Suppressive Effects of Docosahexaenoic Acid Intake on Increased Seizure Susceptibility after Growth Due to Febrile Seizures in Infancy.

Febrile seizures are seizures accompanied by a fever and frequently occur in children six months to five years of age. Febrile seizures are classified as simple or complex, and complex febrile seizures increase the risk of temporal lobe epilepsy after growth. Therefore, it is important to interfere with epileptogenesis after febrile seizures to prevent post-growth epilepsy. The present study challenged nutritional intervention using docosahexaenoic acid (DHA). Febrile seizures were induced in mice at the age of 10 d using a heat chamber, and seizure sensitivity was examined using pentylenetetrazol (PTZ) administration after growth. PTZ increased the seizure score and shortened the latency in the complex febrile seizure group compared to the control, hyperthermia and simple febrile seizure groups. Mice in the complex febrile seizure group showed abnormal electroencephalograms pre- and post-PTZ administration. Therefore, seizure susceptibility increases the episodes of complex febrile seizures. DHA supplementation after febrile seizures clearly suppressed the increased seizure susceptibility due to complex febrile seizures experienced in infancy. DHA also attenuated microglial activation after complex febrile seizures. Taken together, DHA suppressed microglial activation following complex febrile seizures, which may contribute to protecting the brain from post-growth seizures. The intake of DHA in infancy may protect children from high fever-induced developmental abnormalities.

Maternal intake of docosahexaenoic acid decreased febrile seizure sensitivity by increasing estrogen synthesis in offspring.

Febrile seizures, which are convulsion in children, are caused by an abrupt increase in body temperature. They are sometimes recurrent, and the more seizures are triggered, the higher the risk of epilepsy and psychiatric disorders increase after growing up. Prevention of febrile seizure is considered to be one of the effective countermeasures in protecting the future health of children; however, pharmacological prevention in the developmental stage is not realistic from the health aspects of the offspring. Docosahexaenoic acid (DHA) is an important nutrient especially during pregnancy and childhood and is reported to suppress several types of epilepsy. The purpose of this study was to examine the effect of DHA intake during pregnancy and infancy on febrile seizures in mice. We used a heat chamber for febrile seizure induction in offspring at the age of from 10 to 11 days old. Intake of DHA during pregnancy and infancy significantly increased the amount of DHA in the brain of offspring. Although DHA had no effect on seizure severity, DHA significantly prolonged the seizure latency and increased body temperature at which the first seizure occurred, indicating that maternal DHA intake decreases febrile seizure sensitivity. Brain estrogen levels significantly increased by DHA intake and administration of an inhibitor for cytochrome P450 aromatase, which is a rate-limiting enzyme for estrogen synthesis, clearly decreased seizure latency and body temperature at which the first seizure occurred. Taken together, DHA could reduce susceptibility to febrile seizures owing to increases in brain estrogen contents. DHA intake during pregnancy and infancy is of significance in protecting infant from seizures as well as conserving health after growth.

Word scanning in native and non-native languages: insights into reading with declined accommodation.

This study investigated the effects of declined accommodation on reading performance in non-native and native languages. Eighteen native Japanese speakers participated: eight presbyopes and ten non-presbyopes. In the experiment, participants were asked to scan, or sequentially read six-word items presented in two-line texts, identify a non-word target as quickly as possible, and indicate its location. In addition to the participant type (presbyopes/non-presbyopes) and language of the reading material (Japanese/English), viewing distance (35 cm/70 cm) and contrast (18%/100%) were manipulated. The results showed that the presbyopes exhibited worse reading performance than the non-presbyopes at closer distances irrespective of the language. Notably, the inferiority of the presbyopes' reading performance was more pronounced when they read in a non-native language than in their native language. It should be noted that differences in reading performance between the presbyopes and non-presbyopes were subtle for high-contrast words at longer viewing distances, indicating that age- or cohort-related perceptual, motor, and cognitive differences were almost negligible, but accommodation mattered. These results suggest that the effect of accommodation decline is influenced by the language of the reading material.

