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.     

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.     

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.     

Bimodal activation of SMC ATPase by intra- and inter-molecular interactions.

Structural maintenance of chromosomes (SMC) proteins play fundamental roles in higher-order chromosome dynamics from bacteria to humans. It has been proposed that the Bacillus subtilis SMC (BsSMC) homodimer is composed of two anti-parallel coiled-coil arms, each having an ATP-binding domain at its distal end. It remains totally unknown, however, how the two-armed structure supports ATP-dependent actions of BsSMC. By constructing a number of mutant derivatives including 'single-armed' BsSMC, we show here that the central hinge domain provides a structural flexibility that allows opening and closing of the two arms. This unique structure brings about bimodal regulation of the SMC ATPase cycle. Closing the arm can trigger ATP hydrolysis by allowing an end-end interaction within a dimer (intramolecular mode). When bound to DNA, ATP promotes a dimer-dimer interaction, which in turn activates their DNA-dependent ATPase activity (intermolecular mode). Our results reveal a novel mechanism of ATPase regulation and provide mechanistic insights into how eukaryotic SMC protein complexes could mediate diverse chromosomal functions, such as chromosome condensation and sister chromatid cohesion.     

Enhanced binding of morphine and nalorphine to opioid delta receptor by glucuronate and sulfate conjugations at the 6-position

Effect of the modification of morphine and nalorphine by glucuronate and sulfate conjugations at the 3- and 6-positions on the binding to opioid receptors was examined in a particulate fraction of rat brain. Competing potencies of both drugs against [3H]morphine and [3H]leucine enkephalin bindings were extremely decreased by either glucuronate or sulfate conjugation at the 3-position. On the other hand, the potencies of morphine and nalorphine against [3H]leucine enkephalin binding were considerably enhanced by the conjugations at the 6-position, whereas the potencies against [3H]morphine binding were decreased. These altered interactions of the conjugates at the 6-position with the two ligands were attributed to their enhanced binding to delta-receptor and reduced binding to mu-receptor by Hill plot and modified Scatchard analysis. Resulted comparable and simultaneous interactions with mu- and delta- receptors were assumed to be a cause of the enhanced mu-receptor-directed analgesia of morphine and elevated same receptor-directed antagonistic effect of nalorphine, which have been found previously in our laboratory.     

Structure and expression of human B cell stimulatory factor-2 (BSF-2/IL-6) gene.

The chromosomal DNA segment of human B cell stimulatory factor-2 (BSF-2/IL-6) was isolated and characterized by nucleotide sequence analysis. The human BSF-2/IL-6 gene consists of five exons and four introns and its organization shows a distinctive similarity to granulocyte colony-stimulating factor gene. The two genes have the same number of exons and introns and the size of each exon is strikingly similar. The BSF-2/IL-6 mRNA was found to be constitutively expressed in a human T cell leukemia virus-1 transformed T cell line, TCL-Na1, a bladder cell carcinoma line, T24, and an amnion derived cell line, FL. The BSF-2/IL-6 mRNA was also found to be inducible with interleukin-1 beta in an astrocytoma line, U373 and a glioblastoma line, SK-MG-4. S1 mapping and primer extension analyses showed the presence of multiple initiation sites and the preferential utilization of a different initiation site for each individual tissue tested.     

Analyses of azopigments obtained from the delta fraction of bilirubin from mammalian plasma (mammalian biliprotein).

Azopigments were obtained from the delta fraction of bilirubin (mammalian biliprotein) in cholestatic sera of men, rats and guinea pigs by diazo reaction with diazotized p-iodoaniline and analysed by t.l.c. Delta bilirubin of men and rats generated both unconjugated and glucuronide-conjugated azodipyrroles, whereas that of guinea pigs, in which the predominant form of conjugated bilirubin in serum was bilirubin monoglucuronide, generated only unconjugated azodipyrrole. We further analysed the azopigments by reversed-phase h.p.l.c. to distinguish their endovinyl and exovinyl isomers. The results indicated (a) that covalent binding of bilirubin to protein occurs exclusively on the conjugated dipyrrolic (either endovinyl or exovinyl) half of the parent conjugated bilirubin, (b) that both bilirubin monoglucuronide and bilirubin diglucuronide generate delta bilirubin, the latter yielding a 'conjugated' form of delta bilirubin that preserves the glucuronic acid moiety on the dipyrrolic half not bound covalently to protein, and (c) that therefore at least four forms of delta bilirubin exist in jaundiced sera of men and rats.