Sequential Ubiquitination of Ribosomal Protein uS3 Triggers the Degradation of Non-functional 18S rRNA

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Mutant analyses reveal different functions of fgfr1 in medaka and zebrafish despite conserved ligand-receptor relationships

Medaka (Oryzias latipes) is a small freshwater teleost that provides an excellent developmental genetic model complementary to zebrafish. Our recent mutagenesis screening using medaka identified headfish (hdf) which is characterized by the absence of trunk and tail structures with nearly normal head including the midbrain-hindbrain boundary (MHB). Positional-candidate cloning revealed that the hdf mutation causes a functionally null form of Fgfr1. The fgfr1hdf is thus the first fgf receptor mutant in fish. Although FGF signaling has been implicated in mesoderm induction, mesoderm is induced normally in the fgfr1hdf mutant, but subsequently, mutant embryos fail to maintain the mesoderm, leading to defects in mesoderm derivatives, especially in trunk and tail. Furthermore, we found that morpholino knockdown of medaka fgf8 resulted in a phenotype identical to the fgfr1hdf mutant, suggesting that like its mouse counterpart, Fgf8 is a major ligand for Fgfr1 in medaka early embryogenesis. Intriguingly, Fgf8 and Fgfr1 in zebrafish are also suggested to form a major ligand-receptor pair, but their function is much diverged, as the zebrafish fgfr1 morphant and zebrafish fgf8 mutant acerebellar (ace) only fail to develop the MHB, but develop nearly unaffected trunk and tail. These results provide evidence that teleost fish have evolved divergent functions of Fgf8-Fgfr1 while maintaining the ligand-receptor relationships. Comparative analysis using different fish is thus invaluable for shedding light on evolutionary diversification of gene function.     

Schizosaccharomyces pombe switches mating type by the synthesis-dependent strand-annealing mechanism

Schizosaccharomyces pombe cells can switch between two mating types, plus (P) and minus (M). The change in cell type occurs due to a replication-coupled recombination event that transfers genetic information from one of the silent-donor loci, mat2P or mat3M, into the expressed mating-type determining mat1 locus. The mat1 locus can as a consequence contain DNA encoding either P or M information. A molecular mechanism, known as synthesis-dependent strand annealing, has been proposed for the underlying recombination event. A key feature of this model is that only one DNA strand of the donor locus provides the information that is copied into the mat1. Here we test the model by constructing strains that switch using two different mutant P cassettes introduced at the donor loci, mat2 and mat3. We show that in such strains wild-type P-cassette DNA is efficiently generated at mat1 through heteroduplex DNA formation and repair. The present data provide an in vivo genetic test of the proposed molecular recombination mechanism.     

Lipopolysaccharide enhances the production of nicotine-induced prostaglandin E2 by an increase in cyclooxygenase-2 expression in osteoblasts

Previous studies have indicated that lipopolysaccharide(LPS)from Gram-negative bacteria inplaque induces the release of prostaglandin E_2(PGE_2),which promotes alveolar bone resorption in periodontitis,and that tobacco smoking might be an important risk factor for the development and severity of periodontitis.We determined the effect of nicotine and LPS on alkaline phosphatase(ALPase)activity,PGE_2 production,and the expression of cyclooxygenase(COX-1,COX-2),PGE_2 receptors Ep1-4,and macrophage colonystimulating factor(M-CSF)in human osteoblastic Saos-2 cells.The cells were cultured with 10~(-3)M nicotinein the presence of 0,1,or 10μg/ml LPS,or with LPS alone.ALPase activity decreased in cells cultured withnicotine or LPS alone,and decreased further in those cultured with both nicotine and LPS,whereas PGE_2production significantly increased in the former and increased further in the latter.By itself,nicotine did notaffect expression of COX-1,COX-2,any of the PGE_2 receptors,or M-CSF,but when both nicotine and LPSwere present,expression of COX-2,Ep3,Ep4,and M-CSF increased significantly.Simultaneous addition of10~(-4)M indomethacin eliminated the effects of nicotine and LPS on ALPase activity,PGE_2 production,and M-CSF expression.Phosphorylation of protein kinase A was high in cells cultured with nicotine and LPS.Theseresults suggest that LPS enhances the production of nicotine-induced PGE_2 by an increase in COX-2 expres-sion in osteoblasts,that nicotine-LPS-induced PGE_2 interacts with the osteoblast Ep4 receptor primarily inautocrine or paracrine mode,and that the nicotine-LPS-induced PGE_2 then decreases ALPase activity andincreases M-CSF expression.     

DNA adenine methylation changes dramatically during establishment of symbiosis

The DNA adenine methylation status on specific 5'-GANTC-3' sites and its change during the establishment of plant-microbe interactions was demonstrated in several species of alpha-proteobacteria. Restriction landmark genome scanning (RLGS), which is a high-resolution two dimensional DNA electrophoresis method, was used to monitor the genomewide change in methylation. In the case of Mesorhizobium loti MAFF303099, real RLGS images obtained with the restriction enzyme MboI, which digests at GATC sites, almost perfectly matched the virtual RLGS images generated based on genome sequences. However, only a few spots were observed when the restriction enzyme HinfI was used, suggesting that most GANTC (HinfI) sites were tightly methylated and specific sites were unmethylated. DNA gel blot analysis with the cloned specifically unmethylated regions (SUMs) showed that some SUMs were methylated differentially in bacteroids compared to free-living bacteria. SUMs have also been identified in other symbiotic and parasitic bacteria. These results suggest that DNA adenine methylation may contribute to the establishment and/or maintenance of symbiotic and parasitic relationships.     