Pepper veinal mottle virus in Japan is closely related to isolates from other Asian countries, but more distantly to most of those from Africa.

Pepper veinal mottle virus (PVMV) is known to infect chilli pepper and belongs to the Chilli veinal mottle virus phylogroup of potyviruses. PVMV has recently appeared in Japan. In this study, we report six complete genomic sequences of PVMV isolates from chilli pepper (i.e. Capsicum annuum) in Okinawa Islands in Japan, and we determined the evolutionary relationships between Japanese isolates and the isolates reported earlier from African and Asian countries. Complete genomic sequences of the six Japanese PVMV isolates were 9760 nucleotides in length, excluding the nucleotide primer sequences used for amplifying 5' end of the genomes. The major findings of this study are as follows: (1) all the Japanese isolates of PVMV have similar biological and molecular characteristics, indicating the presence of only one population in Japan; (2) there are at least three major phylogenetic groups of PVMV worldwide; (3) PVMV probably originated in East Africa; and (4) all the Asian isolates are closely related to the Ghanaian isolate.

DNA base excision repair and nucleotide excision repair synergistically contribute to survival of stationary-phase cells of the fission yeast Schizosaccharomyces pombe.

Defects of genome maintenance may causally contribute to aging. In general, base excision repair (BER) is involved in the repair of subtle base lesions and AP sites, and bulky helix-distorting lesions are restored by nucleotide excision repair (NER). Here, we measured the chronological lifespan (CLS) of BER- and NER-deficient mutants of the fission yeast Schizosaccharomyces pombe, and observed the aging process of cells. The CLS of the nth1 (gene for DNA glycosylase/AP lyase) mutant and the rad16 (a homolog of human XPF) mutant were slightly shorter than that of the wild-type (WT) strain. However, survival of the nth1Δ rad16Δ double mutant was significantly reduced after entry into the stationary phase. Deletion of rad16 in an AP endonuclease mutant apn2Δ also accelerated chronological aging. These results indicate that BER and NER synergistically contribute to genome maintenance in non-dividing cells. Reactive oxygen species (ROS) accumulated in cells during the stationary phase, and nth1Δ rad16Δ cells produced more ROS than WT cells. High mutation frequencies and nuclear DNA fragmentation were observed in nth1Δ rad16Δ stationary-phase cells concurrent with apoptotic-like cell death. Calorie restriction significantly reduced the level of ROS in the stationary phase and extended the CLS of nth1Δ rad16Δ cells. Therefore, ROS production critically affects the survival of the DNA repair mutant during chronological aging.

Comparison of the efficacy of three topical antiseptic solutions for the prevention of catheter colonization: a multicenter randomized controlled study.

BACKGROUND: To compare the efficacy of three antiseptic solutions [0.5%, and 1.0% alcohol/chlorhexidine gluconate (CHG), and 10% aqueous povidone-iodine (PVI)] for the prevention of intravascular catheter colonization, we conducted a randomized controlled trial in patients from 16 intensive care units in Japan. METHODS: Adult patients undergoing central venous or arterial catheter insertions were randomized to have one of three antiseptic solutions applied during catheter insertion and dressing changes. The primary endpoint was the incidence of catheter colonization, and the secondary endpoint was the incidence of catheter-related bloodstream infections (CRBSI). RESULTS: Of 1132 catheters randomized, 796 (70%) were included in the full analysis set. Catheter-tip colonization incidence was 3.7, 3.9, and 10.5 events per 1000 catheter-days in 0.5% CHG, 1% CHG, and PVI groups, respectively (p = 0.03). Pairwise comparisons of catheter colonization between groups showed a significantly higher catheter colonization risk in the PVI group (0.5% CHG vs. PVI: hazard ratio, HR 0.33 [95% confidence interval, CI 0.12-0.95], p = 0.04; 1.0% CHG vs. PVI: HR 0.35 [95% CI 0.13-0.93], p = 0.04). Sensitivity analyses including all patients by multiple imputations showed consistent quantitative conclusions (0.5% CHG vs. PVI: HR 0.34, p = 0.03; 1.0% CHG vs. PVI: HR 0.35, p = 0.04). No significant differences were observed in the incidence of CRBSI between groups. CONCLUSIONS: Both 0.5% and 1.0% alcohol CHG are superior to 10% aqueous PVI for the prevention of intravascular catheter colonization. TRIAL REGISTRATION: Japanese Primary Registries Network; No.: UMIN000008725 Registered on 1 September 2012.

