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1.  Predicting Visual Outcomes for Macula-Off Rhegmatogenous Retinal Detachment with Optical Coherence Tomography 
Journal of Ophthalmology  2014;2014:269837.
Purpose. We evaluated the ability of novel optical coherence tomography (OCT) parameters to predict postoperative best-corrected visual acuity (BCVA) in macula-off rhegmatogenous retinal detachment (RRD) eyes. Methods. We reviewed the medical records of 56 consecutive eyes with macula-off RRD. Clinical findings were analyzed including the relationship between preoperative OCT findings and 6-month postoperative BCVA. Results. Six-month postoperative BCVA was significantly correlated with preoperative findings including retinal height at the fovea, total and inner layer cross-sectional macular area within 2 mm of the fovea, and preoperative BCVA (P < 0.001, P < 0.001, P = 0.001, and P < 0.001, resp.). Multiple regression analysis revealed that the duration of macular detachment and total cross-sectional macular area were independent factors predicting 6-month postoperative BCVA (P = 0.024 and P = 0.041, resp.). Conclusions. Measuring preoperative total cross-sectional area of the macular layer within 2 mm of the fovea with OCT is a useful and objective way to predict postoperative visual outcome in eyes with macula-off RRD.
PMCID: PMC4276691  PMID: 25574380
2.  Risky locations for out-of-hospital cardiopulmonary arrest in a typical urban city 
The aim of this study is to clarify the circumstances including the locations where critical events resulting in out-of-hospital cardiopulmonary arrest (OHCPA) occur.
Materials and Methods:
Subjects of this population-based observational case series study were the clinical records of patients with nontraumatic and nonneck-hanging OHCPA.
Of all 1546 cases, 10.3% occurred in a public place (shop, restaurant, workplace, stations, public house, sports venue, and bus), 8.3% on the street, 73.4% in a private location (victim's home, the homes of the victims’ relatives or friends or cheap bedrooms, where poor homeless people live), and 4.1% in residential institutions. In OHCPA occurring in private locations, the frequency of asystole was higher and the outcome was poorer than in other locations. A total of 181 OHCPA cases (11.7%) took place in the lavatory and 166 (10.7%) in the bathroom; of these, only 7 (3.9% of OHCPA in the lavatory) and none in the bath room achieved good outcomes. The frequencies of shockable initial rhythm occurring in the lavatory and in bath room were 3.7% and 1.1% (lower than in other locations, P = 0.011 and 0.002), and cardiac etiology in OHCPA occurring in these locations were 46.7% and 78.4% (the latter higher than in other locations, P < 0.001).
An unignorable population suffered from OHCPA in private locations, particularly in the lavatory and bathroom; their initial rhythm was usually asystole and their outcomes were poor, despite the high frequency of cardiac etiology in the bathroom. We should try to treat OHCPA victims and to prevent occurrence of OHCPA in these risky spaces by considering their specific conditions.
PMCID: PMC4231265  PMID: 25400390
Bathroom; lavatory; OHCPA; out-of-hospital cardiopulmonary arrest; the relation between outcome and location
3.  Transfer kinetics of perfluorooctane sulfonate from water and sediment to a marine benthic fish, the marbled flounder (Pseudopleuronectes yokohamae) 
The authors investigated the kinetics of transfer of perfluorooctane sulfonate (PFOS) from water, suspended sediment, and bottom sediment to a marine benthic fish, the marbled flounder (Pseudopleuronectes yokohamae). Fish were exposed in 3 treatments to PFOS in combinations of these exposure media for 28 d and then depurated for 84 d. A major part (37–66%) of PFOS in the fish was in the carcass (i.e., whole body minus muscle and internal organs). Three first-order-kinetic models that differed in exposure media, that is, 1) sum of dissolved and particulate phases and sediment; 2) dissolved phase, particulate phase, and sediment; and 3) dissolved phase only, were fitted to the data assuming common rate constants among the treatments. The uptake efficiency of dissolved PFOS at the respiratory surfaces was estimated to be 3.2% that of oxygen, and the half-life of PFOS in the whole body to be 29 d to 31 d. The better fit of models 1 and 2 and the values of the estimated uptake rate constants suggested that the PFOS in suspended and bottom sediments, in addition to that dissolved in water, contributed to the observed body burden of the fish. Based on an evaluation of several possible contributing factors to the uptake of PFOS from suspended and bottom sediments, the authors propose that further investigation is necessary regarding the mechanisms responsible for the uptake. Environ Toxicol Chem 2013;32:2009–2017. © 2013 The Authors. Environmental Toxicology and Chemistry Published by Wiley Periodicals, Inc., on behalf of SETAC. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial License, which permits use, distribution, and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
PMCID: PMC3881517  PMID: 23636803
Bioconcentration; Persistent organic pollutants; Respiratory uptake; Aquatic organism; Particle
4.  Simultaneous Dislocation of Radiocapitellar and Distal Radioulnar Joint 
Case Reports in Orthopedics  2013;2013:106567.
