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.

Clinical introduction of Monte Carlo treatment planning for lung stereotactic body radiotherapy.

The purpose of this study was to investigate the impact of Monte Carlo (MC) calculations and optimized dose definitions in stereotactic body radiotherapy (SBRT) for lung cancer patients. We used a retrospective patient review and basic virtual phantom to determine dose prescriptions. Fifty-three patients underwent SBRT. A basic virtual phantom had a gross tumor volume (GTV) of 10.0 mm with equivalent water density of 1.0 g/cm3, which was surrounded by equivalent lung surrounding the GTV of 0.25 g/cm3. D95 of the planning target volume (PTV) and D99 of the GTV were evaluated with different GTV sizes (5.0 to 30.0 mm) and different lung densities (0.05 to 0.45 g/cm3). Prescribed dose was defined as 95% of the PTV should receive 100% of the dose (48 Gy/4 fractions) using pencil beam (PB) calculation and recalculated using MC calculation. In the patient study, average doses to the D95 of the PTV and D99 of the GTV using the MC calculation plan were 19.9% and 10.2% lower than those by the PB calculation plan, respectively. In the phantom study, decreased doses to the D95 of the PTV and D99 of the GTV using the MC calculation plan were accompanied with changes GTV size from 30.0to 5.0 mm, which was decreased from 8.4% to 19.6% for the PTV and from 17.4%to 27.5% for the GTV. Similar results were seen with changes in lung density from 0.45 to 0.05 g/cm3, with doses to the D95 of the PTV and D99 of the GTV were decreased from 12.8% to 59.0% and from 7.6% to 44.8%, respectively. The decrease in dose to the PTV with MC calculation was strongly dependent on lung density. We suggest that dose definition to the GTV for lung cancer SBRT be optimized using MC calculation. Our current clinical protocol for lung SBRT is based on a prescribed dose of 44 Gy in 4 fractions to the GTV using MC calculation.

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.

Long-term management of chronic obstructive pulmonary disease: a survey of collaboration among physicians involved in pulmonary rehabilitation in Japan.

BACKGROUND: This study evaluated the implementation of pulmonary rehabilitation (PR), and the extent of the collaboration between primary care and chest physicians involved in the management of chronic obstructive pulmonary disease (COPD) in Japan. METHODS: The survey was conducted in 2006 via post and facsimile and included all medical institutions approved by the Japan Respiratory Society. RESULTS: In total, 176 institutions responded (response rate, 27%); a PR program was conducted at 55.1% of these institutions throughout Japan, but with regional differences. The mean duration of each session in an outpatient setting was 30 min with 2 sessions per week, and the mean length of hospitalization was 2-3 weeks. Although 33% of the hospitals adopted PR programs, on a scale from none (0) to maximum achievement (100), the accomplishment score was 48. Similarly, the mean satisfaction level score for collaboration was 44. The main problem arising with regards to chest physicians' referral to general physicians was the reluctance of patients or family members (88%). Chest physicians believed that general physicians should perform early screening of patients and manage early exacerbations, including educating patients of the need to discontinue smoking. CONCLUSIONS: Most chest physicians in Japan were not satisfied with the status of long-term COPD management. PR for COPD patients and collaboration between primary care physicians and specialists remain problematic in Japan. Moreover, there are widespread regional differences in terms of implementation. Sharing and implementing appropriate clinical information with primary care physicians according to current clinical guidelines should be emphasized.

Concerns with the health check-up system for chronic obstructive pulmonary disease on two Japanese islands.

OBJECTIVE: Early diagnosis is a key factor in the management of chronic obstructive pulmonary disease (COPD). Although mass screening is widely used, little is known about its accuracy and efficacy. This study investigated whether using spirometry during mass screening to detect COPD among community residents might be ineffective because of variability in the training and experience of examiners. PARTICIPANTS AND METHODS: Both spirometry and a self-written questionnaire-based survey, including questions designed to detect respiratory symptoms, were conducted on community residents. Two separate studies were conducted on islanders living in similar environments. Study I was performed from 2004 to 2007 on Hachijyo Island residents, while study II, with a similar study design, was performed in 2003 on Inno Island residents. RESULTS: In study I, 3,592 subjects underwent examination over the 4-year study period; of these, 378 subjects underwent repeated examinations. Approximately 25% of the subjects had respiratory symptoms. Acceptable spirometry recordings were obtained for 62.0% (2004) to 84.1% (2006) of the subjects. In study II, 167 of the 254 subjects (65.7%) had respiratory symptoms. Acceptable assessment recordings were achieved in 254 subjects (95.5%). The suitability of the recordings was influenced by the extent/level of training of the examiners and the accompanying thoracic specialists. CONCLUSION: We concluded that the effectiveness of health check-ups for COPD evaluation using spirometry was greatly influenced by the quality of the examiners, even when the subjects had respiratory symptoms. Thus, we recommend caution when screening for early signs of COPD during health check-ups.

