NBS1 recruits RAD18 via a RAD6-like domain and regulates Pol η-dependent translesion DNA synthesis.

Translesion DNA synthesis, a process orchestrated by monoubiquitinated PCNA, is critical for DNA damage tolerance. While the ubiquitin-conjugating enzyme RAD6 and ubiquitin ligase RAD18 are known to monoubiquitinate PCNA, how they are regulated by DNA damage is not fully understood. We show that NBS1 (mutated in Nijmegen breakage syndrome) binds to RAD18 after UV irradiation and mediates the recruitment of RAD18 to sites of DNA damage. Disruption of NBS1 abolished RAD18-dependent PCNA ubiquitination and Polη focus formation, leading to elevated UV sensitivity and mutation. Unexpectedly, the RAD18-interacting domain of NBS1, which was mapped to its C terminus, shares structural and functional similarity with the RAD18-interacting domain of RAD6. These domains of NBS1 and RAD6 allow the two proteins to interact with RAD18 homodimers simultaneously and are crucial for Polη-dependent UV tolerance. Thus, in addition to chromosomal break repair, NBS1 plays a key role in translesion DNA synthesis.

Preproghrelin gene polymorphisms in obese Japanese women. Minor homozygotes are light eaters, do not prefer protein or fat, and apparently have a poor appetite.

Preproghrelin gene single-nucleotide polymorphisms are possible predisposing factors to obesity and other metabolic syndromes. To study the correlation between genotypes and obesity, we recruited 117 obese Japanese women (BMI, 25.0-41.1; average, 31.1). Minor homozygotes for five preproghrelin gene polymorphisms, namely, -1500C>G (rs3755777), -1062G>C (rs26311), -994C>T (rs26312) (promoter region), Leu72Met (rs696217) (exon 2), and +3056T>C (rs2075356) (intron 2), had high values of total and visceral fat areas, waist circumference, and BMI, indicating significant correlation of the polymorphisms with obesity and fat metabolism. Here, we studied the relationship between the genotypes and dietary tendency. Self-administered Diet History Questionnaire showed that total food intake, sugar, and dairy product intake were low in +3056C/C women. Their energy, protein, fat, and meat intake was also low. Energy balance calculation showed considerably reduced fat and protein consumption. Dietary habits were surveyed using Sakata's Questionnaire on Eating Behavior. Of the genotypes, -1062C/C women showed low scores for "motivation for eating" and "eating because of stress or something else." Thus, surprisingly, it was revealed that minor homozygotes for preproghrelin gene polymorphisms were light eaters, did not prefer fat or protein, and apparently had a poor appetite, although they were predisposed to obesity.

Caffeine abolishes the ultraviolet-induced REV3 translesion replication pathway in mouse cells.

When a replicative DNA polymerase stalls upon encountering a photoproduct on the template strand, it is relieved by other low-processivity polymerase(s), which insert nucleotide(s) opposite the lesion. Using an alkaline sucrose density gradient sedimentation technique, we previously classified this process termed UV-induced translesion replication (UV-TLS) into two types. In human cancer cells or xeroderma pigmentosum variant (XP-V) cells, UV-TLS was inhibited by caffeine or proteasome inhibitors. However, in normal human cells, the process was insensitive to these reagents. Reportedly, in yeast or mammalian cells, REV3 protein (a catalytic subunit of DNA polymerase ζ) is predominantly involved in the former type of TLS. Here, we studied UV-TLS in fibroblasts derived from the Rev3-knockout mouse embryo (Rev3KO-MEF). In the wild-type MEF, UV-TLS was slow (similar to that of human cancer cells or XP-V cells), and was abolished by caffeine or MG-262. In 2 cell lines of Rev3KO-MEF (Rev3(-/-)p53(-/-)), UV-TLS was not observed. In p53KO-MEF, which is a strict control for Rev3KO-MEF, the UV-TLS response was similar to that of the wild-type. Introduction of the Rev3 expression plasmid into Rev3KO-MEF restored the UV-TLS response in selected stable transformants. In some transformants, viability to UV was the same as that in the wild-type, and the death rate was increased by caffeine. Our findings indicate that REV3 is predominantly involved in UV-TLS in mouse cells, and that the REV3 translesion pathway is suppressed by caffeine or proteasome inhibitors.