A 45-year-old male presented to the emergency room of our institution complaining of severe pain around the left elbow. While playing volleyball, he slipped down with his left arm hit between the floor and his body. He complaind of strong pain from left elbow to hand, and active motion of elbow and wrist joint was impossible. His forearm was held in supinated position. On X-ray examination, radius head was deviated to anterior lateral side, and distal end of radius was dislocated to dorsal side. Tenderness was prominent at the site of radial head and distal radioulnar joint. Surgical treatment was performed using triceps tendon strip. Good functional recovery was gained.
PMCID: PMC3806512  PMID: 24194995
5.  Myocarditis with fulminant type 1 diabetes mellitus diagnosed by cardiovascular magnetic resonance imaging: a case report 
BMC Research Notes  2013;6:347.
Fulminant type 1 diabetes is a non-autoimmune disorder characterized by sudden onset. This complication is rarely associated with myocarditis, suggesting an involvement of viral infection. We report a patient with myocarditis who was admitted for fulminant type 1 diabetes and diagnosed using a combination of non-invasive techniques.
Case presentation
We describe the case of a 25-year-old Japanese man with fulminant type 1 diabetes complicated by myocarditis. The patient was admitted with flu-like symptoms and diabetic ketoacidosis, followed by chest pain the next day. Myocardial damage was suspected based on ST-segment elevation on electrocardiogram and elevation of cardiac enzymes. However, coronary angiography revealed no abnormality in the coronary arteries. We diagnosed myocarditis by a combination of echocardiography, cardiovascular magnetic resonance imaging (CMR), as well as Thallium-201 and Iodine-123 beta-methyl iodophenyl pentadecanoic acid (Tl-201 BMIPP and I-123 BMIPP) and myocardial imaging. More importantly, CMR revealed diffuse enhancement in the subepicardium of the left ventricle with late gadolinium enhancement, consistent with myocardial edema. The patient was successfully treated, received a two-week education program on diabetes and discharged without complication.
The rapid onset and flu-like symptoms strongly suggest the involvement of viral infection in the pathogenesis of fulminant type 1 diabetes and myocarditis. While cardiac muscle biopsy is routinely performed, this case demonstrates that a combination of non-invasive techniques, especially CMR, may successfully diagnose myocarditis in patients with fulminant type 1 diabetes.
PMCID: PMC3766228  PMID: 24060110
Fulminant type 1 diabetes; Myocarditis; Diabetes; Cardiovascular magnetic resonance imaging; Diabetic ketoacidosis
6.  Etiology of out-of-hospital cardiac arrest diagnosed via detailed examinations including perimortem computed tomography 
The spectrum of the etiology of out-of-hospital cardiopulmonary arrest (OHCPA) has not been established. We have performed perimortem computed tomography (CT) during cardiopulmonary resuscitation.
To clarify the incidence of non-cardiac etiology (NCE), actual distribution of the causes of OHCPA via perimortem CT and its usefulness.
Settings and Design:
Population-based observational case series study.
Materials and Methods:
We reviewed the medical records of 1846 consecutive OHCPA cases and divided them into two groups: 370 showing an obvious cause of OHCPA with NCE (trauma, neck hanging, terminal stage of malignancy, and gastrointestinal bleeding) and others.
