Analysis of Nuclear Accumulation of Influenza Nucleoprotein Antigen in the Presence of p-Fluorophenylalanine

When p-fluorophenylalanine (FPA) was added to influenza virus RI/5+-infected cells 4 hr after infection, virus-specific proteins were synthesized but infectious progeny virus was not produced. In these cells, synthesis of viral RNA was strongly inhibited and nucleoprotein (NP) antigen was found predominantly in the nucleus in contrast to untreated cells in which NP antigen was distributed throughout the whole cell. The intracellular location and migration of NP were examined by isotope labeling followed by fractionation of infected cells. In untreated cells, a large portion of the NP was present in the cytoplasm and most of it was detected in the form of ribonucleoprotein (RNP). In contrast, in FPA-treated cells little viral RNP was detectable and NP was present predominantly in the nucleus in a nonassembled, soluble form. When FPA was removed from the culture, synthesis of viral RNA was soon restored and a large amount of viral RNP appeared in the cytoplasm; this was followed by the production of infectious virus. The results of the experiments suggest that the NP synthesized in the presence of FPA is not assembled into viral RNP because of the lack of available RNA, and such NP migrates readily into the nucleus and accumulates there.     

Prophages integrating into prophages: A mechanism to accumulate type III secretion effector genes and duplicate Shiga toxin-encoding prophages in Escherichia coli

Bacteriophages (or phages) play major roles in the evolution of bacterial pathogens via horizontal gene transfer. Multiple phages are often integrated in a host chromosome as prophages, not only carrying various novel virulence-related genetic determinants into host bacteria but also providing various possibilities for prophage-prophage interactions in bacterial cells. In particular, Escherichia coli strains such as Shiga toxin (Stx)-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) strains have acquired more than 10 prophages (up to 21 prophages), many of which encode type III secretion system (T3SS) effector gene clusters. In these strains, some prophages are present at a single locus in tandem, which is usually interpreted as the integration of phages that use the same attachment (att) sequence. Here, we present phages integrating into T3SS effector gene cluster-associated loci in prophages, which are widely distributed in STEC and EPEC. Some of the phages integrated into prophages are Stx-encoding phages (Stx phages) and have induced the duplication of Stx phages in a single cell. The identified attB sequences in prophage genomes are apparently derived from host chromosomes. In addition, two or three different attB sequences are present in some prophages, which results in the generation of prophage clusters in various complex configurations. These phages integrating into prophages represent a medically and biologically important type of inter-phage interaction that promotes the accumulation of T3SS effector genes in STEC and EPEC, the duplication of Stx phages in STEC, and the conversion of EPEC to STEC and that may be distributed in other types of E. coli strains as well as other prophage-rich bacterial species.     

Structural characterization and dynamics of globotetraosylceramide in vascular endothelial cells under TNF-α stimulation

In several vascular inflammatory reactions (i.e. immunity and thrombosis) inflammatory mediators lead to the activation of vascular endothelial cells (EC). To date, a number of functional molecules induced on the surface of activated-EC have been identified. We report here that Globotetraosylceramide (Gb4), a glycosphingolipid expressed in EC, is a novel inducible molecule on EC activated by TNF-α. The cell surface expression of Gb4 is increased in a time-dependent manner under TNF-α stimulation, which shows distinct expression kinetics of major proteins induced by TNF-α on EC. MALDI-TOF-MS analysis revealed that the enhanced Gb4 predominantly contains C24:0 fatty acid in the ceramide moiety. Isolated caveolae/lipid raft-enriched detergent insoluble membrane domains in activated-EC predominantly contain this molecular species of Gb4. Gb4 containing C16:0 fatty acid in the ceramide moiety, which is known to constitute the major species of Gb4 in plasma, is also found as a major molecular species in EC. These observations indicate that Gb4, especially with very long fatty acid, is enhanced in EC during its inflammatory reaction, and suggest the potential utility of Gb4 as a biomarker for monitoring inflammation status of EC involving its related diseases.     

Development of the bovine ileal mucosa

The development of the bovine ileal mucosa was studied with particular reference to maturation during the fetal and neonatal period. In this region, by 4-5 months of fetal development, vacuolation of the epithelial cells had occurred on the villi, and the goblet and absorptive cells in the crypts were present. By 6-9 months, the villi were longer and more numerous than in the previous stages. At the same time, the vacuolated cells could be seen predominantly on the upper half of each villus. The absorptive cells and goblet cells were more distinct in the crypt and lower half of each villus. Moreover, the goblet cells showed differences in mucin, while in the submucosa the lymphoid follicles were seen to have enlarged to become a prominent feature of the Peyer's patches at this stage. At birth, in suckled animals, the ileal cells on the lower area of each villus and in the crypt appeared more like mature cells. In contrast, there were numerous inclusion bodies in epithelial cells on the upper half of each villus. They appeared in the apical portion of the cytoplasm as vacuoles with stainable or dense contents. By 1 week, however, epithelial cells no longer contained inclusion bodies, and absorptive and goblet cell populations had begun to emerge from the crypts. These histological results suggest that the bovine ileal mucosa has two distinct turning points during its development in the fetus and the neonate. Initially all the mucosal structures are present in fetuses at 6-7 months of gestation, and then the vacuolated cells covering the ileal villi are replaced by mature, nonpinocytosing epithelium which emerges from the crypts on or before the 7th day after birth (ileal closure).     

