The induction of H3K9 methylation by PIWIL4 at the p16Ink4a locus

The field of epigenetics has made progress by the identification of the small RNA-mediated epigenetic modification. However, little is known about the key proteins. Here, we report that the human PIWI-like family is a candidate protein that is involved in the pathway responsible for chromatin remodeling. The PIWI-like family proteins, expressed as the Flag-fusion proteins, formed a bulky body and localized to the nuclear periphery. Transient transfection of PIWI-like 4 (PIWIL4), only member of the PIWI-like family that was ubiquitously expressed in human tissues, induced histone H3 lysine 9 methylation at the p16(Ink4a) (CDKN2A) locus. The elevated level of histone methylation resulted in the downregulation of the p16(Ink4a) gene. These results suggest PIWIL4 plays important roles in the chromatin-modifying pathway in human somatic cells.     

A Spaceflight Experiment for the Study of Gravimorphogenesis and Hydrotropism in Cucumber Seedlings

peg , on the transition zone between hypocotyl and root. Our spaceflight experiment verified that the lateral positioning of a peg in cucumber seedlings is modified by gravity. It has been suggested that auxin plays an important role in the gravity-controlled positioning of a peg on the ground. Furthermore, cucumber seedlings grown in microgravity developed a number of the lateral roots that grew towards the water-containing substrate in the culture vessel, whereas on the ground they oriented perpendicular to the primary root growing down. The response of the lateral roots in microgravity was successfully mimicked by clinorotation of cucumber seedlings on the three dimensional clinostat. However, this bending response of the lateral roots was observed only in an aeroponic culture of the seedlings but not in solid medium. We considered the response of the lateral roots in microgravity and on clinostat as positive hydrotropism that could easily be interfered by gravitropism on the ground. This system with cucumber seedlings is thus a useful model of spaceflight experiment for the study of the gravimorphogenesis, root hydrotropism and their interaction. Received 13 September 1999/ Accepted in revised form 12 October 1999     

Improving effect of ethyl eicosapentanoate on statin-induced rhabdomyolysis in Eisai hyperbilirubinemic rats

The effect of ethyl eicosapentanoate (EPA-E) on statin-induced rhabdomyolysis was investigated by co-administration of EPA-E and pravastatin (PV), as a typical statin, to Eisai hyperbilirubinemic rats (EHBR). It was confirmed that the plasma PV concentration was not affected by simultaneous administration of EPA-E, and there was no cumulative increase of PV during prolonged co-administration of EPA-E and PV. Muscular degeneration was prominent (incidence 5/5; average grade 3.5 (range 2-4)) in EHBR treated with PV alone at 200 mg/kg/day for 14 days, but co-administration of EPA-E at doses of 100, 300, and 1000 mg/kg/day decreased the average grades to 1.4 (range 0.3-3.0), 0.5 (0.2-1.0), and 0.6 (0.0-1.7), respectively. Creatine phosphokinase (CPK) and myoglobin levels in plasma were well correlated with the grade of skeletal muscle degeneration. Thus, EPA-E appears to reduce the severity of statin-induced rhabdomyolysis.     

In vitro abzyme evolution to optimize antibody recognition for catalysis

Enzymes have evolved their ability to use binding energies for catalysis by increasing the affinity for the transition state of a reaction and decreasing the affinity for the ground state. To evolve abzymes toward higher catalytic activity, we have reconstructed an enzyme-evolutionary process in vitro. Thus, a phage-displayed combinatorial library from a hydrolytic abzyme, 6D9, generated by the conventional in vivo method with immunization of the transition-state analog (TSA), was screened against a newly devised TSA to optimize the differential affinity for the transition state relative to the ground state. The library format successfully afforded evolved variants with 6- to 20-fold increases in activity (kcat) as compared with 6D9. Structural analysis revealed an advantage of the in vitro evolution over the in vivo evolution: an induced catalytic residue in the evolved abzyme arises from double mutations in one codon, which rarely occur in somatic hypermutation in the immune response.     

Amyloid β-Mediated Zn2+ Influx into Dentate Granule Cells Transiently Induces a Short-Term Cognitive Deficit

We examined an idea that short-term cognition is transiently affected by a state of confusion in Zn²⁺ transport system due to a local increase in amyloid-β (Aβ) concentration. A single injection of Aβ (25 pmol) into the dentate gyrus affected dentate gyrus long-term potentiation (LTP) 1 h after the injection, but not 4 h after the injection. Simultaneously, 1-h memory of object recognition was affected when the training was performed 1 h after the injection, but not 4 h after the injection. Aβ-mediated impairments of LTP and memory were rescued in the presence of zinc chelators, suggesting that Zn²⁺ is involved in Aβ action. When Aβ was injected into the dentate gyrus, intracellular Zn²⁺ levels were increased only in the injected area in the dentate gyrus, suggesting that Aβ induces the influx of Zn²⁺ into cells in the injected area. When Aβ was added to hippocampal slices, Aβ did not increase intracellular Zn²⁺ levels in the dentate granule cell layer in ACSF without Zn²⁺, but in ACSF containing Zn²⁺. The increase in intracellular Zn²⁺ levels was inhibited in the presence of CaEDTA, an extracellular zinc chelator, but not in the presence of CNQX, an AMPA receptor antagonist. The present study indicates that Aβ-mediated Zn²⁺ influx into dentate granule cells, which may occur without AMPA receptor activation, transiently induces a short-term cognitive deficit. Extracellular Zn²⁺ may play a key role for transiently Aβ-induced cognition deficits.     