A large intermediate domain of vertebrate REV3 protein is dispensable for ultraviolet-induced translesion replication.

DNA polymerase ζ (pol ζ) is involved in translesion replication (translesion synthesis, TLS) and plays an essential role in embryogenesis. In adults, pol ζ triggers mutation as a result of error-prone TLS and causes carcinogenesis. The catalytic subunit of pol ζ, REV3, is evolutionarily conserved from yeast and plants to higher eukaryotes. However, the structures are notably different: unlike that in yeast REV3, a large intermediate domain is inserted in REV3 of humans and mice. The domain is mostly occupied with noncommittal structures (random coil…etc.); therefore, its role and function are yet to be resolved. Previously, we reported deficient levels of ultraviolet (UV)-induced TLS in fibroblasts derived from the Rev3-knockout mouse embryo (Rev3KO-MEF). Here, we constructed a mouse Rev3-expressing plasmid with a deleted intermediate domain (532-1793 a.a,) and transfected it into Rev3KO-MEF. The isolated stable transformants showed comparable levels of UV-sensitivity and UV-TLS activity to those in wild-type MEF, detected using an alkaline sucrose density gradient sedimentation. These results indicate that the intermediate domain is nonessential for UV-induced translesion replication in cultured mouse cells.

Proteasome inhibitors remarkably prevent translesion replication in cancer cells but not normal cells.

When a replicative DNA polymerase encounters a lesion on the template strand and stalls, it is replaced with another polymerase(s) with low processivity that bypasses the lesion to continue DNA synthesis. This phenomenon is known as translesion replication or replicative bypass. Failing this, the cell is increasingly likely to undergo apoptosis. In this study, we found that proteasome inhibitors prevent translesion replication in human cancer cells but not in normal cells. Three proteasome inhibitors, MG-132, lactacystin, and MG-262, inhibited UV-induced translesion replication in a wide range of cancer cell lines, including HeLa, HGC-27, MCF-7, HepG2, WiDr, a malignant melanoma, an acute lymphoblastic leukemia, and a multiple myeloma cell line; irrespective of cell origin, histological type, or p53 status. In contrast, these inhibitors had little or no influence on normal fibroblasts (NB1RGB and TIG-1) or a normal liver mesenchymal (LI90) cell line. Among the DNA-damaging antineoplastic agents, cisplatin caused a UV-type translesion reaction; the proteasome inhibitors delayed cisplatin-induced translesion replication in cancer cell lines but had only a weak effect on normal cell lines. Therefore, translesion replication would be an effective target of proteasome inhibitors for cancer chemotherapy by which cancer cells can be efficiently sensitized to DNA-damaging antineoplastic agents, such as cisplatin.

A novel Rad18 function involved in protection of the vertebrate genome after exposure to camptothecin.

In Saccharomyces cerevisiae, Rad18 functions in post-replication repair pathways, such as error-free damage bypass involving Rad30 (Poleta) and error-prone damage bypass involving Rev3/7 (Polzeta). Chicken DT40 RAD18(-/-) cells were found to be hypersensitive to camptothecin (CPT), while RAD30(-/-) and REV3(-/-) cells, which are defective in translesion DNA synthesis, were not. RAD18(-/-) cells also showed higher levels of H2AX phosphorylation and chromosomal aberrations, particularly chromosomal gaps and breaks, upon exposure to CPT. Detailed analysis by alkaline sucrose density gradient centrifugation revealed that RAD18(-/-) and wild type cells exhibited similar rates of elongation of newly synthesized DNA in the presence or absence of low concentrations of CPT but that DNA breaks frequently occurred on both parental and nascent strands within 1h after a brief exposure to an elevated concentration of CPT, with more breaks induced in RAD18(-/-) cells than in wild type cells. These data suggest a previously unanticipated role for Rad18 in dealing with replication forks upon encountering DNA lesions induced by CPT.

Hyperlactemia can predict the prognosis of liver resection.

