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.     

Catalytic function of a tyrosyl residue in tryptophanase

Tryptophanase has an essential tyrosyl residue/active site which can be modified by tetranitromethane. Pyridoxal 5'-phosphate can prevent this modification efficiently, whereas pyridoxal 5'-phosphate N-oxide cannot, indicating that the free pyridinium N is required for the interaction of the coenzyme with the tyrosyl residue, probably via a hydrogen bond. The weakened binding of the coenzyme to the modified enzyme was confirmed on gel filtration, the modified enzyme being dissociated from the coenzyme seven-fold faster than the native enzyme. Furthermore, absorption spectral analyses demonstrated that the modified enzyme can catalyze the transaldimination step, but fails to abstract the alpha-H of substrates. The tyrosyl residue, therefore, not only participates in coenzyme binding, but also contributes to alpha-H labilization.     

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.     

Fungi in bathwater and sludge of bathroom drainpipes

Samples of bathwater from 14 homes and 22 public bathhouses and sludge in drainpipes from 19 house-hold bathrooms were plated out onto potato dextrose agar supplemented with chloramphenicol. Several media were used to study colony morphology of the isolates and the thermotolerance and alkaline tolerance of each isolate were examined.Eleven sludge samples produced 12 isolates of Exophiala jeanselmei, 2 of E. dermatitidis and 1 of E. moniliae. Five household bathwater samples produced 2 isolates of E. jeanselmei, 4 of E. dermatitidis and 1 of E. alcalophila. One isolate of E. jeanselmei, 2 of E. dermatitidis, 3 of E. moniliae and 2 of unidentified Exophiala species were recovered from 6 samples of the bathwater dissolving Chinese medicine in the bathtubs of public bathhouses. One isolate of E. jeanselmei was recovered from the 15 samples of bathwater from public bathhouses. Bathwater and sludge in bathroom drainpipes may be an important habitat of Exophiala species.     

Complete deficiency of 20 KDa homologous restriction factor (HRF20) and restoration with purified HRF20

20 KDa homologous restriction factor (HRF20) is a membrane glycoprotein which inhibits formation of membrane attack complexes of homologous complement. Erythrocytes from a patient who is completely deficient in HRF20 were readily hemolyzed by homologous complement activated by sucrose or by acidification as in paroxysmal nocturnal hemoglobinuria (PNH). After incubating PNH erythrocytes (PNH-E) with purified HRF20, the cells were analyzed by flow cytometry using a monoclonal antibody to HRF20 and shown to have the antigen absorbed. These PNH-E acquired resistance to hemolysis by homologous complement suggesting that HRF20 may be successfully used for treatment of these patients.     

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.