MCMBP promotes the assembly of the MCM2-7 hetero-hexamer to ensure robust DNA replication in human cells.

The MCM2-7 hetero-hexamer is the replicative DNA helicase that plays a central role in eukaryotic DNA replication. In proliferating cells, the expression level of the MCM2-7 hexamer is kept high, which safeguards the integrity of the genome. However, how the MCM2-7 hexamer is assembled in living cells remains unknown. Here, we revealed that the MCM-binding protein (MCMBP) plays a critical role in the assembly of this hexamer in human cells. MCMBP associates with MCM3 which is essential for maintaining the level of the MCM2-7 hexamer. Acute depletion of MCMBP demonstrated that it contributes to MCM2-7 assembly using nascent MCM3. Cells depleted of MCMBP gradually ceased to proliferate because of reduced replication licensing. Under this condition, p53-positive cells exhibited arrest in the G1 phase, whereas p53-null cells entered the S phase and lost their viability because of the accumulation of DNA damage, suggesting that MCMBP is a potential target for killing p53-deficient cancers.

The RIF1-long splice variant promotes G1 phase 53BP1 nuclear bodies to protect against replication stress.

Human cells lacking RIF1 are highly sensitive to replication inhibitors, but the reasons for this sensitivity have been enigmatic. Here, we show that RIF1 must be present both during replication stress and in the ensuing recovery period to promote cell survival. Of two isoforms produced by alternative splicing, we find that RIF1-Long alone can protect cells against replication inhibition, but RIF1-Short is incapable of mediating protection. Consistent with this isoform-specific role, RIF1-Long is required to promote the formation of the 53BP1 nuclear bodies that protect unrepaired damage sites in the G1 phase following replication stress. Overall, our observations show that RIF1 is needed at several cell cycle stages after replication insult, with the RIF1-Long isoform playing a specific role during the ensuing G1 phase in damage site protection.

The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice.

Protein knockdown using the auxin-inducible degron (AID) technology is useful to study protein function in living cells because it induces rapid depletion, which makes it possible to observe an immediate phenotype. However, the current AID system has two major drawbacks: leaky degradation and the requirement for a high dose of auxin. These negative features make it difficult to control precisely the expression level of a protein of interest in living cells and to apply this method to mice. Here, we overcome these problems by taking advantage of a bump-and-hole approach to establish the AID version 2 (AID2) system. AID2, which employs an OsTIR1(F74G) mutant and a ligand, 5-Ph-IAA, shows no detectable leaky degradation, requires a 670-times lower ligand concentration, and achieves even quicker degradation than the conventional AID. We demonstrate successful generation of human cell mutants for genes that were previously difficult to deal with, and show that AID2 achieves rapid target depletion not only in yeast and mammalian cells, but also in mice.

RIF1 controls replication initiation and homologous recombination repair in a radiation dose-dependent manner.

RIF1 controls both DNA replication timing and the DNA double-strand break (DSB) repair pathway to maintain genome integrity. However, it remains unclear how RIF1 links these two processes following exposure to ionizing radiation (IR). Here, we show that inhibition of homologous recombination repair (HRR) by RIF1 occurs in a dose-dependent manner and is controlled via DNA replication. RIF1 inhibits both DNA end resection and RAD51 accumulation after exposure to high doses of IR. Contrastingly, HRR inhibition by RIF1 is antagonized by BRCA1 after a low-dose IR exposure. At high IR doses, RIF1 suppresses replication initiation by dephosphorylating MCM helicase. Notably, the dephosphorylation of MCM helicase inhibits both DNA end resection and HRR, even without RIF1. Thus, our data show the importance of active DNA replication for HRR and suggest a common suppression mechanism for DNA replication and HRR at high IR doses, both of which are controlled by RIF1.This article has an associated First Person interview with the first author of the paper.

Control of homologous recombination by the HROB-MCM8-MCM9 pathway.