Functional similarities of a thermostable protein-disulfide oxidoreductase identified in the archaeon <Emphasis Type="Italic">Pyrococcus horikoshii</Emphasis> to bacterial DsbA enzymes

We have isolated and characterized a gene for a putative protein-disulfide oxidoreductase (phdsb) in the archaeon Pyrococcus horikoshii. The open reading frame of phdsb encodes a protein of 170 amino acids with an NH₂-terminal extension similar to the bacterial signal peptides. The putative mature region of PhDsb includes a sequence motif, Cys-Pro-His-Cys (CPHC), that is conserved in members of the bacterial DsbA family, but otherwise the archaeal and bacterial sequences do not show substantial similarity. A recombinant protein corresponding to the predicted mature form of PhDsb behaved as a monomer and manifested oxidoreductase activities in vitro similar to those of DsbA of Escherichia coli. The catalytic activity of PhDsb was thermostable and was shown by mutation analysis to depend on the NH₂-terminal cysteine residue of the CPHC motif. Thus, in spite of their low overall sequence similarities, DsbA-like proteins of archaea and bacteria appear to be highly similar in terms of function.     

Population dynamics,productivity and biomass allocation ofZizania latifolia in an aquatic-terrestrial ecotone

The population and production ecology of aZizania latifolia stand at a sheltered shore of the Hitachi-Tone River were investigated. Shoot emergence was observed twice a year; the fist was a synchronized shoot emergence in April and the second was from August to October. Aboveground biomass was mostly occupied by leaves and peaked at 1500 g dry weight m⁻² in August. The belowground biomass also reached its peak, 750 g dry weight m⁻², in August. The secondary shoots were small in spite of their high density. Leaves were produced continuously throughout the season. The leaf life span was as short as 55.6 days for cohorts that emerged from May through to September. Total annual net production ofZ. latifolia could be more than 3400 g dry weight m⁻². Shoot clusters of several centimeters were observed in April. The following self-thinning caused a regular distribution of the remaining shoots in August. Most shoots produced in August to October were found near a shoot persisting since April. They showed more concentrated distribution than shoots in April. A large biomass allocation to leaves and the ability to produce many clump shoots during the late growing period may facilitate dominance ofZ. latifolia in relatively sheltered sites.     

Nuclear marginalization of host cell chromatin associated with expansion of two discrete virus-induced subnuclear compartments during baculovirus infection

Chromatin structure is strictly regulated during the cell cycle. DNA viruses occasionally disturb the spatial organization of the host cell chromatin due to formation of the viral DNA replication compartment. To examine chromatin behavior in baculovirus-infected cells, we constructed recombinant plasmids expressing fluorescent protein-tagged histone H4 molecules and visualized the intracellular localization of chromatin by their transient expression in live infected cells. Similar to other DNA viruses, the baculovirus Bombyx mori nucleopolyhedrovirus induced marginal relocation of chromatin within the nuclei of BmN cells, simultaneously with expansion of the viral DNA replication compartment, the virogenic stroma (VS). In the late stage of infection, however, the peristromal region (PR), another virus-induced subnuclear compartment, was also excluded from the chromatin-localizing area. Provided that late-gene products such as PR proteins (e.g., envelope proteins of the occlusion-derived virus) were expressed, blockage of viral DNA synthesis failed to inhibit chromatin relocation, despite abrogation of VS expansion. Instead, chromatin became marginalized concomitantly with PR expansion, suggesting that the PR contributes directly to chromatin replacement. In addition, chromatin was excluded from relatively large subnuclear structures that were induced in uninfected cells by cotransfection with four baculovirus genes, ie1, lef3, p143, and hr. Omission of any of the four genes, however, failed to result in formation of the large structures or chromatin exclusion. This correlation between compartmentalization and chromatin exclusion suggests the possibility that a chromatin-exclusive property of viral molecules, at least in part, supports nuclear compartmentalization of virus-infected cells.     

Akt is an endogenous inhibitor toward tumor necrosis factor-related apoptosis inducing ligand-mediated apoptosis in rheumatoid synovial cells

Akt is known to be activated in the rheumatoid synovial tissues. We examined here functional role of Akt during tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis in rheumatoid synovial cells. Rheumatoid synovial cells in vitro were rapidly committed to apoptosis in response to TRAIL in mitochondria-dependent manner whereas Akt and extracellular signal-regulated kinase (ERK) were also phosphorylated. TRAIL-mediated apoptosis in synovial cells was significantly increased through inactivation of Akt by LY294002, however, that process was not so changed by adding ERK inhibitor, PD98059. Platelet-derived growth factor (PDGF) clearly phosphorylated both Akt and ERK in synovial cells, and PDGF pretreatment markedly suppressed TRAIL-mediated synovial cell apoptosis. The use of not PD98059 but LY294002 abrogated PDGF-mediated inhibitory effect toward TRAIL-induced apoptosis in synovial cells. The above protective effect of Akt was confirmed by the use of short interfering RNA (siRNA)-directed inhibition of Akt. Our data suggest that Akt is an endogenous inhibitor during TRAIL-mediated synovial cell apoptotic pathway, which may explain that synovial cells in situ of the rheumatoid synovial tissues are resistant toward apoptotic cell death in spite of death receptor expression.