Of a total OHCPA, perimortem CT was performed in 57.5% and 62.5% were finally diagnosed as NCE: Acute aortic dissection (AAD) 8.07%, pulmonary thrombo-embolization (PTE) 1.46%, hypoxia due to pneumonia 5.25%, asthma and acute worsening of chronic obstructive pulmonary disease 2.06%, cerebrovascular disorder (CVD) 4.48%, airway obstruction 7.64%, and submersion 5.63%. The rates of patients who survived to hospital discharge were 6-14% in patients with NCE. Out of the 1476 cases excluding obvious NCE of OHCPA, 66.3% underwent perimortem CT, 14.6% of cases without obvious NCE and 22.1% of cases with perimortem CT were confirmed as having some NCE.
Of the total OHCPA the incidences of NCE was 62.5%; the leading etiologies were AAD, airway obstruction, submersion, hypoxia and CVD. The rates of cases converted from cardiac etiology to NCE using perimortem CT were 14.6% of cases without an obvious NCE.
PMCID: PMC3665077  PMID: 23723616
Etiology of out-of-hospital cardiac arrest; out-of-hospital cardiac arrest with non-cardiac etiology; perimortem computed tomography; perimortem imaging
7.  Th1 biased response to a novel Porphyromonas gingivalis protein aggravates bone resorption caused by this oral pathogen 
In previous studies we showed that biasing the immune response to Porphyromonas gingivalis antigens to the Th1 phenotype increases inflammatory bone resorption caused by this organism. Using a T cell screening strategy we identified eight P. gingivalis genes coding for proteins that appear to be involved in T-helper cell responses. In the present study we characterized the protein, encoded by PG_1841 gene and evaluated its relevance in the in bone resorption caused by P. gingivalis because subcutaneous infection of mice with this organism resulted in the induction of Th1 biased response to the recombinant PG1841 antigen molecule. Using an immunization regime that strongly biases toward the Th1 phenotype followed by challenge with P. gingivalis in dental pulp tissue, we demonstrate that mice pre-immunized with rPG1841 developed severe bone loss compared with control immunized mice. Pre-immunization of mice with the antigen using a Th2 biasing regime resulted in no exacerbation of the disease.
These results support the notion that selected antigens of P. gingivalis are involved in a biased Th1 host response that leads to the severe bone loss caused by this oral pathogen.
PMCID: PMC3607305  PMID: 18457976
8.  Blood transfusion therapy for traumatic cardiopulmonary arrest 
Blood transfusion therapy (BTT), which represents transplantation of living cells, poses several risks. Although BTT is necessary for trauma victims with hemorrhagic shock, it may be futile for patients with blunt traumatic cardiopulmonary arrest (BT-CPA).
Materials and Methods:
We retrospectively examined the medical records of consecutive patients with T-CPA. The study period was divided into two periods: The first from 1995-1998, when we used packed red cells (PRC) regardless of the return of spontaneous circulation (ROSC), and the second from 1999-2004, when we did not use PRC before ROSC. The rates of ROSC, admission to the ICU, and survival-to-discharge were compared between these two periods.
We studied the records of 464 patients with BT-CPA (175 in the first period and 289 in the second period). Although the rates of ROSC and admission to the ICU were statistically higher in the first period, there was no statistical difference in the rate of survival-to-discharge between these two periods. In the first period, the rate of ROSC was statistically higher in the non-BTT group than the BTT group. However, for cases in which ROSC was performed and was successful, there were no statistical differences in the rate of admission and survival-to-discharge between the first and second group, and between the BTT and non-BTT group.
Our retrospective consecutive study shows the possibility that BTT before ROSC for BT-CPA and a treatment strategy that includes this treatment improves the success rate of ROSC, but not the survival rate. BTT is thought to be futile as a treatment for BT-CPA before ROSC.