DNA repair by homologous recombination (HR) is essential for genomic integrity, tumor suppression, and the formation of gametes. HR uses DNA synthesis to repair lesions such as DNA double-strand breaks and stalled DNA replication forks, but despite having a good understanding of the steps leading to homology search and strand invasion, we know much less of the mechanisms that establish recombination-associated DNA polymerization. Here, we report that C17orf53/HROB is an OB-fold-containing factor involved in HR that acts by recruiting the MCM8-MCM9 helicase to sites of DNA damage to promote DNA synthesis. Mice with targeted mutations in Hrob are infertile due to depletion of germ cells and display phenotypes consistent with a prophase I meiotic arrest. The HROB-MCM8-MCM9 pathway acts redundantly with the HELQ helicase, and cells lacking both HROB and HELQ have severely impaired HR, suggesting that they underpin two major routes for the completion of HR downstream from RAD51. The function of HROB in HR is reminiscent of that of gp59, which acts as the replicative helicase loader during bacteriophage T4 recombination-dependent DNA replication. We therefore propose that the loading of MCM8-MCM9 by HROB may similarly be a key step in the establishment of mammalian recombination-associated DNA synthesis.

Analysis of the Stay Time of Patients in Gunma University Heavy Ion Medical Center (GHMC) Using RFID Technology.

We observed the stay time of patients and staff in Gunma University Heavy Ion Medical Center. The stay time of patients with the prostatic cancer and the facing time with radiotherapy technicians in treatment rooms were significantly reduced as times goes by. This decreasing in time has an implication in scheduling algorithm development: for patients. RFID technology can be a potential method to track both staff and patients and thereby to assess the resource utilization efficiency.

Chromatin modification and NBS1: their relationship in DNA double-strand break repair.

The importance of chromatin modification, including histone modification and chromatin remodeling, for DNA double-strand break (DSB) repair, as well as transcription and replication, has been elucidated. Phosphorylation of H2AX to γ-H2AX is one of the first responses following DSB detection, and this histone modification is important for the DSB damage response by triggering several events, including the accumulation of DNA damage response-related proteins and subsequent homologous recombination (HR) repair. The roles of other histone modifications such as acetylation, methylation and ubiquitination have also been recently clarified, particularly in the context of HR repair. NBS1 is a multifunctional protein that is involved in various DNA damage responses. Its recently identified binding partner RNF20 is an E3 ubiquitin ligase that facilitates the monoubiquitination of histone H2B, a process that is crucial for recruitment of the chromatin remodeler SNF2h to DSB damage sites. Evidence suggests that SNF2h functions in HR repair, probably through regulation of end-resection. Moreover, several recent reports have indicated that SNF2h can function in HR repair pathways as a histone remodeler and that other known histone remodelers can also participate in DSB damage responses. On the other hand, information about the roles of such chromatin modifications and NBS1 in non-homologous end joining (NHEJ) repair of DSBs and stalled fork-related damage responses is very limited; therefore, these aspects and processes need to be further studied to advance our understanding of the mechanisms and molecular players involved.

Functional Role of NBS1 in Radiation Damage Response and Translesion DNA Synthesis.

Nijmegen breakage syndrome (NBS) is a recessive genetic disorder characterized by increased sensitivity to ionizing radiation (IR) and a high frequency of malignancies. NBS1, a product of the mutated gene in NBS, contains several protein interaction domains in the N-terminus and C-terminus. The C-terminus of NBS1 is essential for interactions with MRE11, a homologous recombination repair nuclease, and ATM, a key player in signal transduction after the generation of DNA double-strand breaks (DSBs), which is induced by IR. Moreover, NBS1 regulates chromatin remodeling during DSB repair by histone H2B ubiquitination through binding to RNF20 at the C-terminus. Thus, NBS1 is considered as the first protein to be recruited to DSB sites, wherein it acts as a sensor or mediator of DSB damage responses. In addition to DSB response, we showed that NBS1 initiates Polη-dependent translesion DNA synthesis by recruiting RAD18 through its binding at the NBS1 C-terminus after UV exposure, and it also functions after the generation of interstrand crosslink DNA damage. Thus, NBS1 has multifunctional roles in response to DNA damage from a variety of genotoxic agents, including IR.

