Molecular characterization of ENU mouse mutagenesis and archives

The large-scale mouse mutagenesis with ENU has provided forward-genetic resources for functional genomics. The frozen sperm archive of ENU-mutagenized generation-1 (G1) mice could also provide a "mutant mouse library" that allows us to conduct reverse genetics in any particular target genes. We have archived frozen sperm as well as genomic DNA from 9224 G1 mice. By genome-wide screening of 63 target loci covering a sum of 197 Mbp of the mouse genome, a total of 148 ENU-induced mutations have been directly identified. The sites of mutations were primarily identified by temperature gradient capillary electrophoresis method followed by direct sequencing. The molecular characterization revealed that all the identified mutations were point mutations and mostly independent events except a few cases of redundant mutations. The base-substitution spectra in this study were different from those of the phenotype-based mutagenesis. The ENU-based gene-driven mutagenesis in the mouse now becomes feasible and practical.     

Inference of S-system models of genetic networks using a cooperative coevolutionary algorithm

MOTIVATION: To resolve the high-dimensionality of the genetic network inference problem in the S-system model, a problem decomposition strategy has been proposed. While this strategy certainly shows promise, it cannot provide a model readily applicable to the computational simulation of the genetic network when the given time-series data contain measurement noise. This is a significant limitation of the problem decomposition, given that our analysis and understanding of the genetic network depend on the computational simulation. RESULTS: We propose a new method for inferring S-system models of large-scale genetic networks. The proposed method is based on the problem decomposition strategy and a cooperative coevolutionary algorithm. As the subproblems divided by the problem decomposition strategy are solved simultaneously using the cooperative coevolutionary algorithm, the proposed method can be used to infer any S-system model ready for computational simulation. To verify the effectiveness of the proposed method, we apply it to two artificial genetic network inference problems. Finally, the proposed method is used to analyze the actual DNA microarray data.     

Properties of blocking and non-blocking monoclonal antibodies specific for human macrophage galactose-type C-type lectin (MGL/ClecSF10A/CD301)

Monoclonal antibodies (mAbs) specific for the human macrophage galactose-type calcium-type lectin (MGL) were established. The recombinant extracellular domain of MGL was used to immunize a mouse, and 10 hybridoma clones were obtained. Binding of recombinant MGL to asialo-bovine submaxillary mucin was shown to be blocked by mAbs MLD-1, 4 and 6. Immunoprecipitation of MGL from lysates of COS-1 cells transfected with MGL cDNA (form 6A) was achieved with mAbs MLD-1, 4, 7, 8 and 16. Chimeric recombinant proteins between human MGL and mouse MGL1 were used to determine the location of the epitopes for these mAbs. mAbs MLD-8, 13, 15 and 16 interacted with the amino terminal side of the conserved WVDGTD sequence immediately upstream of QPD, whereas mAbs MLD-7, 12 and 17 interacted with the other side. mAbs MLD-1, 4, and 6 apparently required both sides of this boundary. mAbs MLD-15 and 16 were shown to recognize the protein products of alternatively spliced mRNA 6A/8A and 6C/8A, having deletions at the boundary of exons 7 and 8, in addition to full length and other spliced forms of MGL (6A, 6B and 6C), whereas the other mAbs bound only full length and forms 6A, 6B and 6C.     

Production of seedlings from ovules excised at the zygote stage in Lilium spp.

The present study was undertaken to establish a culture system for ovules excised at the zygote stage in Lilium spp. Ovules of Lilium × `Connecticut King' and L. × `Enchantment' were excised together with placental tissue 3, 5, and 10 days after pollination (DAP) and cultured on B5 medium and half-strength B5 medium containing sucrose at different concentrations. In vitro embryo development in ovules cultured at 3 DAP was influenced by the basal media and the sucrose concentration. The half-strength B5 medium with 9% sucrose was the best condition, but only a few ovules isolated from placental tissue developed into seedlings. Application of embryo culture, in which embryos were excised from ovules after 14 weeks of ovule-with-plancetal-tissue culture, greatly improved the production of seedlings. The present study indicates that a two-step culture procedure, ovule-with-placental-tissue culture and embryo culture, make it possible to produce seedlings from ovules just after fertilization.     

Tmem64 Modulates Calcium Signaling during RANKL-Mediated Osteoclast Differentiation

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Highlights? Tmem64-deficient mice show increased bone volume ? Tmem64 deficiency reduces [Ca²⁺]_i oscillation in response to RANKL stimulation ? Tmem64 interacts with SERCA2 ? Tmem64 positively regulates osteoclast formation via SERCA2/Ca²⁺ signaling     

Characteristic Cerebrospinal Fluid Cytokine/Chemokine Profiles in Neuromyelitis Optica,Relapsing Remitting or Primary Progressive Multiple Sclerosis

Background

Differences in cytokine/chemokine profiles among patients with neuromyelitis optica (NMO), relapsing remitting multiple sclerosis (RRMS), and primary progressive MS (PPMS), and the relationships of these profiles with clinical and neuroimaging features are unclear. A greater understanding of these profiles may help in differential diagnosis.

