N- and O-methylation of sphingomyelin markedly affects its membrane properties and interactions with cholesterol

We have prepared palmitoyl sphingomyelin (PSM) analogs in which either the 2-NH was methylated to NMe, the 3-OH was methylated to OMe, or both were methylated simultaneously. The aim of the study was to determine how such modifications in the membrane interfacial region of the molecules affected interlipid interactions in bilayer membranes. Measuring DPH anisotropy in vesicle membranes prepared from the SM analogs, we observed that methylation decreased gel-phase stability and increased fluid phase disorder, when compared to PSM. Methylation of the 2-NH had the largest effect on gel-phase instability (T(m) was lowered by ~7°C). Atomistic molecular dynamics simulations showed that fluid phase bilayers with methylated SM analogs were more expanded but thinner compared to PSM bilayers. It was further revealed that 3-OH methylation dramatically attenuated hydrogen bonding also via the amide nitrogen, whereas 2-NH methylation did not similarly affect hydrogen bonding via the 3-OH. The interactions of sterols with the methylated SM analogs were markedly affected. 3-OH methylation almost completely eliminated the capacity of the SM analog to form sterol-enriched ordered domains, whereas the 2-NH methylated SM analog formed sterol-enriched domains but these were less thermostable (and thus less ordered) than the domains formed by PSM. Cholestatrienol affinity to bilayers containing methylated SM analogs was also markedly reduced as compared to its affinity for bilayers containing PSM. Molecular dynamics simulations revealed further that cholesterol's bilayer location was deeper in PSM bilayers as compared to the location in bilayers made from methylated SM analogs. This study shows that the interfacial properties of SMs are very important for interlipid interactions and the formation of laterally ordered domains in complex bilayers.     

Preparation and characterization of polyurethane foams using a palm oil-based polyol

Polyurethane (PU) foams were prepared using a palm oil-based polyol (PO-p). At the first stage, palm oil was converted to monoglycerides as a new type of polyol by glycerolysis. A yield of the product reached 70% at reaction temperature of 90 degrees C by using an alkali catalyst and a solvent. At the second stage, PU foams were prepared from mixtures of the polyol and polyethylene glycol (PEG) or diethylene glycol (DEG) and an isocyanate compound. Characterization of the foams was carried out by thermal and mechanical analyses. The analyses showed that the chain motion of polyurethane becomes more flexible at the higher PO-p content in the whole polymer, which indicates that the monoglyceride molecules work as soft segments. The study here may lead to a development of a new type of polyurethane foams using palm oil as a raw material.     

Hamster PIWI proteins bind to piRNAs with stage-specific size variations during oocyte maturation

In animal gonads, transposable elements are actively repressed to preserve genome integrity through the PIWI-interacting RNA (piRNA) pathway. In mice, piRNAs are abundantly expressed in male germ cells, and form effector complexes with three distinct PIWIs. The depletion of individual Piwi genes causes male-specific sterility with no discernible phenotype in female mice. Unlike mice, most other mammals have four PIWI genes, some of which are expressed in the ovary. Here, purification of PIWI complexes from oocytes of the golden hamster revealed that the size of the PIWIL1-associated piRNAs changed during oocyte maturation. In contrast, PIWIL3, an ovary-specific PIWI in most mammals, associates with short piRNAs only in metaphase II oocytes, which coincides with intense phosphorylation of the protein. An improved high-quality genome assembly and annotation revealed that PIWIL1- and PIWIL3-associated piRNAs appear to share the 5′-ends of common piRNA precursors and are mostly derived from unannotated sequences with a diminished contribution from TE-derived sequences, most of which correspond to endogenous retroviruses. Our findings show the complex and dynamic nature of biogenesis of piRNAs in hamster oocytes, and together with the new genome sequence generated, serve as the foundation for developing useful models to study the piRNA pathway in mammalian oocytes.     

Nucleotide sequence of a Sendai virus genome region covering the entire M gene and the 3'' proximal 1013 nucleotides of the F gene.