Although hyperlactemia is known to accompany hepatic failure and metabolic acidosis, few reports examined the relationships between lactate concentrations and outcome after liver resection. We examined the ability of arterial plasma lactate concentration to predict the patient outcome after hepatectomy. The relationships of arterial lactate and base excess (BE) measured on admission to the intensive care unit (ICU) after hepatectomy to postoperative outcome were investigated in 151 consecutive patients. Lactate level was significantly higher in nonsurvivors than in survivors (P < 0.001), and in patients with postoperative complications than in those without complications (P < 0.001). Base excess was significantly reduced in nonsurvivors (P < 0.001) and in patients with postoperative complications (P = 0.004). The area under the receiver-operator curve of lactate to mortality was 0.86, whereas that of BE to the mortality was 0.82. Moderate correlation was observed between the lactate level at ICU admission and the highest total bilirubin concentration measured within 14 days after the surgery (r = 0.61), whereas the correlation between BE and bilirubin levels was lower (r = 0.35). Using multivariate analysis, the lactate level independently predicted mortality (P = 0.008) and morbidity (P = 0.013). Lactate (P < 0.001) and BE (P = 0.0068) levels both independently predicted the highest bilirubin concentration. The arterial plasma lactate concentration measured on admission to ICU seemed an excellent predictor of patient outcome after liver resection.

Incidence and outcomes of ventilator-associated pneumonia in Japanese intensive care units: the Japanese nosocomial infection surveillance system.

OBJECTIVES: To determine the incidence of ventilator-associated pneumonia (VAP) among intensive care unit (ICU) patients in Japan and to assess the impact of VAP on patient outcomes, including mortality, length of stay, and duration of mechanical ventilation. DESIGN: Multicenter cohort study. SETTING: Twenty-eight ICUs in multidisciplinary Japanese hospitals with more than 200 beds. PATIENTS: A total of 21,909 patients 16 years or older who were admitted to an ICU between June 2002 and June 2004, stayed in the ICU for 24 to 1,000 hours, and were not transferred to another ICU. RESULTS: The overall infection rates for nosocomial pneumonia and VAP were 6.5 cases per 1,000 patient-days and 12.6 cases per 1,000 ventilator-days, respectively. The standardized mortality rates for the patients with VAP was 1.3 (95% confidence interval [CI], 1.1-1.6): 1.1 (95% CI, 0.9-1.4) for the cases due to drug-susceptible pathogens and 1.5 (95% CI, 1.1-1.9) for the cases due to drug-resistant pathogens. After adjusting for Acute Physiology and Chronic Health Evaluation II score, the mean length of stay for the patients with VAP caused by drug-susceptible pathogens (15.2 days [95% CI, 14.6-15.8]) and by drug-resistant pathogens (17.8 days [95% CI, 17.0-18.6]) was significantly longer than that in the patients without nosocomial infection (6.8 days [95% CI, 6.7-6.9]). The mean duration of mechanical ventilation in the patients with VAP caused by drug-susceptible pathogens (12.0 days [95% CI, 11.5-12.5]) and drug-resistant pathogens (14.1 days [95% CI, 13.5-14.8]) was significantly longer than that in the patients without nosocomial infection (4.7 days [95% CI, 4.6-4.8]). CONCLUSION: The incidence of VAP is substantial among ICU patients in Japan. The potential impact of VAP on patient outcomes emphasizes the importance of preventive measures against VAP, especially for VAP caused by drug-resistant pathogens.

Measurement of DNA synthesis and strand breaks using alkaline sucrose density gradient centrifugation.

Alkaline sucrose density gradient (ASDG) centrifugation is probably an only method to detect elongation of "pulse-labeled" replication products in cells. If the cells are pulse-labeled after being exposed to some DNA-damaging agents, their "post-replication repair" can be measured by ASDG technique. With non-damaged cells, normal replication in replicon size can be observed, too. In addition, the method is also applicable to measure single strand breaks. We have modified this classical method to reproducibly detect very long single-stranded DNA at the megabase level. Here, the protocols are optimized to DT40 cells.