PMCID: PMC3589857  PMID: 23493056
Blood transfusion therapy; return of spontaneous circulation; survival to discharge; traumatic cardiopulmonary arrest
9.  Effects of Enteric-coated Lactoferrin Tablets Containing Lactobacillus brevis subsp. coagulans on Fecal Properties, Defecation Frequency and Intestinal Microbiota of Japanese Women with a Tendency for Constipation: a Randomized Placebo-controlled Crossover Study 
The effects of oral administration of enteric-coated tablets containing lactoferrin (LF; 100 mg/tablet) and heat-killed Lactobacillus brevis subsp. coagulans FREM BP-4693 (LB; 6×109 bacteria/tablet) on fecal properties were examined in 32 Japanese women (20–60 years of age) with a tendency for constipation (defecation frequency at equal to or less than 10 times/2 weeks) by a double-blind placebo-controlled crossover design. A significant increase in defecation days per week was obserbed in the subjects who ingested the tablets containing LF and LB compared with the placebo group. The number of bifidobacteria in feces also significantly increased compared with the placebo group. In an in vitro study, LF and tryptic hydrolysate of LF, but not peptic hydrolysate of LF, upregulated the growth of Bifidobacterium longum ATCC15707 when added to the culture. These results demonstrate the capability of the enteric-coated tablets containing LF and LB in improving intestinal function and suggest that they have a growth promoting function for bifidobacteria.
PMCID: PMC4034292  PMID: 24936358
double-blind placebo-controlled trial; lactoferrin; Lactobacillus brevis subsp. coagulans; probiotics; intestinal microbiota; enteric-coated tablets
10.  Complications of bystander cardiopulmonary resuscitation for unconscious patients without cardiopulmonary arrest 
Insufficient knowledge of the risks and complications of cardiopulmonary resuscitation (CPR) may be an obstructive factor for CPR, however, particularly for patients who are not clearly suffering out of hospital cardiopulmonary arrest (OH-CPA). The object of this study was to clarify the potential complication, the safety of bystander CPR in such cases.
Materials and Methods:
This study was a population-based observational case series. To be enrolled, patients had to have undergone CPR with chest compressions performed by lay persons, had to be confirmed not to have suffered OHCPA. Complications of bystander CPR were identified from the patients’ medical records and included rib fracture, lung injury, abdominal organ injury, and chest and/or abdominal pain requiring analgesics. In our emergency department, one doctor gathered information while others performed X-ray and blood examinations, electrocardiograms, and chest and abdominal ultrasonography.
A total of 26 cases were the subjects. The mean duration of bystander CPR was 6.5 minutes (ranging from 1 to 26). Nine patients died of a causative pathological condition and pneumonia, and the remaining 17 survived to discharge. Three patients suffered from complications (tracheal bleeding, minor gastric mucosal laceration, and chest pain), all of which were minimal and easily treated. No case required special examination or treatment for the complication itself.
The risk and frequency of complications due to bystander CPR is thought to be very low. It is reasonable to perform immediate CPR for unconscious victims with inadequate respiration, and to help bystanders perform CPR using the T-CPR system.
PMCID: PMC3299150  PMID: 22416146
Cardiopulmonary arrest; cardiopulmonary resuscitation; complication; education
11.  18beta-Glycyrrhetinic Acid Inhibits Periodontitis Via Glucocorticoid-Independent NF–κB Inactivation In IL-10 Deficient Mice 
Journal of periodontal research  2010;45(6):757-763.
Background and objective
18beta-glycyrrhetinic acid (GA) is a natural anti-inflammatory compound derived from licorice root extract (Glycyrrhiza glabra). The effect of GA on experimental periodontitis and its mechanism of action were determined in the present study.
Periodontitis was induced by oral infection with Porphyromonas gingivalis W83 in IL-10 deficient mice. The effect of GA, which was delivered by subcutaneous injections in either prophylactic or therapeutic regimens, on alveolar bone loss and gingival gene expressions was determined on day 42 after initial infection. The effect of GA on LPS-stimulated macrophages, T cell proliferation, and osteoclastogenesis was also examined in vitro.