Increased oxidative stress in AOA3 cells disturbs ATM-dependent DNA damage responses.

Ataxia telangiectasia (AT) is caused by a mutation in the ataxia-telangiectasia-mutated (ATM) gene; the condition is associated with hyper-radiosensitivity, abnormal cell-cycle checkpoints, and genomic instability. AT patients also show cerebellar ataxia, possibly due to reactive oxygen species (ROS) sensitivity in neural cells. The ATM protein is a key regulator of the DNA damage response. Recently, several AT-like disorders have been reported. The genes responsible for them are predicted to encode proteins that interact with ATM in the DNA-damage response. Ataxia with oculomotor apraxia types 1-3 (AOA1, 2, and 3) result in a neurodegenerative and cellular phenotype similar to AT; however, the basis of this phenotypic similarity is unclear. Here, we show that the cells of AOA3 patients display aberrant ATM-dependent phosphorylation and apoptosis following γ-irradiation. The ATM-dependent response to H2O2 treatment was abrogated in AOA3 cells. Furthermore, AOA3 cells had reduced ATM activity. Our results suggest that the attenuated ATM-related response is caused by an increase in endogenous ROS in AOA3 cells. Pretreatment of cells with pyocyanin, which induces endogenous ROS production, abolished the ATM-dependent response. Moreover, AOA3 cells had decreased homologous recombination (HR) activity, and pyocyanin pretreatment reduced HR activity in HeLa cells. These results indicate that excess endogenous ROS represses the ATM-dependent cellular response and HR repair in AOA3 cells. Since the ATM-dependent cell-cycle checkpoint is an important block to carcinogenesis, such inactivation of ATM may lead to tumorigenesis as well as neurodegeneration.

Histone chaperone FACT regulates homologous recombination by chromatin remodeling through interaction with RNF20.

The E3 ubiquitin ligase RNF20 regulates chromatin structure through ubiquitylation of histone H2B, so that early homologous recombination repair (HRR) proteins can access the DNA in eukaryotes during repair. However, it remains unresolved how RNF20 itself approaches the DNA in the presence of chromatin structure. Here, we identified the histone chaperone FACT as a key protein in the early steps of HRR. Depletion of SUPT16H, a component of FACT, caused pronounced defects in accumulations of repair proteins and, consequently, decreased HRR activity. This led to enhanced sensitivity to ionizing radiation (IR) and mitomycin-C in a fashion similar to RNF20-deficient cells, indicating that SUPT16H is essential for RNF20-mediated pathway. Indeed, SUPT16H directly bound to RNF20 in vivo, and mutation at the RING-finger domain in RNF20 abolished its interaction and accumulation, as well as that of RAD51 and BRCA1, at sites of DNA double-strand breaks (DSBs), whereas the localization of SUPT16H remained intact. Interestingly, PAF1, which has been implicated in transcription as a mediator of FACT and RNF20 association, was dispensable for DNA-damage-induced interaction of RNF20 with SUPT16H. Furthermore, depletion of SUPT16H caused pronounced defects in RNF20-mediated H2B ubiquitylation and thereby, impaired accumulation of the chromatin remodeling factor SNF2h. Consistent with this observation, the defective phenotypes of SUPT16H were effectively counteracted by enforced nucleosome relaxation. Taken together, our results indicate a primary role of FACT in RNF20 recruitment and the resulting chromatin remodeling for initiation of HRR.

Prospective multicenter case-control study of telemedicine for home medical care.

We evaluated the safety and efficacy of the combined use of remote medical services for patients with stroke, cancer, neuromuscular diseases, and other conditions, who are being cared for at home. This study was conducted as a part of a multicenter joint trial supported by the Health and Labour Sciences Research Grant for the 'Comparative Study of the home telemedicine in Japan'.

[Attenuation of phrenic nerve palsy associated with brachial plexus block by modified supra costal approach under fluoroscopic guidance].