Mitochondrial superoxide dismutase deficiency accelerates chronological aging in the fission yeast Schizosaccharomyces pombe.

A mitochondrial superoxide dismutase (SOD2) is the first line of antioxidant defense against mitochondrial superoxide. Even though the involvement of SOD2 in lifespan has been studied extensively in several organisms, characterization of the aging process has not been performed for the sod2 mutant (sod2Δ) of a prominent model Schizosaccharomyces pombe. In this study, we measured the chronological lifespan of sod2Δ cells by their ability to survive in long-term culture. SOD2 deficiency drastically decreased cell viability in the stationary phase. The mutation frequency of nuclear DNA in sod2Δ was elevated in the stationary phase, and cellular proteins and nuclear DNA were extensively degraded, concurrent with cell death. The sod2 gene in wild-type cells could be induced by an increase in endogenous oxidative stresses, after which, SOD2 activity was substantially elevated during the stationary phase. Culture in a lower glucose concentration (calorie restriction) prominently extended the sod2Δ lifespan. Therefore, S. pombe SOD2 plays a critical role in longevity through its upregulation in the non-dividing phase.

Nuclear dynamics of topoisomerase IIß reflects its catalytic activity that is regulated by binding of RNA to the C-terminal domain.

DNA topoisomerase II (topo II) changes DNA topology by cleavage/re-ligation cycle(s) and thus contributes to various nuclear DNA transactions. It is largely unknown how the enzyme is controlled in a nuclear context. Several studies have suggested that its C-terminal domain (CTD), which is dispensable for basal relaxation activity, has some regulatory influence. In this work, we examined the impact of nuclear localization on regulation of activity in nuclei. Specifically, human cells were transfected with wild-type and mutant topo IIß tagged with EGFP. Activity attenuation experiments and nuclear localization data reveal that the endogenous activity of topo IIß is correlated with its subnuclear distribution. The enzyme shuttles between an active form in the nucleoplasm and a quiescent form in the nucleolus in a dynamic equilibrium. Mechanistically, the process involves a tethering event with RNA. Isolated RNA inhibits the catalytic activity of topo IIß in vitro through the interaction with a specific 50-residue region of the CTD (termed the CRD). Taken together, these results suggest that both the subnuclear distribution and activity regulation of topo IIß are mediated by the interplay between cellular RNA and the CRD.

Development of plants resistant to Papaya leaf distortion mosaic virus by intergeneric hybridization between Carica papaya and Vasconcellea cundinamarcensis.

In this study, we confirmed that Vasconcellea cundinamarcensis resists Papaya leaf distortion mosaic virus (PLDMV), and used it to produce intergeneric hybrids with Carica papaya. From the cross between C. papaya and V. cundinamarcensis, we obtained 147 seeds with embryos. Though C. papaya is a monoembryonic plant, multiple embryos were observed in all 147 seeds. We produced 218 plants from 28 seeds by means of embryo-rescue culture. All plants had pubescence on their petioles and stems characteristic of V. cundinamarcensis. Flow cytometry and PCR of 28 plants confirmed they were intergeneric hybrids. To evaluate virus resistance, mechanical inoculation of PLDMV was carried out. The test showed that 41 of 134 intergeneric hybrid plants showed no symptoms and were resistant. The remaining 93 hybrids showed necrotic lesions on the younger leaves than the inoculated leaves. In most of the 93 hybrids, the necrotic lesions enclosed the virus and prevented further spread. These results suggest that the intergeneric hybrids will be valuable material for PLDMV-resistant papaya breeding.

