Eotaxin-3/CC chemokine ligand 26 is a functional ligand for CX3CR1

Eotaxin-3/CCL26 is a functional ligand for CCR3 and abundantly produced by IL-4-/IL-13-stimulated vascular endothelial cells. CCL26 also functions as a natural antagonist for CCR1, CCR2, and CCR5. In this study, we report that CCL26 is yet a functional ligand for CX3CR1, the receptor for fractalkine/CX3CL1, which is expressed by CD16(+) NK cells, cytotoxic effector CD8(+) T cells, and CD14(low)CD16(high) monocytes. Albeit at relatively high concentrations, CCL26 induced calcium flux and chemotaxis in mouse L1.2 cells expressing human CX3CR1 but not mouse CX3CR1 and competed with CX3CL1 for binding to CX3CR1. In chemotaxis assays using human PBMCs, CCL26 attracted not only eosinophils but also CD16(+) NK cells, CD45RA(+)CD27(-)CD8(+) T cells, and CD14(low)CD16(high) monocytes. Intraperitoneal injection of CCL26 into mice rapidly recruited mouse eosinophils and intravenously transferred human CD16(+) NK cells into the peritoneal cavity. IL-4-stimulated HUVECs produced CCL26 and efficiently induced adhesion of cells expressing CX3CR1. Real-time PCR showed that skin lesions of psoriasis consistently contained CX3CL1 mRNA but not CCL26 mRNA, whereas those of atopic dermatitis contained CCL26 mRNA in all samples but CX3CL1 mRNA in only about half of the samples. Nevertheless, the skin lesions from both diseases consistently contained CX3CR1 mRNA at high levels. Thus, CCL26 may be partly responsible for the recruitment of cells expressing CX3CR1 in atopic dermatitis particularly when the expression of CX3CL1 is low. Collectively, CCL26 is another agonist for CX3CR1 and may play a dual role in allergic diseases by attracting eosinophils via CCR3 and killer lymphocytes and resident monocytes via CX3CR1.     

Ionotropic glutamate receptor AMPA 1 is associated with ovulation rate

Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system by opening ion channels upon the binding of glutamate. Despite the essential roles of glutamate in the control of reproduction and anterior pituitary hormone secretion, there is a limited understanding of how glutamate receptors control ovulation. Here we reveal the function of the ionotropic glutamate receptor AMPA-1 (GRIA1) in ovulation. Based on a genome-wide association study in Bos taurus, we found that ovulation rate is influenced by a variation in the N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain of GRIA1, in which serine is replaced by asparagine. GRIA1(Asn) has a weaker affinity to glutamate than GRIA1(Ser), both in Xenopus oocytes and in the membrane fraction of bovine brain. This single amino acid substitution leads to the decreased release of gonadotropin-releasing hormone (GnRH) in immortalized hypothalamic GT1-7 cells. Cows with GRIA1(Asn) have a slower luteinizing hormone (LH) surge than cows with GRIA1(Ser). In addition, cows with GRIA1(Asn) possess fewer immature ovarian follicles before superovulation and have a lower response to hormone treatment than cows with GRIA1(Ser). Our work identified that GRIA1 is a critical mediator of ovulation and that GRIA1 might be a useful target for reproductive therapy.     

Structure of an archaeal non-discriminating glutamyl-tRNA synthetase: a missing link in the evolution of Gln-tRNAGln formation

The molecular basis of the genetic code relies on the specific ligation of amino acids to their cognate tRNA molecules. However, two pathways exist for the formation of Gln-tRNA^Gln. The evolutionarily older indirect route utilizes a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) that can form both Glu-tRNA^Glu and Glu-tRNA^Gln. The Glu-tRNA^Gln is then converted to Gln-tRNA^Gln by an amidotransferase. Since the well-characterized bacterial ND-GluRS enzymes recognize tRNA^Glu and tRNA^Gln with an unrelated α-helical cage domain in contrast to the β-barrel anticodon-binding domain in archaeal and eukaryotic GluRSs, the mode of tRNA^Glu/tRNA^Gln discrimination in archaea and eukaryotes was unknown. Here, we present the crystal structure of the Methanothermobacter thermautotrophicus ND-GluRS, which is the evolutionary predecessor of both the glutaminyl-tRNA synthetase (GlnRS) and the eukaryotic discriminating GluRS. Comparison with the previously solved structure of the Escherichia coli GlnRS-tRNA^Gln complex reveals the structural determinants responsible for specific tRNA^Gln recognition by GlnRS compared to promiscuous recognition of both tRNAs by the ND-GluRS. The structure also shows the amino acid recognition pocket of GluRS is more variable than that found in GlnRS. Phylogenetic analysis is used to reconstruct the key events in the evolution from indirect to direct genetic encoding of glutamine.     