Impact of intensive-care-unit(ICU)-acquired ventilator-associated pneumonia(VAP) on hospital mortality: a matched-paired case-control study.

The effect of ICU-acquired ventilator-associated pneumonia (VAP) on hospital mortality is still a controversial issue in many countries. The aim of this study was to evaluate the effect of ICU-acquired VAP on hospital mortality in a Japanese university hospital. Our study population was comprised of patients aged 16 years or older who were admitted to our ICU and received mechanical ventilation for more than 48 hours during a period of 42 months as of December 2003. To evaluate whether VAP was an independent risk factor for hospital mortality after controlling for other clinical factors, patients with fatal outcomes (cases) were compared to those who survived (controls). From 587 eligible patients, we analyzed 75 cases and 150 controls who were successfully matched on sex, age, and the Acute Physiology and Chronic Health Evaluation II (APACHE II) score using conditional logistic regression models. Univariate analysis demonstrated that hemodialysis (odds ratio [OR], 2.24; 95% confidence interval [CI], 1.21-4.15; p = 0.01), surgical site infection (OR, 2.45; 95% CI, 1.22-4.91; p = 0.01), and VAP (OR, 2.69; 95% CI, 1.55-4.69; p < 0.001) were significantly associated with hospital mortality. After adjusting for confounding factors, multivariate conditional logistic regression analysis showed that hemodialysis (OR, 2.05; 95% CI, 1.06-3.94; p = 0.03) and VAP (OR, 2.20; 95% CI, 1.10-4.39; p = 0.03) were independently associated with hospital mortality. In conclusion, these data suggest that ICU-acquired VAP significantly affects hospital mortality.

[Use of PCR based diagnosis for common invasive fungal infections in the intensive care unit].

Deep-seated Candida infections and invasive aspergilloma are becoming a serious problem for individuals who need intensive care. The laboratory diagnosis of such infections is sometimes delayed due to relatively slow growth of these yeasts from clinical specimens. Several studies seem to indicate that early detection of deep-seated and invasive fungal infections is possible using genomic amplification methods. In the present study, we used a novel PCR assay that can assay five clinically common species (C. albicans, C. parapsilosis, C. tropicalis, C. glablata, and A. fumigatus) simultaneously. We evaluated the utility of this PCR based diagnosis with seven patients with candidiases. This assay is more sensitive than the culture result in 26 clinical samples (chi(2)=5.16, p < 0.05). In the clinical course of each patient, the number of detected fungal species gradually increased. More than two species were detected from single or several clinical specimens, and these patients would die within 14 days compared with the 61 day period individuals with zero or one species would live (p < 0.005). Before super infections of fungus, an antifungal drug could be applied to a suspected patient in the ICU. To improve sensitivity of this diagnosis from blood samples, we evaluated them after one day incubation at 34 degree. We found a PCR product in 10 of 20 blood samples taken from five children after bone marrow transplantation. One of four negative samples became positive after more than 48 hours of incubation.

Translesion replication in cisplatin-treated xeroderma pigmentosum variant cells is also caffeine-sensitive: features of the error-prone DNA polymerase(s) involved in UV-mutagenesis.