Pharmacokinetics and dosing estimation of meropenem in Japanese patients receiving continuous venovenous hemodialysis.

The pharmacokinetics of meropenem have not yet been examined in Japanese patients receiving Continuous venovenous hemodialysis (CVVHD). The aim of this clinical investigation was to determine the pharmacokinetic parameters of meropenem in critically ill patients receiving CVVHD in order to estimate dosing regimens for the patient population in Japan. The values of pharmacokinetic parameters were 17.5 ± 5.6 l for V1, 1.27 ± 0.38 h(-1) for K12, 0.71 ± 0.40 h(-1) for K21 and 0.17 ± 0.02 h(-1) for K10. Based on these mean parameters (V1, K12, K21 and K10), time above MIC (T > MIC) values were estimated at different MICs using various meropenem regimens. For bacteria with a meropenem MIC of ≤ 2 µg/ml, a dosing regimen of 0.25 g every 24 h achieved more than 40% T > MIC. For a MIC of 4 µg/ml, all the regimens tested, except for 0.25 g every 24 h, achieved more than 40% T > MIC. For a MIC of 16 µg/ml, dosing regimens of 0.5 g every 8 h, 1 g every 12 h, and 1 g every 8 h achieved 40% T > MIC, reaching the pharmacokinetic-pharmacodynamic target range. This is the first study to examine the pharmacokinetics of meropenem under a CVVHD setting in Japan. The pharmacokinetic-pharmacodynamic profile of dosing regimens tested in this study will assist in selecting the appropriate meropenem regimens for patients receiving CVVHD.

[Two cases of Paragonimiasis westermani in a Chinese family diagnosed with the Ouchterlony double diffusion method].

We report two cases of Paragonimus westermani infection in a Chinese family in Japan. A 41-year-old husband and his 40-year-old wife were infected with P. westermani after consuming a homemade Chinese traditional "Drunken Crab." They were a family with two children who had lived in Japan for 19 years. The crabs were Eriocheir japonica sent from the Kyusyu area that they had pickled at home with soy sauce and Chinese liquor for 5 days. Their children did not eat any of the crabs. One month after consuming the crabs, the husband came to our outpatient clinic with fever and chest pain and his wife also presented with a persistent cough. Both patients had a high peripheral blood eosinophil count (husband:18,900/µL, wife:10,600/µL) with pulmonary effusion, nodular shadow, and pneumothorax in chest X-ray findings. Paragonimiasis was suspected from the episode of consuming the crabs. No parasite eggs were seen in their sputum and stool samples. A multiple-dot ELISA was performed with the sera to screen for parasitic infections, but the result was only weakly positive for P. westermani antigen in the husband and a slightly positive reaction in the wife. The diagnosis of P. westermani was achieved with the double diffusion Ouchterlony method using P. westermani antigen and P. miyazakii antigen. Praziquantel administration for three days improved the symptoms in both patients. The Ouchterlony method proved useful in diagnosing paragonimiasis in these cases.

A case of NDM-1-producing Acinetobacter baumannii transferred from India to Japan.

A 52-year-old male Japanese businessman with massive cerebral bleeding was transferred from India to Japan and was admitted to our hospital. Multidrug-resistant Acinetobacter baumannii was isolated from his sputum. The minimum inhibitory concentrations for this strain were as follows: imipenem, 64 µg/ml; meropenem, 32 µg/ml; ciprofloxacin, 16 µg/ml; amikacin, 16 µg/ml; aztreonam, 16 µg/ml; colistin, <1 µg/ml. This A. baumannii strain had both bla NDM-1 and bla OXA-23 by polymerase chain reaction analysis. In Japan, NDM-1-producing bacteria are extremely rare in clinical specimens. To date, three NDM-1-positive cases have been detected in Japan, and this is the first case of A. baumannii-producing NDM-1 in Japan. Our case suggests that NDM-1-producing bacteria could be introduced into our country easily. There is concern that various resistant bacteria may be transferred from epidemic countries as a result of international medical care.