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.

Excessive visceral fat accumulation in advanced chronic obstructive pulmonary disease.

BACKGROUND: Previous studies have suggested links between chronic obstructive pulmonary disease (COPD), cardiovascular disease, and abdominal obesity. Although abdominal visceral fat is thought to be associated with cardiovascular risk factors, the degree of visceral fat accumulation in patients with COPD has not been directly studied. The aim of this study was to investigate the abdominal visceral fat accumulation and the association between visceral fat and the severity and changes in emphysema in COPD patients. METHODS: We performed clinical and laboratory tests, including pulmonary function, dyspnea score, and the six-minute walking test in COPD patients (n = 101) and control, which included subjects with a smoking history but without airflow obstruction (n = 62). We used computed tomography to evaluate the abdominal visceral fat area (VFA), subcutaneous fat area (SFA), and the extent of emphysema. RESULTS: The COPD group had a larger VFA than the control group. The prevalence of non-obese subjects with an increased VFA was greater in the Global Initiative for Chronic Obstructive Lung Disease Stages III and IV than in the other stages of COPD. The extent of emphysema was inversely correlated with waist circumference and SFA. However, VFA did not decrease with the severity of emphysema. VFA was positively correlated with the degree of dyspnea. CONCLUSION: COPD patients have excessive visceral fat, which is retained in patients with more advanced stages of COPD or severe emphysema despite the absence of obesity.

Efficient integrated education for older patients with chronic obstructive pulmonary disease using the Lung Information Needs Questionnaire.

AIM: Education is a key issue for the long-term management of chronic obstructive pulmonary disease in older patients. We tested the hypothesis that integrated care focusing on patient information needs for self-management can improve patient information needs and health outcomes. METHODS: Patients with chronic obstructive pulmonary disease (n=102) were randomized into integrated care (group I) and usual care (group U) groups. Group I underwent a program of educational sessions for 6months (integrated education period), and then repeatedly received an individually tailored education according to the Lung Information Needs Questionnaire score. Both groups were followed up monthly for 6months. RESULTS: A total of 85 patients (mean age: 71.7years) were followed up for 12months. The total Lung Information Needs Questionnaire score was significantly better in group I than in group U at 12months (P<0.03). Activities of daily living scores were significantly improved in group I at 6months (P<0.03). The dyspnea score and the BODE index (body mass index, dyspnea, airflow obstruction and exercise capacity) were significantly improved in group I at 12months (P<0.01 and P<0.02, respectively). During the integrated education period, the frequency of hospitalization was significantly lower in group I than in group U (P=0.033). CONCLUSION: Integrated education for older patients with chronic obstructive pulmonary disease effectively improved patients' information needs, activities of daily living, dyspnea score, BODE index and reduced hospitalizations during the observed period. Geriatr Gerontol Int 2011; 11: 422-430.

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.

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.

Emphysema on imaging is associated with quality of life in elderly patients with chronic obstructive pulmonary disease.

BACKGROUND: Limited data are available on the association between the severity of emphysema or airway narrowing, and health-related quality of life (HRQOL) in patients with chronic obstructive pulmonary disease (COPD), which has been seen to be more prevalent among elderly subjects. The aim of this study was to examine the association between HRQOL, physical parameters and structural alterations in lung of COPD patients. METHODS: Stable COPD patients (n = 125; mean age 71.0) were studied. Both the severity of emphysema, which was expressed as the extent of the low-attenuation area (LAA%), and percentage of the large airway wall area (WA%) on high-resolution computed tomography (HRCT) were compared with various parameters of the generic and HRQOL, respectively, together with pulmonary function tests and exercise capacity. RESULTS: The predicted value of forced expiratory volume in 1 s was significantly associated with both LAA% and WA%, but the diffusion capacity was strongly correlated with LAA% alone. Parameters of the generic and HRQOL, and almost all other parameters appeared to be significantly associated with LAA% alone, whereas no association was observed between WA% and QOL. CONCLUSION: We concluded that the severity of emphysema, but not that of large airway narrowing on HRCT, is associated with both generic and health-related QOL and reduced diffusion capacity. This notion might provide useful information in practice among elderly subjects who are unable to perform a spirometry.