Patients with xeroderma pigmentosum variant (XP-V) have a higher risk to skin cancer and XP-V cells are extremely mutable by ultraviolet (UV). The defective gene encodes a DNA polymerase (Poleta) which catalyzed relatively accurate translesion synthesis past the cyclobutane dimer of UV-lesions instead of the replicative polymerase(s) that stalled just before the lesion. Pulse-chase studies have shown that translesion replication in XP-V cells is delayed, but does not completely cease. Taking these results together, error-prone polymerase(s) are plausively involved in the UV-mutagenesis in XP-V devoid of Poleta. However, less is known about the polymerase(s) in vivo. Using an alkaline sucrose density gradient centrifugation (ASDG) technique, translesion replication is detected in the two XP-V strains XP30RO and XP115LO. As reported by Lehmann et al. [Proc. Natl. Acad. Sci. U.S.A. 72 (1975): 219] in XP-V; (i) smaller replication products were accumulated after UV irradiation; (ii) the elongation of these products was delayed; (iii) the elongation was markedly inhibited by caffeine. XP-V cells UV-irradiated at mid-S phase were normally S-arrested, and no "override" by caffeine (i.e. abrogation of the S-checkpoint) was observed by flow cytometry, suggesting that caffeine does not act via cdc kinase here; (iv) butylphenyldeoxyguanosine (BuPGdR) inhibited elongation of replication products only in UV-irradiated XP-V cells; (v) dideoxycytidine or dideoxyinosine had no effect on this process in either normal or XP-V cells. Next, similar phenomena to UV (all of above i to v) were observed also in cisplatin-treated XP-V cells. Pol eta was indicated to participate in cisplatin-induced translesion replication in normal cells. Summing up the above results, the polymerase(s) which work in translesion replication in XP-V are probably BuPGdR-sensitive, insensitive to dideoxynucleotides and can bypass also cisplatin-lesions. To date, several polymerases capable of lesion-bypass synthesis have been isolated. The features presented here are quite useful for identifying the error-prone polymerase(s) involved in UV-mutagenesis.

Comparison of proportional hazard model and neural network models in a real data set of intensive care unit patients.

There has been increased interest in using neural network model (NNM) for prognosis tasks. However, the performance of NNM has seldom been compared with that of traditional statistical models such as proportional hazard model (PHM) in real data sets. We conducted a comparative study of PHM and two types of NNM, that is, aggregate single point model (ASPM) and multiple point model (MPM), using a real data set of intensive care unit patients. The three models were developed using the 70% training subset and their predictive accuracy were assessed using the 30% testing subset according to classification accuracy, area under receiver operating curve, and concordance index. Overall, the highest predictive accuracy was found in MPM, followed by PHM and ASPM. MPM is likely to have the potential ability to provide more accurate estimation of prognosis than PHM and ASPM

[Nutritional therapy in acute pancreatitis].

In this review, systematic search of literatures for acute pancreatitis and nutrition was performed. Eleven randomized controlled trials (RCT) were found. Eight of them are about parenteral or enteral nutrition, 2 are about immunomodulated nutrition, and one is nutritional method in postoperative period. None of these showed benefit of total parenteral nutrition. Moreover, enteral nutrition via nasojejunal tube after 1-2 days after onset or operation of acute pancreatitis showed safe and more effectiveness than parenteral nutrition even in severe cases. These results indicate no benefit of parenteral nutrition in mild to moderate pancreatitis. Early enteral nutrition via nasojejunal tube can be used even in severe cases without ileus or intestinal ischemia.

[Current guideline for hospital infection control in Japan].

The Committee for Prevention of Nosocomial Infection organized by national university hospitals has developed guideline for preventing hospital acquired infections. This was developed after intensive and systematic reviews of the existing scientific papers, followed by a consensus meeting with presence of the infection control specialists. The guideline consisted of following categories: standard precaution, causality organism, urinary tract infection, ventilator associated pneumonia, surgical site infection, catheter related bloodstream infection, and accidental contamination by needles. We also plan to evaluate the effectiveness of this guideline, and continue to update it by our consistent review of scientific papers.

[Polymorphisms related to SIRS and inflammatory response].

Recently, susceptibility and outcome of disease are proved to depend on some gene polymorphisms. Here, we review of gene polymorphisms and its contributions to systemic inflammatory response syndrome (SIRS) and inflammation. The relationships of many polymorphisms, such as TNF, IL-1, PAI-1, TLR, etc. and infections have been studied. Gene polymorphisms of some cytokines are reported to increase expression and production of these cytokines, prevalence rate, morbidity and mortality. These polymorphism informations will be very useful for the prevention and therapy in infected diseases, and recognition of patients that need intensive therapy. From analysis of each patient's gene polymorphism, therapy will be changed case by case. Judging from the difference of races and polymorphisms, Japanese need multi-center large scaled studies in Japan.

Rev1, Rev3, or Rev7 siRNA Abolishes Ultraviolet Light-Induced Translesion Replication in HeLa Cells: A Comprehensive Study Using Alkaline Sucrose Density Gradient Sedimentation.