BACKGROUND: Brachial plexus block (BPB) frequently accompanies phrenic nerve palsy (PNP). METHODS: Thirty six patients scheduled for upper-limb surgery were allocated to 2 groups; 14 patients undergoing BPB with the supra costal approach (i. e. placing the needle-tip at the middle of the 1st lib), and 22 patients undergoing BPB with the modified supra costal approach (i. e. placing the needle-tip in the visceral or dorsal area of the 1st lib). We evaluated analgesic effects of the block and changes in forced vital capacity (FVC). RESULTS: BPB with both approaches provided sufficient analgesia. After BPB with both approaches, a significant reductions in FVC was observed; however, the reduction after BPB with the modified supra costal approach was significantly lower than that with the supra costal approach. CONCLUSIONS: These results suggest that BPB with the modified supra costal approach provides sufficient analgesia with a significantly lower degree of PNP. We suppose that distribution of local anesthetics is altered by changing the location of the needle-tip on the 1st lib. Amounts of local anesthetics distributing around the phrenic nerve can be reduced by the modified supra costal approach, leading to the significantly less reduction in FVC after BPB.

Effects of raloxifene on brachial arterial endothelial function, carotid wall thickness, and arterial stiffness in osteoporotic postmenopausal women.

The beneficial effects of raloxifene, a selective estrogen receptor modulator, on cardiovascular risks and events have been investigated. Brachial arterial flow-mediated vasodilatation (FMD), carotid intima-media thickness (IMT), and pulse wave velocity (PWV) have been widely used in clinical settings as surrogate markers of atherosclerosis. This study investigated the simultaneous effects of raloxifene on brachial arterial FMD, carotid IMT, and PWV in osteoporotic postmenopausal women. A total of 31 postmenopausal women with osteoporosis or osteopenia were divided into 2 groups: a raloxifene group (n = 15; mean age +/- SD, 66.1 +/- 8.2 years) was treated with raloxifene hydrochloride (60 mg/day) orally for 12 months, and an untreated control group (n = 16; 64.1 +/- 7.8 years). Brachial arterial FMD, carotid IMT, and brachial-ankle PWV (baPWV) were measured at baseline and at 12 months after the start of the study. The brachial arterial FMD increased significantly, from 4.5 +/- 1.8% to 9.2 +/- 3.0%, in the raloxifene group (P < 0.01) but did not change in the control group. Nitroglycerin-induced vasodilatation did not change in either group. The carotid IMT decreased significantly, from 0.82 +/- 0.15 mm to 0.72 +/- 0.11 mm, in the raloxifene group (P < 0.01) but did not change in the control group. The baPWV did not change in either group. In conclusion, raloxifene may have beneficial effects on brachial arterial endothelial function and carotid wall thickness in osteoporotic postmenopausal women.

Pueraria mirifica phytoestrogens improve dyslipidemia in postmenopausal women probably by activating estrogen receptor subtypes.

Impaired lipid metabolism is an important health problem in postmenopausal women with insufficient estrogens, because dyslipidemia is a risk factor for development of atherosclerosis and the incidence of cardiovascular disease markedly increases after menopause. Pueraria mirifica (PM), a Thai herb, has been noticed as a source of phytoestrogens, estrogen-mimicking plant compounds. However, the clinical effects of PM on lipid metabolism and the underlying molecular mechanisms remain undetermined. Therefore, we examined the effects of PM on serum lipid parameters in a randomized, double-blind, placebo-controlled clinical trial. Nineteen postmenopausal women were randomly assigned to receive oral administration of PM powder or placebo. After 2 months of treatment, the PM group showed a significant increase in serum concentrations of high-density lipoprotein (HDL) cholesterol and apolipoprotein (apo) A-1 (34% and 40%, respectively), and a significant decrease in low-density lipoprotein (LDL) cholesterol and apo B (17% and 9%, respectively), compared with baseline measurements. Moreover, significant decreases were observed in the ratios of LDL cholesterol to HDL cholesterol (37%) and apo B to apo A-1 (35%). Next, we determined the effects of PM phytoestrogens on the activation of estrogen receptor (ER)-mediated transactivation by transient expression assays of a reporter gene in cultured cells. Among PM phytoestrogens, miroestrol and coumestrol enhanced both ERalpha- and ERbeta-mediated transactivation, whereas other phytoestrogens, including daidzein and genistein, preferentially enhanced ERbeta-mediated transactivation. In conclusion, PM has a beneficial effect on lipid metabolism in postmenopausal women, which may result from the activation of gene transcription through selective binding of phytoestrogens to ERalpha and ERbeta.