Selective DNA-binding of SP120 (rat ortholog of human hnRNP U) is mediated by arginine-glycine rich domain and modulated by RNA.

A human protein heterogeneous ribonucleoprotein U (hnRNP U) also known as Scaffold attachment factor A (SAF-A) and its orthologous rat protein SP120 are abundant and multifunctional nuclear protein that directly binds to both DNA and RNA. The C-terminal region of hnRNP U enriched with arginine and glycine is essential for the interaction with RNA and the N-terminal region of SAF-A termed SAP domain has been ascribed to the DNA binding. We have reported that rat hnRNP U specifically and cooperatively binds to AT-rich DNA called nuclear scaffold/matrix-associated region (S/MAR) although its detailed mechanism remained unclear. In the present study analysis of hnRNP U deletion mutants revealed for the first time that a C-terminal domain enriched with Arg-Gly (defined here as 'RG domain') is predominantly important for the S/MAR-selective DNA binding activities. RG domain alone directly bound to S/MAR and coexistence with the SAP domain exerted a synergistic effect. The binding was inhibited by netropsin, a minor groove binder with preference to AT pairs that are enriched in S/MAR, suggesting that RG domain interacts with minor groove of S/MAR DNA. Interestingly, excess amounts of RNA attenuated the RG domain-dependent S/MAR-binding of hnRNP U. Taken together, hnRNP U may be the key element for the RNA-regulated recognition of S/MAR DNA and thus contributing to the dynamic structural changes of chromatin compartments.

Methylated histones on mitotic chromosomes promote topoisomerase IIα function for high fidelity chromosome segregation.

DNA Topoisomerase IIα (TopoIIα) decatenates sister chromatids, allowing their segregation in mitosis. Without the TopoIIα Strand Passage Reaction (SPR), chromosome bridges and ultra-fine DNA bridges (UFBs) arise in anaphase. The TopoIIα C-terminal domain is dispensable for the SPR in vitro but essential for mitotic functions in vivo. Here, we present evidence that the Chromatin Tether (ChT) within the CTD interacts with specific methylated nucleosomes and is crucial for high-fidelity chromosome segregation. Mutation of individual αChT residues disrupts αChT-nucleosome interaction, induces loss of segregation fidelity and reduces association of TopoIIα with chromosomes. Specific methyltransferase inhibitors reducing histone H3 or H4 methylation decreased TopoIIα at centromeres and increased segregation errors. Methyltransferase inhibition did not further increase aberrant anaphases in the ChT mutants, indicating a functional connection. The evidence reveals novel cellular regulation whereby TopoIIα specifically interacts with methylated nucleosomes via the αChT to ensure high-fidelity chromosome segregation.

Comparison of free and anonymous testing for HIV and sexually transmitted infections between the University Hospital and Health Center.

We gathered data regarding age, sex, and positivity rates for human immunodeficiency virus (HIV), syphilis, gonococcus, and chlamydia from individuals who underwent free and anonymous sexually transmitted infection (STI) testing conducted at the Jikei University School of Medicine Hospital (our hospital). These data were compared to results of subjects who underwent similar testing at the Minato Health Center and several private facilities of urologists and gynecologists belonging to the Minato Ward Medical Association. The positivity rate of chlamydia was found to be high in female subjects, particularly at the Minato Health Center, with 15 of 194 subjects (7.73 %) testing positive. In our hospital, we only detected 3 of 133 subjects (2.26 %) who were gonococcus positive. On the other hand, at the doctor's facilities, 10 of 188 male subjects (5.32 %) were syphilis positive, and 8 of 185 male subjects (4.32 %) were chlamydia positive, thus showing high positivity rates for both infections. At our hospital, 1 of 231 subjects was positive for gonococcus and 4 of 230 subjects (1.74 %) were positive for chlamydia, thus showing lower positivity rates for both infections. HIV-positive subjects were, however, only confirmed at our hospital, with 2 of 243 subjects (0.82 %) being positive. We were able to diagnose infected patients using free and anonymous STI testing at hospitals, and the same as at doctors' facilities. This result suggests that the hospitals that have many opportunities to diagnose HIV patients may become potential candidates for the development of new consultation facilities, establishment of testing facilities, and enhancement of consultation processes that include STI prevention.