GA administered either prophylactically or therapeutically dramatically reduced infection-induced bone loss in IL-10 deficient mice, which are highly disease-susceptible. Although GA has been reported to exert its anti-inflammatory activity via down-regulation of 11-beta hydroxysteroid dehydrogenase-2 (HSD2), which converts active glucocorticoids (GC) to their inactive forms, GA did not reduce HSD2 gene expression in gingival tissue. Rather, under GC-free conditions, GA potently inhibited LPS-stimulated proinflammatory cytokine production and RANKL-stimulated osteoclastogenesis, both of which are NF–κB-dependent. GA furthermore suppressed LPS- and RANKL-stimulated phosphorylation of NF–κB p105 in vitro.
These findings indicate that GA inhibits periodontitis by inactivation of NF–κB in an IL-10 and GC-independent fashion.
PMCID: PMC3075584  PMID: 20682015
18beta-glycyrrhetinic acid; periodontal disease; NF–κB; IL-10 deficient mouse
12.  Cardiopulmonary arrest on arrival due to penetrating trauma 
The aim of this study was to clarify the outcome of patients with cardiopulmonary arrest on arrival due to penetrating trauma (PT-CPA) and to establish the treatment strategy.
The clinical course of 29 patients with PT-CPA over the past 10 years was examined. We have taken three approaches to these patients: (i) an aggressive treatment strategy; (ii) an in-hospital system supporting this aggressive resuscitation; and (iii) the pre-hospital emergency medical service (EMS) system in our city.
Although the return of spontaneous circulation (ROSC) was established in 59% of patients, only 17% survived for 7 days, 14% were discharged, and 7% were neurologically intact. Of 10 patients showing pulseless electrical activity (PEA) on the scene, ROSC was established in 100% and 30% were discharged; however, of 12 patients showing asystole, ROSC was established in 33% and no patient could be discharged. There was no difference in the time interval from the arrival at the emergency department to ROSC between discharged patients and patients who died. The time interval from collapse to arrival at the emergency department in discharged patients and patients who went to the intensive care unit was shorter than that of patients who died in the emergency department with and without ROSC.
We cannot decide to give up and terminate resuscitation in any PT-CPA patients and cannot define salvageable patients. However, our data show that 30-min resuscitation is thought to be relevant and that we should not give up on resuscitation because of the time interval without ROSC after arrival at the hospital.
PMCID: PMC3025245  PMID: 20353643
Cardiopulmonary arrest; Penetrating trauma; Emergency medical service system; Resuscitation; Emergency department; Thoracotomy
13.  Poor tolerance of beta-blockers by elderly patients with heart failure 
Despite the well-understood importance of beta-blocker therapy in heart failure, it is sometimes not possible to use beta-blockers in elderly patients due to poor tolerance. In this report, we describe the case of an 83-year-old patient with severe systolic heart failure complicated by aortic valve stenosis and atrial fibrillation. A simple therapeutic approach involving discontinuation of beta-blockers remarkably alleviated the symptoms such as left ventricular ejection fraction, and improved the chest radiography and laboratory findings; further, atrial fibrillation converted to sinus rhythm. It is important to carefully administer beta-blocker therapy to elderly patients with heart failure, especially after considering cardiac output.
PMCID: PMC2998243  PMID: 21152238
elderly; octogenarians; beta-blockers; heart failure
14.  Treatment of hepatic encephalopathy by on-line hemodiafiltration: a case series study 
It is thought that a good survival rate of patients with acute liver failure can be achieved by establishing an artificial liver support system that reliably compensates liver function until the liver regenerates or a patient undergoes transplantation. We introduced a new artificial liver support system, on-line hemodiafiltration, in patients with acute liver failure.
This case series study was conducted from May 2001 to October 2008 at the medical intensive care unit of a tertiary care academic medical center. Seventeen consecutive patients who admitted to our hospital presenting with acute liver failure were treated with artificial liver support including daily on-line hemodiafiltration and plasma exchange.