Microdose clinical trial: quantitative determination of fexofenadine in human plasma using liquid chromatography/electrospray ionization tandem mass spectrometry

A sample treatment procedure and high-sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for quantitative determination of fexofenadine in human plasma was developed for a microdose clinical trial with a cold drug, i.e., a non-radioisotope-labeled drug. Fexofenadine and terfenadine, as internal standard, were extracted from plasma samples using a 96-well solid-phase extraction plate (Oasis HLB). Quantitation was performed on an ACQUITY UPLC system and an API 5000 mass spectrometer by multiple reaction monitoring. Chromatographic separation was achieved on an XBridge C18 column (100 mm x 2.1 mm i.d., particle size 3.5 microm) using acetonitrile/2 mM ammonium acetate (91:9, v/v) as the mobile phase at a flow rate of 0.6 ml/min. The analytical method was validated in accordance with the FDA guideline for validation of bioanalytical methods. The calibration curve was linear in the range of 10-1000 pg/ml using 200 microl of plasma. Analytical method validation for the clinical dose, for which the calibration curve was linear in the range of 1-500 ng/ml using 20 microl of plasma, was also conducted. Each method was successfully applied for making determinations in plasma using LC/ESI-MS/MS after administration of a microdose (100 microg solution) and a clinical dose (60 mg dose) in eight healthy volunteers.     

Optimization and validation of a high-performance liquid chromatographic method with UV detection for the determination of pyrrole-imidazole polyamides in rat plasma

A simple and sensitive high-performance liquid chromatography (HPLC) method utilizing UV detection was developed for the determination of plasma pyrrole (Py)-imidazole (Im) polyamides in rats and applied to the pharmacokinetic study of compounds. After deproteinization of plasma with methanol, Py-Im polyamides were analyzed with a reversed-phase TSK-GEL ODS-80TM (4.6 mmx15.0 cm TOSOH Co., Japan) column maintained at 40 degrees C. The mobile phase solvent A was 0.1% acetic acid and the solvent B was HPLC-grade acetonitrile (0-10 min, A: 100-20%, B: 0-80% linear gradient; 10-15 min, A: 40%, B: 60%). The flow rate was 1.0 ml/min. The detection wavelength was set at 310 nm. The method was used to determine the plasma concentration time profiles of Py-Im polyamides after intravenous injection.     

Retrotransposon silencing by DNA methylation can drive mammalian genomic imprinting

Among mammals, only eutherians and marsupials are viviparous and have genomic imprinting that leads to parent-of-origin-specific differential gene expression. We used comparative analysis to investigate the origin of genomic imprinting in mammals. PEG10 (paternally expressed 10) is a retrotransposon-derived imprinted gene that has an essential role for the formation of the placenta of the mouse. Here, we show that an orthologue of PEG10 exists in another therian mammal, the marsupial tammar wallaby (Macropus eugenii), but not in a prototherian mammal, the egg-laying platypus (Ornithorhynchus anatinus), suggesting its close relationship to the origin of placentation in therian mammals. We have discovered a hitherto missing link of the imprinting mechanism between eutherians and marsupials because tammar PEG10 is the first example of a differentially methylated region (DMR) associated with genomic imprinting in marsupials. Surprisingly, the marsupial DMR was strictly limited to the 5′ region of PEG10, unlike the eutherian DMR, which covers the promoter regions of both PEG10 and the adjacent imprinted gene SGCE. These results not only demonstrate a common origin of the DMR-associated imprinting mechanism in therian mammals but provide the first demonstration that DMR-associated genomic imprinting in eutherians can originate from the repression of exogenous DNA sequences and/or retrotransposons by DNA methylation.     

Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications

We developed novel methods for phosphopeptide enrichment using aliphatic hydroxy acid-modified metal oxide chromatography (MOC). Titania and zirconia were successfully applied to enrich phosphopeptides with the aid of aliphatic hydroxy acids, such as lactic acid and beta-hydroxypropanoic acid, to reduce the interaction between acidic non-phosphopeptides and the metal oxides. These methods removed the vast majority of non-phosphopeptides from phosphoprotein standard digests, and large numbers of phosphopeptides could be readily identified. The methods were coupled with nano-LC-MS/MS systems without difficulty. Recovery of phosphopeptides in MOC varied greatly from peptide to peptide, ranging from a few percent to 100%, and the average was almost 50%. Repeatability and linearity were satisfactory. In an examination of the cytoplasmic fraction of HeLa cells, more than 1000 phosphopeptides were identified using lactic acid-modified titania MOC and beta-hydroxypropanoic acid-modified zirconia MOC, respectively. The overlap between phosphopeptides enriched by these two methods was 40%, and the combined results provided 1646 unique phosphopeptides. To our knowledge, this is the first successful application of a single MOC-based approach to phosphopeptide enrichment from complex biological samples such as cell lysates.