When a replicative DNA polymerase stalls upon encountering a lesion on the template strand, it is relieved by other low-processivity polymerase(s), which insert nucleotide(s) opposite the lesion, extend by a few nucleotides, and dissociate from the 3'-OH. The replicative polymerase then resumes DNA synthesis. This process, termed translesion replication (TLS) or replicative bypass, may involve at least five different polymerases in mammals, although the participating polymerases and their roles have not been entirely characterized. Using siRNAs originally designed and an alkaline sucrose density gradient sedimentation technique, we verified the involvement of several polymerases in ultraviolet (UV) light-induced TLS in HeLa cells. First, siRNAs to Rev3 or Rev7 largely abolished UV-TLS, suggesting that these 2 gene products, which comprise Polζ, play a main role in mutagenic TLS. Second, Rev1-targeted siRNA also abrogated UV-TLS, indicating that Rev1 is also indispensable to mutagenic TLS. Third, Polη-targeted siRNA also prevented TLS to a greater extent than our expectations. Forth, although siRNA to Polι had no detectable effect, that to Polκ delayed UV-TLS. To our knowledge, this is the first study reporting apparent evidence for the participation of Polκ in UV-TLS.

Preproghrelin gene polymorphisms in obese Japanese: Association with diabetes mellitus in men and with metabolic syndrome parameters in women.

SUMMARY: Preproghrelin gene polymorphisms (SNPs) are possible predisposing factors to obesity and metabolic syndrome. We analysed SNPs in obese Japanese individuals and studied the correlation with diabetes and metabolic syndrome. We recruited 235 subjects (BMI > 28.3) from individuals undergoing periodic medical check-up at Saku Central Hospital. Their SNPs were genotyped using PCR-RFLP method. Frequencies of 5 SNPs in the preproghrelin gene -1500C>G (rs3755777), -1062G>C (rs26311), -994C>T (rs26312), Leu72Met (+408C>A) (rs696217), and +3056T>C (rs2075356) were compared with healthy individuals (data from HapMap Project or Asian population studies). Associations between these SNPs and clinical parameters were investigated. The phenotypes evidently differed between men and women. In men, higher fasting glucose and HbA1c values were observed in the +3056C/C minor homozygotes without leptin or insulin accumulation. The +408C -- +3056C haplotype was more frequent in the diabetic subgroup, in which diagnosis was based on fasting glucose, 75gOGTT, and HbA1c values, than normal subgroup. In contrast, in women, a significant correlation was observed between fat metabolism and obesity. The -1062C/C minor homozygotes had higher values of C-peptide, insulin, total and visceral fat area, waist circumference and BMI. The 72Met/Met minor homozygotes showed reduced leptin, total, HDL and LDL cholesterol concentrations and increased value of visceral fat area. Further, in the other SNPs, the minor homozygotes showed a similar trend, and the heterozygotes had intermediate values. Preproghrelin gene polymorphisms in obese Japanese may be predisposing factors to diabetes mellitus in men and to obesity via aberrant fat metabolism in women.:

Epidemiological approach to nosocomial infection surveillance data: the Japanese Nosocomial Infection Surveillance System.

Surveillance of nosocomial infection is the foundation of infection control. Nosocomial infection surveillance data ought to be summarized, reported, and fed back to health care personnel for corrective action. Using the Japanese Nosocomial Infection Surveillance (JANIS) data, we determined the incidence of nosocomial infections in intensive care units (ICUs) of Japanese hospitals and assessed the impact of nosocomial infections on mortality and length of stay. We also elucidated individual and environmental factors associated with nosocomial infections, examined the benchmarking of infection rates and developed a practical tool for comparing infection rates with case-mix adjustment. The studies carried out to date using the JANIS data have provided valuable information on the epidemiology of nosocomial infections in Japanese ICUs, and this information will contribute to the development of evidence-based infection control programs for Japanese ICUs. We conclude that current surveillance systems provide an inadequate feedback of nosocomial infection surveillance data and, based on our results, suggest a methodology for assessing nosocomial infection surveillance data that will allow infection control professionals to maintain their surveillance systems in good working order.