Regulation of DNA Topoisomerase IIbeta through RNA-dependent association with heterogeneous nuclear ribonucleoprotein U (hnRNP U).

Recent studies suggest that DNA topoisomerase IIbeta (topo IIbeta) is involved in transcriptional activation of certain genes, which assumes accurate targeting of the enzyme to its action site. The target selection may be achieved by cooperation with unknown regulatory factors. To seek out such factors, we looked for proteins associated with the enzyme in differentiating cerebellar neurons. Antibody against topo IIbeta co-precipitated RNA-binding proteins including PSF, NonO/p54nrb, as well as hnRNP U/SAF-A/SP120. Reconstitution experiments with tag-purified proteins showed that topo IIbeta associates stoichiometrically with SP120 in the presence of RNA that was co-purified with SP120. The most effective RNA species for the complex formation was a subset of cellular polyadenylated RNAs. The C-terminal 187-residue domain of SP120 was necessary and sufficient for the association with both topo IIbeta and the endogenous RNA. The RNA isolated from the tag-purified SP120 inhibited the relaxation of supercoiled DNA by topo IIbeta. When the enzyme associates with SP120, however, the inhibition was abolished and the catalytic property was modulated to more processive mode, which may prolong its residence time at the genomic target site. Furthermore, the presence of SP120 was required for the stable expression of topo IIbeta in vivo. Thus, SP120 regulates the enzyme in dual ways.

The genome sequence of pepper vein yellows virus (family Luteoviridae, genus Polerovirus).

The complete genome of pepper vein yellows virus (PeVYV) was sequenced using random amplification of RNA samples isolated from vector insects (Aphis gossypii) that had been given access to PeVYV-infected plants. The PeVYV genome consisted of 6244 nucleotides and had a genomic organization characteristic of members of the genus Polerovirus. PeVYV had highest amino acid sequence identities in ORF0 to ORF3 (75.9 - 91.9%) with tobacco vein distorting polerovirus, with which it was only 25.1% identical in ORF5. These sequence comparisons and previously studied biological properties indicate that PeVYV is a distinctly different virus and belongs to a new species of the genus Polerovirus.

Regulation of catalytic activity and nucleolar localization of rat DNA topoisomerase IIα through its C-terminal domain.

Type II DNA topoisomerase (topo II) catalyzes double-stranded DNA cleavage and re-ligation, thus solving problems in DNA topology. Vertebrates have two isozymes (α and ß). Recently, the C-terminal regulatory domain (CRD), which regulates catalytic activity and subnuclear localization by associating with RNA, was identified within the C-terminal domain (CTD) of rat topo IIß. In contrast, it is unclear whether a ß CRD-like domain is present in the CTD of topo IIα. In this study, we aimed to identify an RNA-mediated regulatory domain in the rat topo IIα CTD. First, we exchanged the CTDs of rat topo IIα (amino acids 1,192-1,528) and ß (1,201-1,614) and examined the two chimeras' in vitro catalytic activities. Interestingly, the relaxation activities of topo IIα WT enzyme and both of the CTD-swapped mutants were inhibited in the presence of isolated cellular RNA, suggesting that the α CTD is involved in the RNA-mediated regulation of catalytic activity in topo IIα. The results of on-bead assays using a CTD-deleted mutant of rat topo IIα indicated that the RNA-mediated inhibition of the relaxation activity was caused by an interaction between the α CTD and RNA. Further, to identify the domain within the CTD that is associated with subnuclear localization of rat topo IIα, we transiently expressed EGFP-tagged CTD deletion mutants in human cells. The data indicated that the 1,192-1,289 region of rat topo IIα was required for targeting the enzyme to nucleoli. Finally, a relaxation assay using 1-1,289 and Δ1,192-1,289 truncated mutants indicated that the 1,192-1,289 region is involved in RNA-mediated inhibition. These results indicated that the CTD of rat topo IIα, containing the 1,192-1,289 region, is involved in the regulation of catalytic activity by associating with RNA, as well as in the localization to nucleoli in interphase cells.

