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.     

Yeast plasmids resembling 2 micron DNA: regional similarities and diversities at the molecular level.

The nucleotide sequence of two Zygosaccharomyces plasmids, pSB2 (5,415 base pairs), isolated from Zygosaccharomyces bailii, and pSM1 (5,416 base pairs), isolated from Zygosaccharomyces fermentati Naganishi, was determined. The predicted amino acid sequences of open reading frames among six yeast plasmids that resemble 2 microns DNA indicated regional sequence similarities among FLP proteins. Greater similarities were seen among Zygosaccharomyces plasmids (pSB2, pSB3, pSR1, and pSM1) than other combinations. A putative recognition site for the FLP enzyme of a Zygosaccharomyces plasmid also showed some conservation, especially in the 4 nucleotides flanking the central spacer region. From comparative studies of the sequences of putative genes of each plasmid, we propose an apparent phylogenetic relationship among yeast plasmids resembling 2 micron DNA. Among the Zygosaccharomyces plasmids, pSB2 and pSR1 are most closely related, since not only were the FLP enzymes of these two plasmids most closely related, but also the amino acid sequence of the putative P gene of pSR1 showed clear homology with that of open reading frame B of pSB2.     

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     

Immunohistochemical localization of atrial natriuretic polypeptide (ANP) in human atrial and ventricular myocardiocytes

To date, there have been few immunohistochemical investigations of atrial natriuretic polypeptide (ANP) in human cardiac tissue, especially the ventricles. In this study, myocardial tissue was obtained from two sources: the bilateral atria and ventricles at autopsy; and biopsy tissues from the right auricle and left ventricle of a patient with myocardial infarction undergoing surgery. These tissues were examined by the avidin-biotin immunoperoxidase technique using three kinds of primary ANP-antibodies. ANP-immunoreactivity was observed in the perinuclear region of myocytes of all tissues examined. The intensity of the reaction was stronger in atrial tissue, weaker in ventricular tissue. In the later tissue, the positive-staining myocytes were not part of the pulse-conducting system. Although the tissues we studied were not obtained from normal hearts, our data demonstrates that ANP-reactivity can be detected in ventricular myocytes outside the pulse-conducting system.