[Multiple linear regression analysis for predictability of the extent of spinal anesthesia by plain bupivacaine].

BACKGROUND: Predictability of the extent of spinal anesthesia by plain bupivacaine has been controversial. METHODS: Two hundred and twenty-eight patients undergoing elective surgery with spinal anesthesia were enrolled in this retrospective study. Using gender, age, height, body mass index (BMI), chosen spinal interspace for spinal tap (L2-3 or L3-4), and dose of plain bupivacaine as independent variables, we performed stepwise multiple linear regression analysis to examine predictability of the extent of sensory blockade produced by spinal anesthesia using plain bupivacaine. RESULTS: Following equation was obtained. Extent of sensory blockade = 14.9 + (male : 0.540, female: -0.540) -0.0774 x height + 0.124 x BMI + (L2-3 : 0.345, L3-4: -0.345): r2 = 0.0604. P values of gender, height and BMI were less than 0.05; however, r2 of each variable was very low. CONCLUSIONS: Results of this retrospective study imply the unpredictability of the extent of spinal anesthesia produced by plain bupivacaine.

[Severe hypotension as a complication of intramyometrial injection of vasopressin: a case report].

A thirty-year-old woman was scheduled for laparoscopic myomectomy. After insertion of an epidural catheter at the L4-5 interspace, general anesthesia was induced with thiopental 250 mg followed by vecuronium 8mg intravenously to facilitate tracheal intubation. General anesthesia was maintained with sevoflurane and nitrous oxide. Just after intramyometrial injection of vasopressin, blood pressure decreased from 122/66 to 45/25 mmHg, and heart rate decreased from 52 to 45 beats x min(-1). The patient was ventilated with 100% oxygen, and we administered atropine 0.25 mg and ephedrine 16 mg intravenously. Blood pressure increased to 150/100 mmHg and heart rate increased to 135 beats x min(-1). Since electrocardiogram showed ST-segment depression and premature ventricular contraction, we administered nicorandil 3 mg followed by continuous infusion at a rate of 3 mg x hr(-1), and lidocaine 60 mg, intravenously. The ST depression and premature ventricular contraction disappeared immediately. To decrease blood pressure and heart rate, we increased inspiratory concentrations of sevoflurane and nitrous oxide and administered local anesthetics via epidural catheter, and hemodynamic parameters became gradually stable. We estimate that severe hypotension observed in this case is associated with intramyometrial injection of vasopressin. Increased blood concentration of vasopressin might cause vasoconstriction of coronary artery, increases in afterload, and/or direct myocardial depression resulting in decreased cardiac output.

Time course of systolic and diastolic blood pressure decreases during the preintubation period of anesthesia induction: modeling with a logistic function.

