Epstein-Barr virus nuclear antigen leader protein induces expression of thymus- and activation-regulated chemokine in B cells

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) plays a critical role in transformation of primary B lymphocytes to continuously proliferating lymphoblastoid cell lines (LCLs). To identify cellular genes in B cells whose expression is regulated by EBNA-LP, we performed microarray expression profiling on an EBV-negative human B-cell line, BJAB cells, that were transduced by a retroviral vector expressing the EBV EBNA-LP (BJAB-LP cells) and on BJAB cells that were transduced with a control vector (BJAB-vec cells). Microarray analysis led to the identification of a cellular gene encoding the CC chemokine TARC as a novel target gene that was induced by EBNA-LP. The levels of TARC mRNA expression and TARC secretion were significantly up-regulated in BJAB-LP compared with BJAB-vec cells. Induction of TARC was also observed when a subline of BJAB cells was converted by a recombinant EBV. Among the EBV-infected B-cell lines with the latency III phenotype that were tested, the LCLs especially secreted significantly high levels of TARC. The level of TARC secretion appeared to correlate with the level of full-length EBNA-LP expression. These results indicate that EBV infection induces TARC expression in B cells and that EBNA-LP is one of the viral gene products responsible for the induction.     

The SUMO pathway is required for selective degradation of DNA topoisomerase IIbeta induced by a catalytic inhibitor ICRF-193(1)

DNA topoisomerase I and II have been shown to be modified with a ubiquitin-like protein SUMO in response to their specific inhibitors called 'poisons'. These drugs also damage DNA by stabilizing the enzyme-DNA cleavable complex and induce a degradation of the enzymes through the 26S proteasome system. A plausible link between sumoylation and degradation has not yet been elucidated. We demonstrate here that topoisomerase IIbeta, but not its isoform IIalpha, is selectively degraded through proteasome by exposure to the catalytic inhibitor ICRF-193 which does not damage DNA. The beta isoform immunoprecipitated from ICRF-treated cells was modified by multiple modifiers, SUMO-2/3, SUMO-1, and polyubiquitin. When the SUMO conjugating enzyme Ubc9 was conditionally knocked out, the ICRF-induced degradation of topoisomerase IIbeta did not occur, suggesting that the SUMO modification pathway is essential for the degradation.     

The occurrence and run timing of naturally spawning chum salmon in northern Japan

Since the late 20th century, the biomass of Pacific salmon Oncorhynchus spp. has increased. Hokkaido, northern Japan, is one of the main areas of chum salmon O. keta production in the North Pacific and intensive hatchery programs support the recent high abundance. However, proper management of naturally spawning populations is necessary to conserve healthy stocks of this species. In 2008, we started a program to assess the naturally spawning chum salmon populations in Hokkaido. Of the total of approximately 1,500 rivers in Hokkaido, 238 rivers with lengths of longer than 8 km (excluding those rivers used for hatchery broodstock collection) were surveyed in 2008 and 2009. The number of non-enhanced rivers found to contain naturally reproducing chum salmon was 59 (31.4% of surveyed rivers) and 50 (37.6% of surveyed rivers) rivers in 2008 and 2009, respectively. Including the rivers where hatchery broodstock were collected and rivers shorter than 8 km that contain naturally spawning chum salmon, chum salmon ascended at least 191 and 175 rivers in Hokkaido in 2008 and 2009, respectively. Repeated foot surveys indicated that the run timings of naturally spawning chum salmon may be affected by coastal commercial fisheries. This study showed that naturally spawning chum salmon remain in many rivers in Hokkaido where hatchery programs have been intensively conducted.     

Molecular defect in human erythropoietic protoporphyria with fatal liver failure

We investigated the molecular basis of ferrochelatase in a Japanese patient with erythropoietic protoporphyria (EPP), complicated by fatal liver failure, and defined a novel point mutation in the ferrochelatase gene. cDNAs were synthesized using Epstein-Barr-virus-transformed lymphoblastoid cells from the proband. cDNA clones encoding ferrochelatase in the proband were isolated by amplification using the polymerase chain reaction. There were two sizes of ferrochelatase cDNAs; one was normal in size, the other being smaller. Sequence analysis of the abnormally sized cDNA clones revealed that they lacked exon 9 of the ferrochelatase gene. Genomic DNA analysis demonstrated that the proband had the abnormal allele and that it contained a G to A point mutation at the first position of the donor site of intron 9. An identical mutation was detected in the affected family members of the proband by allele-specific oligonucleotide hybridization analysis. EPP is inherited in an autosomal dominant manner in this family.     

Structural Features of Membrane Fusion between Influenza Virus and Liposome as Revealed by Quick-Freezing Electron Microscopy

The structure of membrane fusion intermediates between the A/PR/8(H1N1) strain of influenza virus and a liposome composed of egg phosphatidylcholine, cholesterol, and glycophorin was studied using quick-freezing electron microscopy. Fusion by viral hemagglutinin protein was induced at pH 5.0 and 23°C. After a 19-s incubation under these conditions, small protrusions with a diameter of 10–20 nm were found on the fractured convex faces of the liposomal membranes, and small pits complementary to the protrusions were found on the concave faces. The protrusions and pits corresponded to fractured parts of outward bendings of the lipid bilayer or “microprotrusions of the lipid bilayer.” At the loci of the protrusions and pits, liposomal membranes had local contacts with viral membranes. In many cases both the protrusions and the pits were aligned in regular polygonal arrangements, which were thought to reflect the array of hemagglutinin spikes on the viral surface. These structures were induced only when the medium was acidic with the virus present. Based on these observations, it was concluded that the microprotrusions of the lipid bilayer are induced by hemagglutinin protein. Furthermore, morphological evidence for the formation of the “initial fusion pore” at the microprotrusion was obtained. The protrusion on the convex face sometimes had a tiny hole with a diameter of <4 nm in the center. The pits transformed into narrow membrane connections <10 nm in width, bridging viruses and liposomes. The structures of the fusion pore and fusion neck with larger sizes were also observed, indicating growth of the protrusions and pits to distinct fusion sites. We propose that the microprotrusion of the lipid bilayer is a fusion intermediate induced by hemagglutinin protein, and suggest that the extraordinarily high curvature of this membrane structure is a clue to the onset of fusion. The possible architecture of the fusion intermediate is discussed with regard to the localization of intramembrane particles at the microprotrusion.     

Effect of opioids on delayed neuronal death in the gerbil hippocampus

The effect of opioids on delayed neuronal death was evaluated in the gerbil hippocampus. Male Mongolian gerbils were subjected to transient forebrain ischemia and neuronal density was evaluated in the hippocampus 7 days following ischemia. When hypothermia during and after ischemia was prevented treatment with morphine, U-50488H, or naloxone provided no significant protection. In contrast, a spontaneous drop in rectal temperature to 32°C at the end of ischemia produced near-complete protection of CA₁ pyramidal neurons. No opioids modulate the protective effect of hypothermia.