Cloning of the Membrane-Bound Aldehyde Dehydrogenase Gene of Acetobacter polyoxogenes and Improvement of Acetic Acid Production by Use of the Cloned Gene

A genomic clone bank of Acetobacter polyoxogenes NBI1028 constructed in Escherichia coli by use of the expression vector pUC18 was screened with antibody raised against membrane-bound aldehyde dehydrogenase (ALDH; 75 kilodaltons [kDa]) from A. polyoxogenes NBI1028. A clone that synthesized a 41-kDa protein cross-reactive with anti-ALDH antibody was isolated. For cloning of the full-length ALDH structural gene, a cosmid gene bank was screened by Southern blot hybridization with the cloned DNA as a probe, and subcloning from the positive cosmid clone was performed with shuttle vector pMV24. Plasmid pAL25, containing the full-length ALDH structural gene, was isolated and expressed in both E. coli and Acetobacter aceti to produce a fused protein (78 kDa) with a short NH₂-terminal β-galactosidase peptide. pAL25 conferred ALDH production on a mutant of A. aceti lacking the enzyme activity. Transformation of A. aceti subsp. xylinum NBI2099 with pAL25 caused 2- and 1.4-fold increases in the production rate and in the maximum concentration of acetic acid in submerged fermentation, respectively.     

Characterization of the glycosphingolipids of pig cortical bone and cartilage

Neutral glycosphingolipids and gangliosides were extracted from pig cortical bone and cartilage. To ensure the completeness of extraction, the cortical bone was demineralized and reextracted. Globotriaosylceramide and globoside were noted to be present at high content in the cortical bone. It contained glucosylceramide, lactosylceramide, globotriaosylceramide and globoside as neutral glycosphingolipids at a ratio of 1:0.7:3.1:2.7. In articular cartilage, the ratio was 1:0.7:0.4:0.8. GM3 and GD3 were the major gangliosides in both these tissues. GM3, GM1, GD3, GD1 and GT1 were present at ratios of 1:0.9:0.9:0.1:0.1 in the cortical bone and 1:0:1.2:0.06:0.02 in the cartilage. Neutral glycosphingolipids could be extracted from the cortical bone without the need for demineralization, while most of the gangliosides were extracted after this treatment, implying the occurrence of interactions between gangliosides and minerals in the bone.     

Changes of DNA methylation level during pre-and postnatal periods in mice

DNA methylation in an adult mammalian body shows tissue-specificity. But when and how the specificity is established in the process of development has not yet been elucidated. Here we have investigated age-dependent changes in the amount of 5-methyldeoxycytidine (5mdC) that DNA of various mouse tissues contains during the late-fetal and postnatal periods, using high-performance liquid chromatography. The tissue-specificity in the 5mdC level was observed in the late-fetal stage, and the level continued to change during the subsequent periods. The most pronounced alterations were observed in brain and liver, where similar biphasic changes were seen, but at different ages. At maturation, the 5mdC levels were high in thymus, spleen and brain, intermediate in lung, and low in liver and sperm. The data demonstrate the importance of the peri- and postnatal periods in establishment of tissue-specificity in 5mdC content.     

Experiments inducing prospective polar body nuclei to participate in embryogenesis of the sawfly Athalia rosae (Hymenoptera)

Summary Mature eggs dissected from ovaries of unmated females of Athalia rosae (Hymenoptera: Tenthredinidae), if placed on a filter-paper soaked with distilled water, are activated and develop to haploid males. Occasionally, however, diploid females develop from these artificially activated eggs. Treatment of mature unfertilized eggs dissected from diploid females with ice-cold temperatures immediately before activation and with a high temperature (36° C) upon and immediately after activation resulted in the production of diploid males, diploid females, triploid females and gynandromorphs at high frequency. The same treatment of mature unfertilized eggs dissected from triploid females resulted in the production of only triploid survivors. These results, together with the results on the segregation of a marker mutation, yellow fatbody (yfb), appear to indicate that meiotic divisions were complete in the treated eggs, and that all four nuclei became potentially capable of participating in development with or without automictic fusion.Studies on the sawfly, Athalia rosae (Insecta, Hymenoptera, Tenthredinidae), part V