New methods for species and sex determination in three sympatric Mustelids, Mustela itatsi, Mustela sibirica and Martes melampus

We developed novel species and sex determination methods for three Japanese mustelid species. We used DDX3Y to determine sex and generated a primer set to amplify both DDX3X and DDX3Y DNA in Mustela itatsi, M. sibirica and Martes melampus. To determine species and sex simultaneously, we generated fluorescence-labelled primers that give different fragment lengths at D-loop, DDX3X and DDX3Y of these three species using a DNA sequencer.     

Chronic Alterations in Monoaminergic Cells in the Locus Coeruleus in Orexin Neuron-Ablated Narcoleptic Mice

Narcolepsy patients often suffer from insomnia in addition to excessive daytime sleepiness. Narcoleptic animals also show behavioral instability characterized by frequent transitions between all vigilance states, exhibiting very short bouts of NREM sleep as well as wakefulness. The instability of wakefulness states in narcolepsy is thought to be due to deficiency of orexins, neuropeptides produced in the lateral hypothalamic neurons, which play a highly important role in maintaining wakefulness. However, the mechanism responsible for sleep instability in this disorder remains to be elucidated. Because firing of orexin neurons ceases during sleep in healthy animals, deficiency of orexins does not explain the abnormality of sleep. We hypothesized that chronic compensatory changes in the neurophysiologica activity of the locus coeruleus (LC) and dorsal raphe (DR) nucleus in response to the progressive loss of endogenous orexin tone underlie the pathological regulation of sleep/wake states. To evaluate this hypothesis, we examined firing patterns of serotonergic (5-HT) neurons and noradrenergic (NA) neurons in the brain stem, two important neuronal populations in the regulation of sleep/wakefulness states. We recorded single-unit activities of 5-HT neurons and NA neurons in the DR nucleus and LC of orexin neuron-ablated narcoleptic mice. We found that while the firing pattern of 5-HT neurons in narcoleptic mice was similar to that in wildtype mice, that of NA neurons was significantly different from that in wildtype mice. In narcoleptic mice, NA neurons showed a higher firing frequency during both wakefulness and NREM sleep as compared with wildtype mice. In vitro patch-clamp study of NA neurons of narcoleptic mice suggested a functional decrease of GABAergic input to these neurons. These alterations might play roles in the sleep abnormality in narcolepsy.     

Effects of type III antifreeze protein on sperm and embryo cryopreservation in rabbit

We investigated the effects of antifreeze protein (AFP) III supplementation on the cryopreservation of rabbit sperm cells and embryos. Ejaculated semen was collected from male Japanese white (JW) rabbits and divided into four AFP-supplemented groups (0.1 μg/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml) and one control group with no AFP-supplementation. The semen samples were treated with egg-yolk HEPES extender containing 6% acetamide before the sperm was cooled from room temperature to 5 °C, then packed into sperm straws. The straws were frozen in steam of liquid nitrogen (LN₂) and then preserved in the LN₂. The motility of the sperm after thawing in 37 °C water was analyzed. The percentage of rapidly motile sperm in the 1 μg/ml AFP group was significantly higher than in the control group. Morulae were collected from female JW rabbits and divided into three AFP-supplemented groups (100 ng/ml, 500 ng/ml, 1000 ng/ml) and one control group. The morulae, immersed in an embryo-freezing solution (M199-HEPES containing 20% ethylene glycol, 20% dimethylsulfoxide, 10% fetal bovine serum and 0.25 M sucrose), were packed into open pulled embryo straws and vitrified in LN₂. The frozen embryos were thawed in the embryo-freezing solution, and the rates of embryo survival and development to blastocyte stage were analyzed after incubation for 72 h. The development rate of the embryos in the 500 ng/ml AFP group was significantly higher than in the control group, but that in the 1000 ng/ml AFP group was significantly lower. In conclusion, the appropriate dose of AFP III increased the number of rapidly motile sperm and embryo survival following freezing and thawing. The results suggest that supplementation with AFP III can increase the efficiency of cryopreservation of rabbit sperm cells and embryos.     