Methods/Principal Findings

We measured 27 cytokines/chemokines and growth factors in CSF collected from 20 patients with NMO, 26 with RRMS, nine with PPMS, and 18 with other non-inflammatory neurological diseases (OND) by multiplexed fluorescent bead-based immunoassay. Interleukin (IL)-17A, IL-6, CXCL8 and CXCL10 levels were significantly higher in NMO patients than in OND and RRMS patients at relapse, while granulocyte-colony stimulating factor (G-CSF) and CCL4 levels were significantly higher in NMO patients than in OND patients. In NMO patients, IL-6 and CXCL8 levels were positively correlated with disability and CSF protein concentration while IL-6, CXCL8, G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN-γ were positively correlated with CSF neutrophil counts at the time of sample collection. In RRMS patients, IL-6 levels were significantly higher than in OND patients at the relapse phase while CSF cell counts were negatively correlated with the levels of CCL2. Correlation coefficients of cytokines/chemokines in the relapse phase were significantly different in three combinations, IL-6 and GM-CSF, G-CSF and GM-CSF, and GM-CSF and IFN-γ, between RRMS and NMO/NMOSD patients. In PPMS patients, CCL4 and CXCL10 levels were significantly higher than in OND patients.

Conclusions

Our findings suggest distinct cytokine/chemokine alterations in CSF exist among NMO, RRMS and PPMS. In NMO, over-expression of a cluster of Th17- and Th1-related proinflammatory cytokines/chemokines is characteristic, while in PPMS, increased CCL4 and CXCL10 levels may reflect on-going low grade T cell and macrophage/microglia inflammation in the central nervous system. In RRMS, only a mild elevation of proinflammatory cytokines/chemokines was detectable at relapse.     

Effect of the age of tobacco leaves on photosynthesis and photorespiration

Relationships among the activities of enzymes related to photosynthesisand photorespiration, and ¹⁴CO₂ photosynthetic products wereinvestigated with individual tobacco leaves attached to thestalk from the bottom to the top. P-glycolate phosphatase ofthe chloroplasts and glycolate oxidase of the peroxisomes hadtheir maximum activities in the 25th leaf from the dicotyledons.Maximum photorespiration was similarly distributed. The highestratio of serine-¹⁴C to glycine-¹⁴C in the photosynthesates andmaximum glycolate formation were also observed in the 25th leaf.Glutamateglyoxylate aminotransferase, serine hydroxymethyltransferaseand glycine decarboxylase were more active in the upper leaves.RuDP carboxylase had nearly constant activity in all leaves,except for the youngest in which activity decreased. MaximumCO₂ photosynthesis and enzyme activity for the C₄ dicarboxylicacid cycle occurred in the upper, youngest leaf. Distributionof photosynthetic CO² fixation among the leaves did not coincidewith RuDP carboxylase activity. The photosynthetic capacityappeared to be better related to the distribution pattern forenzymes of the C₄ dicarboxylic acid pathway, i.e. PEP carboxylase,pyruvate Pi dikinase and 3-PGA phosphatase in the upper leaves.The results suggest that the C₄ dicarboxylic acid pathway participates,to some extent, in photosynthesis in young leaves of tobacco,a dicotyledonous plant. ¹This work was reported at the Annual Meeting (1970) of theJapanese Plant Physiologists in Kobe. ²The Central Research Institute, Japan Monopoly Corporation1-28-3, Nishishinagawa, Shinagawaku, Tokyo, 141 Japan. (Received November 2, 1972; )     

Intra-strain biological and epidemiological characterization of plum pox virus

Plum pox virus (PPV) is one of the most important plant viruses causing serious economic losses. Thus far, strain typing based on the definition of 10 monophyletic strains with partially differentiable biological properties has been the sole approach used for epidemiological characterization of PPV. However, elucidating the genetic determinants underlying intra-strain biological variation among populations or isolates remains a relevant but unexamined aspect of the epidemiology of the virus. In this study, based on complete nucleotide sequence information of 210 Japanese and 47 non-Japanese isolates of the PPV-Dideron (D) strain, we identified five positively selected sites in the PPV-D genome. Among them, molecular studies showed that amino acid substitutions at position 2,635 in viral replicase correlate with viral titre and competitiveness at the systemic level, suggesting that amino acid position 2,635 is involved in aphid transmission efficiency and symptom severity. Estimation of ancestral genome sequences indicated that substitutions at amino acid position 2,635 were reversible and peculiar to one of two genetically distinct PPV-D populations in Japan. The reversible amino acid evolution probably contributes to the dissemination of the virus population. This study provides the first genomic insight into the evolutionary epidemiology of PPV based on intra-strain biological variation ascribed to positive selection.