After 4.9 ± 0.7 (mean ± SD) on-line hemodiafiltration sessions, 16 of 17 (94.1%) patients completely recovered from hepatic encephalopathy and maintained consciousness for 16.4 ± 3.4 (7-55) days until discontinuation of artificial liver support (a total of 14.4 ± 2.6 [6-47] on-line hemodiafiltration sessions). Significant correlation was observed between the degree of encephalopathy and number of sessions of on-line HDF required for recovery of consciousness. Of the 16 patients who recovered consciousness, 7 fully recovered and returned to society with no cognitive sequelae, 3 died of complications of acute liver failure except brain edema, and the remaining 6 were candidates for liver transplantation; 2 of them received living-related liver transplantation but 4 died without transplantation after discontinuation of therapy.
On-line hemodiafiltration was effective in patients with acute liver failure, and consciousness was maintained for the duration of artificial liver support, even in those in whom it was considered that hepatic function was completely abolished.
PMCID: PMC2898817  PMID: 20492684
15.  Outcome of Patients with Localized Prostate Cancer Treated by Radiotherapy After Confirming the Absence of Lymph Node Invasion 
Management of lymph nodes in radiotherapy for prostate cancer is an issue for curative intent. To find the influence of lymph nodes, patients with T1–T3 prostate cancer and surgically confirmed negative nodes were treated with radiotherapy.
After lymphadenectomy, 118 patients received photon beam radiotherapy with 66 Gy to the prostate. No adjuvant treatment was performed until biochemical failure. After failure, hormone therapy was administered. Follow-up period was 57 months (mean).
Biochemical failure occurred in 47 patients. Few failures were observed in patients with low (24%) and intermediate risks (14%). In contrast, 64% of high-risk patients experienced failure, 97% of whom showed until 36 months. Most patients with failure responded well to hormone therapy. After 15 months (mean), a second biochemical failure occurred in 21% of patients who had the first failure, most of them were high risk. Factors involving failure were high initial and nadir prostate-specific antigen, advanced stage, short prostate-specific antigen-doubling time and duration between radiation and first failure. Failure showed an insufficient reduction in prostate-specific antigen after radiotherapy. Factor for second failure was prostate-specific antigen-doubling time at first failure.
Half of high-risk patients experienced biochemical failure, indicating one of the causes involves factors other than lymph nodes. Low-, intermediate- and the other half of high-risk patients did not need to take immediate hormone therapy after radiotherapy. After failure, delayed hormone therapy was effective. Prostate-specific antigen parameters were predictive factors for further outcome.
PMCID: PMC2893779  PMID: 20382633
prostate cancer; radiotherapy; biochemical failure; high risk; PSA-doubling time
16.  Evaluation of an algorithm for estimating a patient's life threat risk from an ambulance call 
Utilizing a computer algorithm, information from calls to an ambulance service was used to calculate the risk of patients being in a life-threatening condition (life threat risk), at the time of the call. If the estimated life threat risk was higher than 10%, the probability that a patient faced a risk of dying was recognized as very high and categorized as category A+. The present study aimed to review the accuracy of the algorithm.
Data collected for six months from the Yokohama new emergency system was used. In the system, emergency call workers interviewed ambulance callers to obtain information necessary to assess triage, which included consciousness level, breathing status, walking ability, position, and complexion. An emergency patient's life threat risk was then estimated by a computer algorithm applying logistic models. This study compared the estimated life threat risk occurring at the time of the emergency call to the patients' state or severity of condition, i.e. death confirmed at the scene by ambulance crews, resulted in death at emergency departments, life-threatening condition with occurrence of cardiac and/or pulmonary arrest (CPA), life-threatening condition without CPA, serious but not life-threatening condition, moderate condition, and mild condition. The sensitivity, specificity, predictive values, and likelihood ratios of the algorithm for categorizing A+ were calculated.
The number of emergency dispatches over the six months was 73,992. Triage assessment was conducted for 68,692 of these calls. The study targets account for 88.8% of patients who were involved in triage calls. There were 2,349 cases where the patient had died or had suffered CPA. The sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio and negative likelihood ratio of the algorithm at predicting cases that would result in a death or CPA were 80.2% (95% confidence interval: 78.6% - 81.8%), 96.0% (95.8% - 96.1%), 42.6% (41.1% - 44.0%), 99.2% (99.2% - 99.3%), 19.9 (18.8 - 21.1), and 0.21 (0.19 - 0.22), respectively.