Molecular evidence of natural hybridization between abies veitchii and A. homolepis (Pinaceae) revealed by chloroplast, mitochondrial and nuclear DNA markers

Sub-alpine Abies veitchii and A. homolepis are distributed in the central part of Honshu Island, Japan, and their habitats are segregated vertically. These species sometimes form a mixed forest in the overlapping area of the two species, that is, in the upper limit of the A. homolepis habitat and the lower limit of A. veitchii. These species have been considered to be distantly related because they were classified into different sections by most conventional classifications. No natural hybridization has been reported between the two species. The aim of this study was to demonstrate, through the use of molecular markers, whether natural hybridization takes place between these two species at two experimental sites on Mt. Fuji, where the species occur naturally. DNA markers from paternally inherited chloroplast DNA (cpDNA), maternally inherited mitochondrial DNA (mtDNA) and biparentally inherited nuclear DNA (nDNA), were used for this study. As organelle DNA markers, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) markers were developed to determine the maternal and paternal species for each individual. Two of 334 individuals possessed a cpDNA haplotype derived from A. homolepis and a mtDNA haplotype from A. veitchii. Furthermore, the nDNA of these two individuals was analysed using the random amplified polymorphic DNA (RAPD) assay to investigate their genomic composition. RAPD analysis indicated that the nuclear genomes of the two individuals were derived from both species. We conclude that A. veitchii and A. homolepis produce natural hybrids, and that their systematic relationship should be re-evaluated.     

Disruption of the epilepsy KCNQ2 gene results in neural hyperexcitability

Benign familial neonatal convulsion (BFNC) is a common idiopathic epilepsy with autosomal dominant inheritance. Recently, two novel voltage-dependent potassium channel genes, KCNQ2 and KCNQ3, were identified by positional cloning as being responsible for BFNC. Heterotetramers of the products of these genes form M-channels and regulate the threshold of electrical excitability of neurons. We disrupted the mouse KCNQ2 gene via gene targeting to study the relationship between KCNQ2 and epilepsy. Homozygous pups (KCNQ2 -/-) died within a few hours after birth owing to pulmonary atelectasis that was not due to the status of epileptic seizures, although their development was morphologically normal. Heterozygous mice had decreased expression of KCNQ2 and showed hypersensitivity to pentylenetetrazole, an inducer of seizure. These data indicate that the decreased expression of KCNQ2 might cause a hyperexcitability of the CNS, which accounts for the mechanism of BFNC.     

Novel (p)ppGpp0 suppressor mutations reveal an unexpected link between methionine catabolism and GTP synthesis in Bacillus subtilis

In bacteria, guanosine (penta)tetra-phosphate ([p]ppGpp) is essential for controlling intracellular metabolism that is needed to adapt to environmental changes, such as amino acid starvation. The (p)ppGpp⁰ strain of Bacillus subtilis, which lacks (p)ppGpp synthetase, is unable to form colonies on minimal medium. Here, we found suppressor mutations in the (p)ppGpp⁰ strain, in the purine nucleotide biosynthesis genes, prs, purF and rpoB/C, which encode RNA polymerase core enzymes. In comparing our work with prior studies of ppGpp⁰ suppressors, we discovered that methionine addition masks the suppression on minimal medium, especially of rpoB/C mutations. Furthermore, methionine addition increases intracellular GTP in rpoB suppressor and this effect is decreased by inhibiting GTP biosynthesis, indicating that methionine addition activated GTP biosynthesis and inhibited growth under amino acid starvation conditions in (p)ppGpp⁰ backgrounds. Furthermore, we propose that the increase in intracellular GTP levels induced by methionine is due to methionine derivatives that increase the activity of the de novo GTP biosynthesis enzyme, GuaB. Our study sheds light on the potential relationship between GTP homeostasis and methionine metabolism, which may be the key to adapting to environmental changes.     

Sense-overlapping lncRNA as a decoy of translational repressor protein for dimorphic gene expression

Long noncoding RNAs (lncRNAs) are vastly transcribed and extensively studied but lncRNAs overlapping with the sense orientation of mRNA have been poorly studied. We analyzed the lncRNA DAPALR overlapping with the 5´ UTR of the Doublesex1 (Dsx1), the male determining gene in Daphnia magna. By affinity purification, we identified an RNA binding protein, Shep as a DAPALR binding protein. Shep also binds to Dsx1 5´ UTR by recognizing the overlapping sequence and suppresses translation of the mRNA. In vitro and in vivo analyses indicated that DAPALR increased Dsx1 translation efficiency by sequestration of Shep. This regulation was impaired when the Shep binding site in DAPALR was deleted. These results suggest that Shep suppresses the unintentional translation of Dsx1 by setting a threshold; and when the sense lncRNA DAPALR is expressed, DAPALR cancels the suppression caused by Shep. This mechanism may be important to show dimorphic gene expressions such as sex determination and it may account for the binary expression in various developmental processes.