We determined the sequence of the 2,138 nucleotides in the Sendai virus genome just following the 3' proximal 3,686 nucleotides which we had previously reported (Nucleic Acids Res. 11, 7317-7330, 1983). This covers the entire third gene of 1,173 nucleotides and the 3' proximal 1,013 nucleotides of the fourth gene. Like the NP and P+C genes, both the third and fourth genes start from consensus sequence R1 (3'-UCCCAC(or UA)UUUC) at the 3' end and the third gene terminates with consensus sequence R2 (3'-AUUCUUUUU) at the 5' end. The third gene was identified as M, and the deduced 348 amino acids indicated that the M protein is rich in basic residues and has hydrophobic domains near the C-terminal. The fourth gene, although sequencing is not complete yet, was identified as F, since a large open reading frame found in the gene contains the characteristic sequence of 20 amino acids located at the N-terminal of the F1 protein. Analyses of the amino acid sequence suggested that the structure of the F gene product is NH2-signal peptide-F2-F1-COOH.     

The E3 ubiquitin ligase activity of Trip12 is essential for mouse embryogenesis

Protein ubiquitination is a post-translational protein modification that regulates many biological conditions. Trip12 is a HECT-type E3 ubiquitin ligase that ubiquitinates ARF and APP-BP1. However, the significance of Trip12 in vivo is largely unknown. Here we show that the ubiquitin ligase activity of Trip12 is indispensable for mouse embryogenesis. A homozygous mutation in Trip12 (Trip12(mt/mt)) that disrupts the ubiquitin ligase activity resulted in embryonic lethality in the middle stage of development. Trip12(mt/mt) embryos exhibited growth arrest and increased expression of the negative cell cycle regulator p16. In contrast, Trip12(mt/mt) ES cells were viable. They had decreased proliferation, but maintained both the undifferentiated state and the ability to differentiate. Trip12(mt/mt) ES cells had increased levels of the BAF57 protein (a component of the SWI/SNF chromatin remodeling complex) and altered gene expression patterns. These data suggest that Trip12 is involved in global gene expression and plays an important role in mouse development.     

Three-dimensional analysis of the association of viral particles with mitochondria during the replication of Rice gall dwarf virus

Examination of cultured insect vector cells that had been infected with Rice gall dwarf virus (RGDV), using transmission electron microscopy and confocal microscopy, revealed the presence of clusters of virus-coated mitochondria around viroplasms in which replication and assembly of RGDV occurred, suggesting a role for mitochondria in supplying the energy required for viral morphogenetic processes. Electron tomography revealed that RGDV particles on the surface of mitochondria are arrayed in an orderly but loose manner, unlike tightly packaged particles in vesicular compartments, suggesting the presence of counterpart molecules on the surface of mitochondria. The viral particles in close proximity to mitochondria were aligned along intermediate filaments, which might serve as scaffolds for the anchorage of these particles. RGDV has a putative mitochondrion-targeting sequence on the outer surface of the outer-capsid protein P8. The arrangement of RGDV particles around mitochondria suggests that the region of the P8 protein containing the mitochondrion-targeting sequence might attach to a molecule like a receptor on the outer mitochondrial membrane. Our analysis demonstrates the three-dimensional arrangement and molecular basis for the mitochondrial proximity of RGDV particles during viral replication.     

A distinctive set of genes is upregulated during the inflammation-carcinoma sequence in mouse stomach infected by Helicobacter felis.

Helicobacter pylori infects over half the population worldwide and is a leading cause of chronic gastritis and gastric cancer. However, the mechanism by which this organism induces inflammation and carcinogenesis is not fully understood. In the present study we used insulin-gastrin (INS-GAS) transgenic mice that fully develop gastric adenocarcinoma after infection of H. pylori-related Helicobacter felis. Histological examination revealed that more than half of those mice developed invasive adenocarcinoma after 8 months of infection. These carcinomas were stained by NCC-ST-439 and HECA-452 that recognize 6-sulfated and non-sulfated sialyl Lewis X. Lymphocytic infiltration predominantly to submucosa was observed in most H. felis-infected mice, and this was associated with the formation of peripheral lymph node addressin (PNAd) on high endothelial venule (HEV)-like vessels detected by MECA-79. Time-course analysis of gene expression by using gene microarray revealed upregulation of several inflammation-associated genes including chemokines, adhesion molecules, surfactant protein D (SP-D), and CD74 in the infected stomach. Immunohistochemical analysis demonstrated that SP-D is expressed in hyperplasia and adenocarcinoma whereas CD74 is expressed in adenocarcinoma in situ and invasive carcinoma. These results as a whole indicate that H. felis induces HEV-like vessels and inflammation-associated chemokines and chemokine receptors, followed by adenocarcinoma formation.