A patient's life threat risk was quantitatively assessed at the moment of the emergency call with a moderate level of accuracy.
PMCID: PMC2770982  PMID: 19845937
18.  Class 1 Integron Containing Metallo-β-Lactamase Gene blaVIM-2 in Pseudomonas aeruginosa Clinical Strains Isolated in Japan 
Four blaVIM-2 gene-harboring Pseudomonas aeruginosa strains were identified. These strains possessed a class 1 integron harboring ORF1, blaVIM-2, and aacA4 gene cassettes. The transposon-mediated horizontal spread of the blaVIM-2 gene among these strains was suggested, which increases the threat that the blaVIM-2 gene will disseminate among diverse genera of bacteria.
PMCID: PMC321541  PMID: 14742222
19.  Telomere repeat DNA forms a large non-covalent complex with unique cohesive properties which is dissociated by Werner syndrome DNA helicase in the presence of replication protein A 
Nucleic Acids Research  2000;28(18):3642-3648.
We describe the unique structural features of a large telomere repeat DNA complex (TRDC) of >20 kb generated by a simple PCR using (TTAGGG)4 and (CCCTAA)4 as both primers and templates. Although large, as determined by conventional agarose gel electrophoresis, the TRDC was found to consist of short single-stranded DNA telomere repeat units of between several hundred and 3000 bases, indicating that it is a non-covalent complex comprising short cohesive telomere repeat units. S1 nuclease digestion showed that the TRDC contains both single- and double-stranded portions stable enough to survive glycerol density gradient centrifugation, precipitation with ethanol and gel electrophoresis. Sedimentation analysis suggests that a part of the TRDC is non-linear and consists of a three-dimensional network structure. After treatment with Werner DNA helicase the TRDC dissociated into smaller fragments, provided that human replication protein A was present, indicating that: (i) the TRDC is a new substrate for the Werner syndrome helicase; (ii) the telomere repeat sequence re-anneals rapidly unless unwound single-stranded regions are protected by replication protein A; (iii) the TRDC may provide a new clue to understanding deleterious telomere–totelomere interactions that can lead to genomic instability. Some properties of the TRDC account for the extra-chromosomal telomere repeat (ECTR) DNA that exists in telomerase-negative immortalized cell lines and may be involved in maintaining telomeres.
PMCID: PMC110730  PMID: 10982887
20.  Detection by Epitope-defined Monoclonal Antibodies of Werner DNA Helicases in the Nucleoplasm and Their Upregulation by Cell Transformation and Immortalization  
The Journal of Cell Biology  1999;144(1):1-9.
We prepared several monoclonal antibodies (mAbs) specific for the NH2- and COOH-terminal regions of the DNA helicase (WRN helicase) responsible for Werner's syndrome known as a premature aging disease. With these antibodies, we detected by immunoblot analysis the endogenous WRN helicase of a relative mass of 180 kD in several lines of cultured cells, but not in patient cells with a defined mutation. Immunocytochemical staining of proliferating fibroblasts and tumor cells showed that the major part of WRN helicase is in the nucleoplasm and not in the nucleolus. Similar experiments with a rat mAb specific to the mouse homologue of human WRN helicase yielded an identical conclusion. Although this nucleoplasmic staining was evident in cells in interphase, the condensed chromatin structure in metaphase was not stained by the same mAbs, suggesting that WRN helicases exist perhaps in a soluble form or bound to the unfolded chromatin structure. From quantitative immunoblot analysis, higher levels of WRN helicase were observed in all transformed cells and tumor cells examined than those of normal cells. The expression of WRN helicase was enhanced consistently in fibroblasts and B-lymphoblastoid cells by transformation with SV-40 and Epstein-Barr virus, respectively, suggesting that rapidly proliferating cells require a high copy numbers of WRN helicase.
PMCID: PMC2148112  PMID: 9885239
Werner's syndrome; RecQ DNA helicase; genetic instability; aging; nucleoplasmic localization

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