Extent of emphysema on HRCT affects loss of fat-free mass and fat mass in COPD.

BACKGROUND: Although muscle loss is thought to be a prognostic factor in chronic obstructive pulmonary disease (COPD), its determinants remain unclear. AIM: To verify the hypothesis that fat-free mass (FFM) and fat mass (FM) are associated with the extent of emphysema in COPD patients. PATIENTS AND METHODS: A total of 112 stable, male current or ex-smokers with or without COPD attending a secondary care specialist COPD clinic were studied. FFM and FM were measured by bioelectrical impedance analysis. We also assessed the nutrition status, muscle strength by the handgrip test, exercise tolerance by the 6-minute walking test, airflow limitation and diffusion capacity, the extent of emphysema by high-resolution CT scan, systemic inflammation status using C-reactive protein, and a lipid-related hormone (adiponectin). RESULTS: The FFM index (FFMI), which was defined as the FFM divided by the square of the body height, was significantly correlated with age, the total number of lymphocytes, handgrip strength, distance on 6-minute walking, airflow limitation, diffusion capacity, extent of emphysema, and C-reactive protein. On multivariate analysis, the FFMI was associated with handgrip strength and inversely correlated with the extent of emphysema. The FM index (FMI) was positively correlated with pack-years, and was inversely correlated with the extent of emphysema and concentrations of adiponectin. CONCLUSION: The extent of emphysema was correlated with skeletal muscle loss and also the FM.

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.:

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.

Gender-associated differences in dyspnoea and health-related quality of life in patients with chronic obstructive pulmonary disease.

BACKGROUND AND OBJECTIVES: Recent studies have reported several gender-associated differences among patients with COPD, but gender-associated differences in health-related quality of life (HRQoL) in patients with COPD have not yet been clarified. This study evaluated gender differences in dyspnoea and HRQoL in patients with COPD. METHODS: Study participants were 156 patients with COPD (men 117, women 39); men were individually matched to women by age and FEV(1)% predicted to give a ratio of 3:1 (male : female). Study participants were evaluated for dyspnoea and completed HRQoL questionnaires. An oxygen cost diagram (OCD) was used to assess the degree of dyspnoea and Morale Scale was used to assess subjective well-being. St. George's Respiratory Questionnaire (SGRQ) and SF-36 were used for HRQoL evaluation. The findings in the male and female groups were compared. RESULTS: The OCD and Morale Scale showed significantly lower values for female patients with COPD. Disease-specific HRQoL assessed by SGRQ was significantly worse, except for symptoms, in female patients with COPD. Generic HRQoL assessed by SF-36 was also significantly worse, except for general health and social functioning. Stepwise multiple regression showed OCD, Morale Scale and 6-min walking distance to be significantly associated with total SGRQ score in the male group, and Morale Scale and 6-min walking distance were significant associations in the female group. CONCLUSIONS: Gender differences exist in dyspnoea and HRQoL in patients with COPD. These need to be considered when designing treatment strategies for COPD patients.

A critical role for the ubiquitin-conjugating enzyme Ubc13 in initiating homologous recombination.

The ubiquitin (Ub)-conjugating enzyme Ubc13 is implicated in Rad6/Rad18-dependent postreplication repair (PRR) in budding yeast, but its function in vertebrates is not known. We show here that disruption or siRNA depletion of UBC13 in chicken DT40 or human cells confers severe growth defects due to chromosome instability, and hypersensitivity to both UV and ionizing radiation, consistent with a conserved role for Ubc13 in PRR. Remarkably, Ubc13-deficient cells are also compromised for DNA double-strand break (DSB) repair by homologous recombination (HR). Recruitment and activation of the E3 Ub ligase function of BRCA1 and the subsequent formation of the Rad51 nucleoprotein filament at DSBs are abolished in Ubc13-deficient cells. Furthermore, generation of ssDNA/RPA complexes at DSBs is severely attenuated in the absence of Ubc13. These data reveal a critical and unexpected role for vertebrate Ubc13 in the initiation of HR at the level of DSB processing.