Real-Time PCR Assay for the Diagnosis and Quantification of Co-infections by Diaporthe batatas and Diaporthe destruens in Sweet Potato.

Foot rot disease caused by Diaporthe destruens (formerly Plenodomus destruens) has become a major concern for the production of sweet potato [Ipomoea batatas (L.) Lam.] in Japan. A related fungus Diaporthe batatas, which causes dry rot disease of sweet potato, is native and is widespread in fields in Japan. The similar characteristics of these two pathogens pose a challenge for conventional disease diagnosis. Currently, there are no effective molecular measures for identifying and distinguishing D. destruens and D. batatas. Here, we demonstrate a real-time PCR assay that distinguishes and quantifies D. batatas and D. destruens from co-infected sweet potato. The assay was performed with various simulated DNA combinations of D. batatas and D. destruens ranging from 1:1 to 1:100000. The assay was also used with the ratios of D. batatas: D. destruens: sweet potato DNA ranging from 1:1:1 to 1:1:100000. These assays produced a specific amplification product for each of the pathogens, and quantified the fungal biomass over the entire range tested without detecting false positives. The assay was validated by using infected sweet potato collected from various fields; it showed sufficient sensitivity and specificity to quantify and distinguish D. batatas and D. destruens from these field samples. Thus, our real-time PCR assay would be a useful tool for diagnosis of D. batatas and D. destruens and is expected to provide the foundation for the design of integrated disease management strategies for foot rot disease in sweet potato.

DNA binding activity of the proximal C-terminal domain of rat DNA topoisomerase IIß is involved in ICRF-193-induced closed-clamp formation.

DNA topoisomerase II (topo II) is an essential enzyme that regulates DNA topology by DNA cleavage and re-ligation. In vertebrates, there are two isozymes, α and ß. The C-terminal domain (CTD) of the isozymes, which shows a low degree of sequence homology between α and ß, is involved in each isozyme-specific intracellular behavior. The CTD of topo IIß is supposedly involved in topo II regulation. Topo IIß is maintained in an inactive state in the nucleoli by the binding of RNA to the 50-residue region termed C-terminal regulatory domain (CRD) present in the CTD. Although in vitro biochemical analysis indicates that the CTD of topo IIß has DNA binding activity, it is unclear whether CTD influences catalytic reaction in the nucleoplasm. Here, we show that the proximal CTD (hereafter referred to as pCTD) of rat topo IIß, including the CRD, is involved in the catalytic reaction in the nucleoplasm. We identified the pCTD as a domain with DNA binding activity by in vitro catenation assay and electrophoretic mobility shift assay. Fluorescence recovery after photo-bleaching (FRAP) analysis of pCTD-lacking mutant (ΔpCTD) showed higher mobility in nucleoplasm than that of the wild-type enzyme, indicating that the pCTD also affected the nuclear dynamics of topo IIß. ICRF-193, one of the topo II catalytic inhibitors, induces the formation of closed-clamp intermediates of topo II. Treatment of ΔpCTD with ICRF-193 significantly decreased the efficiency of closed-clamp formation. Altogether, our data indicate that the binding of topo IIß to DNA through the pCTD is required for the catalytic reaction in the nucleoplasm.