STUDY OBJECTIVE: To investigate whether systolic (SBP) and diastolic blood pressure (DBP) decreases during the preintubation period could be expressed as 4-parameter logistic and cubic functions giving S-shaped curves. DESIGN: Prospective, clinical study. SETTING: Operating room of a metropolitan general hospital. PATIENTS: Seven ASA physical status I and II patients scheduled for elective spinal surgery during general anesthesia. INTERVENTIONS: Anesthesia was induced with fentanyl, propofol, and vecuronium injection followed by inhalation of sevoflurane. MEASUREMENTS: The SBP and DBP data were recorded at all beats from fentanyl injection to direct laryngoscopy. The respective changes were analyzed using a logistic function: P(t) = p(L) + (q(L) - p(L))/(1 + exp{[4 m(L)/(q(L) - p(L))][k(L) - t]}) and a cubic function: P(t) = at(3) + bt(2) + ct + d, where parameter p(L) is the upper asymptote, q(L) is the lower asymptote, m(L) is the slope at the inflection point, and k(L) is the time to the inflection point and where a, b, and c are coefficients, and d are constants. Goodness of fit of the two functions was compared using a correlation coefficient and residual mean squares. Each parameter was compared with the corresponding observed data. MAIN RESULTS: Logistic correlation coefficient values for SBP and DBP decreases were larger than the cubic correlation coefficient values (0.990 [Z transformation: 2.64 +/- 0.32] vs 0.981 [Z: 2.32 +/- 0.37] and 0.977 [Z: 2.22 +/- 0.33] vs 0.967 [Z: 2.05 +/- 0.34], respectively; P < 0.05). Logistic residual mean squares values for SBP and DBP decreases were smaller than cubic residual mean squares values (20.6 vs 41.0 and 9.2 vs 13.7 mmHg(2), respectively; P < 0.05). There were significant correlations between p(L) and SBP or DBP after anesthesia induction, between q(L) and SBP or DBP before endotracheal intubation, and between k(L) and time to maximal rate of the SBP or DBP decrease (dP/dt(min)), but no significant correlation between m(L) and dP/dt(min) for SBP or DBP. CONCLUSIONS: Time courses of SBP and DBP decreases during the preintubation period of anesthesia induction are modeled effectively by a logistic function.

Preliminary statistical assessment of intervention by a palliative care team working in a Japanese general inpatient unit.

The effectiveness of intervention by the palliative care team at the University of Tokyo Hospital was assessed using the Support Team Assessment Schedule. During the study, 316 consecutive patients with malignant tumor disease were referred to the palliative care team, which assessed 11 physical symptoms. Results were tested by paired t test to calculate 95% confidence intervals comparing the mean Support Team Assessment Schedule scores for each symptom from the first time to the last time after palliative care intervention. The study concluded that (1) intervention by a palliative care team in general inpatient units can effectively control pain, nausea, and vomiting in patients up until the terminal stage; (2) it is likely that cough is controllable in the terminal stage with intervention by a palliative care team; (3) mouth dryness, anorexia, constipation, diarrhea, fatigue, and ascites are difficult to alleviate in the long term even with palliative intervention.

Defect in parathyroid-hormone-induced luminal calcium absorption in connecting tubules of Klotho mice.

BACKGROUND: Homozygous Klotho mutant mice (KL(-/-) mice) exhibit multiple phenotypes resembling human ageing. Increases in the ratio of urinary calcium to urinary creatinine (uCa/uCr) and in serum Ca concentration and decreases in urinary Cr excretion and serum parathyroid hormone (PTH) concentration were reported; however, precise information about renal Ca handling was not reported in these animals. METHODS: We evaluated the PTH-induced increase in intracellular Ca(2+) concentration ([Ca(2+)]i) in cells of isolated perfused connecting tubules (CNTs) of KL(-/-) mice. We also determined fractional excretion of Ca from the urine and serum samples of the same animals (n = 7), and compared them with KL(+/+) mice and hemi-nephrectomized KL(-+/+) mice (n = 10 in each) as controls. RESULTS: FECa was significantly higher in KL(-/-) mice than in controls (0.67 +/- 0.13 vs 0.20 +/- 0.04%). The PTH (10 nM)-induced increase in [Ca(2+)]i was diminished in KL(-/-) mice (58 +/- 5 vs 231 +/- 15 nM). Addition of 10 nM of 8-(4-chlorophenylthio)-cyclic adenosine 3',5'-monophosphate had a similar effect. The PTH-induced increase had completely disappeared by the removal of Ca from lumen and bath in both groups of animals. Removal of sodium (Na) from the solution increased [Ca(2+)]i to a similar extent in both groups. Conclusion. We conclude that renal Ca excretion estimated by determining FECa was defective in the KL(-/-) mice. Impairment of Ca absorption from the lumen by stimulation of PTH in CNTs is one of the mechanisms of this defect. Activity of the basolateral Na/Ca exchanger was preserved in this strain. Therefore, the pathway downstream after generation of second messengers following stimulation of PTH (such as the sorting of transporters of Ca absorption) might be impaired by disruption of the Klotho gene.