Environmental influence on the measurement of exhaled nitric oxide concentration in school children: special reference to methodology.

INTRODUCTION: Measuring exhaled nitric oxide (eNO) is a noninvasive and useful method for evaluating the correlation between airway inflammation and air pollution. The method is being used in studies; however, the effects of polluted air on eNO values are poorly understood. If polluted air significantly affects eNO concentrations, then it would be hard to evaluate the concentration of eNO, particularly in epidemiological measurements to detect the effects of airway inflammation, such as that in bronchial asthma. Thus, we hypothesized that short-term exposure to air pollution affects eNO values. PURPOSE: To study the effects of environmental nitrogen oxides on the measurement of eNO concentration. SUBJECTS AND METHODS: A total of 19 school children who lived on a large street with heavy traffic with random allocation were studied. Subjects with bronchial asthma were identified with a questionnaire. Suspended particulate matter. including particulate matter with an aerodynamic diameter < or =2.5 microm (PM(2.5)), optical black carbon, nitric oxide (NO), nitric dioxide (NO(2)), and nitrogen oxides (NO(X)), were measured at a fixed place along the street every hour for 11 consecutive days. The concentrations of NO and NO(2) for each subject were measured by an individual 2-pyenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl sampler, and the concentration of eNO was measured with the off-line method. RESULTS: Of 19 subjects, 3 were found to have bronchial asthma. The level of each pollutant for 11 days peaked during the mornings (6;9 a.m.) and evenings (6;9 p.m.) due to traffic jams; average eNO values in healthy subjects and those with asthma were 27.1 +/- 9.7 and 57.7 +/- 18.6 ppb (p=0.098), respectively. It was found that the eNO value remained high when the mean values of various pollutants remained high for 8 hours before the measurements. It was estimated that the mean eNO values increased by 1.08 ppb (95% CI: 0.72;1.45) when the mean NO(X) value for the previous 8 hours reached approximately 10 ppb. CONCLUSION: We conclude that short-term exposure to polluted air of at least 8 hours before measurement affects eNO values. Therefore, caution should be exercised when measuring eNO value in epidemiological studies.

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.

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.

The impact of dyspnoea and leg fatigue during exercise on health-related quality of life in patients with COPD.

OBJECTIVE: COPD patients frequently complain of symptoms such as dyspnoea and leg fatigue during exercise. However, the impact of these symptoms on the health-related quality of life (HRQoL) is not known. This study tested whether dyspnoea and leg fatigue during exercise affects the HRQoL of patients with COPD. METHODS: In a cross-sectional study, 90 patients with stable COPD (mean age, 76.0+/-0.7 years; FEV(1), 1.11+/-0.04 L) completed the St. George's Respiratory Questionnaire (SGRQ), pulmonary function testing, arterial blood gas analysis, and a 6-min walking distance test (6MWD). Dyspnoea and leg fatigue during exercise were quantitated into 12 grades using the Borg scale (0--10). Correlations between the SGRQ and various variables were determined. In a longitudinal study, 22 patients with COPD (mean age, 71.5+/-1.1 years; FEV(1), 1.31+/-0.08 L) completed a pulmonary rehabilitation program, for which correlations between changes in the SGRQ as well as changes in both dyspnoea and leg fatigue, during the 6MWD before and 3 months after pulmonary rehabilitation, were examined. RESULTS: For the cross-sectional study, the total SGRQ score correlated significantly with the walking distance, dyspnoea and leg fatigue during the 6MWD and FEV(1), respectively. Stepwise multiple regression analysis showed that dyspnoea and leg fatigue during the 6MWD were independent variables for HRQoL measured by the SGRQ. For the longitudinal study, changes in the SGRQ correlated significantly with changes in dyspnoea and leg fatigue, before and 3 months after, pulmonary rehabilitation. CONCLUSIONS: Symptoms, such as the degree of dyspnoea and leg fatigue during exercise, are significant variables which influence the HRQoL of patients with COPD. In addition, the improvement in HRQoL following pulmonary rehabilitation may be due to improvements in dyspnoea and leg fatigue in patients with COPD.