends in taz1-d cells disappeared upon deletion of rad50+, but the overhang reappeared following concomitant deletion of pku70+. Our data suggest that the Rad50 complex can process DSB ends and telomere ends in the presence of the Ku heterodimer. However, the Ku heterodimer inhibits processing of DSB ends and telomere ends by alternative nucleases in the absence of the Rad50-Rad32 protein complex. While we have identified Exo1 as the alternative nuclease targeting DNA break sites, the identity of the nuclease acting on the telomere ends remains elusive.