A high-resolution anorectal manometry parameter based on integrated pressurized volume: A study based on 204 male patients with constipation and 26 controls.

BACKGROUND: Conventional anorectal manometric parameters based on linear waves cannot properly predict balloon expulsion (BE) time. We aimed to determine the correlation between integrated pressurized volume (IPV) parameters during simulated evacuation (SE) and BE time in healthy individuals and constipated patients and to assess the correlation between each parameter and symptoms. METHODS: A total of 230 male participants (including 26 healthy volunteers and 204 chronically constipated patients) underwent high-resolution anorectal manometry (HRAM) and BE tests. The IPV was calculated by multiplying the amplitude, distance, and time from the HRAM profile. Receiver operating characteristic curve (ROC) analysis and partial least square regression (PLSR) were performed. KEY RESULTS: ROC analysis indicated that the IPV ratio between the upper 1 cm and lower 4 cm of the anal canal was more effective for predicting BE time (area under the curve [AUC]: 0.74, 95% confidence interval [CI]: 0.67-0.80, P < .01) than the conventional anorectal parameters, including defecation index and rectoanal gradient (AUC: 0.60, 95% CI: 0.52-0.67, P = .01). PLSR analysis of a linear combination of IPV parameters yielded an AUC of 0.79. Moreover, the IPV ratio showed a greater clinical correlation with patient symptoms than conventional parameters. CONCLUSIONS AND INFERENCES: The IPV parameters and the combination of IPV parameters via PLSR were more significantly correlated with BE time than the conventional parameters. Thus, this study presents a useful diagnostic tool for the evaluation of pathophysiologic abnormalities in dyssynergic defecation using IPV and BE time.

Validation of the Korean National Healthcare-associated Infections Surveillance System (KONIS): an intensive care unit module report.

BACKGROUND: National surveillance data should be validated to identify methodological problems within the surveillance programme and data quality issues. AIM: To test the validity of healthcare-associated infection (HAI) rate data from the Korean National Healthcare-associated Infections Surveillance System (KONIS). METHODS: Records from intensive care units of 12 (14.8%) of 81 participating hospitals for January-March 2014 were examined. The validation team reviewed 406 medical records of 110 patients with 114 reported HAIs - including 34 urinary tract infections (UTIs), 57 bloodstream infections (BSIs) and 23 cases of pneumonia (PNEU) - and 296 patients with no reported HAIs during one-day visits conducted in August and September 2014. The reviewers' diagnosis of HAI was regarded as the reference standard; in ambiguous cases, the KONIS Steering Committee confirmed the diagnosis of HAI. FINDINGS: Sensitivity values for UTIs, BSIs and PNEU were 85.3%, 74.0% and 66.7%, and specificity values were 98.7%, 99.1% and 98.7%, respectively. Positive predictive values were 85.3%, 94.7% and 78.3%, and negative predictive values were 98.7%, 94.6% and 97.7%, respectively. Sensitivity for PNEU was lower than that for UTIs and BSIs. The hospitals participating in KONIS infrequently reported conditions that were not HAIs. Sensitivity for BSIs was lower in this study than in KONIS validation studies conducted in 2008 and 2010. CONCLUSIONS: KONIS data are generally reliable; however, sensitivity for BSIs exhibited a decrease. This study shows the need for ongoing validation and continuous training of surveillance personnel to maintain the accuracy of surveillance data.

Serum hCG-ß levels of postovulatory day 12 and 14 with the sequential application of hCG-ß fold change significantly increased predictability of pregnancy outcome after IVF-ET cycle.

PURPOSE: To investigate hCG-ß level on postovulatory day (POD) 12 and its fold increase as predictors for pregnancy outcome after in vitro fertilization (IVF) cycles. METHODS: A retrospective cohort study was performed in total 1408 fresh and 598 frozen cycles between November 2008 and October 2011, which resulted in biochemical pregnancy, early pregnancy loss, or live birth of singleton pregnancy. The serum hCG-ß levels of POD 12 and 14 were compared among biochemical pregnancy, early pregnancy loss, and live birth groups. The cutoff values of POD 12 and 14 hCG-ß levels and the degree of hCG-ß increase from POD 12 to 14 were determined for each pregnancy outcome. RESULTS: POD 12 and 14 hCG-ß levels stratified based on pregnancy outcomes were significantly different among the biochemical pregnancy, early pregnancy loss, and live birth in both fresh and frozen cycles. Serum hCG-ß levels of POD 12 and 14 and the fold increase of hCG-ß levels from POD 12 to 14 significantly predict pregnancy outcomes after fresh and frozen cycles. Among these, the cutoff value of POD 14 hCG-ß had the highest sensitivity and positive predictive value (PPV). In fresh cycles, the cutoff values of POD 12 and 14 serum hCG-ß levels for clinical pregnancies were 30.2 mIU/mL (sensitivity 81.3 %, specificity 79.6 %, and PPV 92.3 %) and 70.5 mIU/mL (sensitivity 88.4 %, specificity 85.2 %, and PPV 94.7 %). In pregnancies with POD 12 serum hCG-ß levels ≥30.2 mIU/mL, the cutoff level of increase of hCG-ß for clinical pregnancy was 2.56 (sensitivity 73.6 %, specificity 72.4 %, and PPV 97.8 %). Sequential application of cutoff values such as POD 12 hCG-ß and fold increase of hCG-ß improved predictability of pregnancy outcome as compared with that of POD 12 hCG-ß alone. The cutoff values of POD 12 and 14 serum hCG-ß levels for live birth were 40.5 mIU/mL (sensitivity 75.2 %, specificity 72.6 %, PPV 78.9 %) and 104.5 mIU/mL (sensitivity 80.3 %, specificity 74.1 %, PPV 80.8 %). In the frozen cycles, the cutoff values of POD 12 and 14 serum hCG-ß level for clinical pregnancy were 31.5 IU/L (sensitivity 80.4 %, specificity 71.1 % and PPV 90 %) and 43.5 mIU/mL (sensitivity 72.6 %, specificity 71.7 %, PPV 77.2 %). In pregnancies with POD 12 serum hCG-ß level ≥31.5 mIU/mL, the cutoff value for fold increase of hCG-ß was 2.38 for clinical pregnancy (sensitivity 81.6 %, specificity 71.4 % and PPV 87.9 %). The cutoff values of POD 12 and 14 for live birth were 43.5 mIU/mL (sensitivity 72.6 %, specificity 71.7 %, PPV 77.2 %) and 101.6 mIU/mL (sensitivity 79.6 %, specificity 71.1 %, PPV 78.4 %). Sequential application of cutoff values for POD 12 hCG-ß level and fold increase of hCG-ß significantly increased PPV for live birth but not clinical pregnancy in frozen cycles. CONCLUSIONS: Early prediction of pregnancy outcome by using POD 12 and 14 cutoff levels and sequential application of cutoff value of fold increase could provide appropriate reference to health care providers to initiate earlier management of high-risk pregnancies and precise follow-up of abnormal pregnancies.

Five-year decreased incidence of surgical site infections following gastrectomy and prosthetic joint replacement surgery through active surveillance by the Korean Nosocomial Infection Surveillance System.

BACKGROUND: Surveillance of healthcare-associated infection has been associated with a reduction in surgical site infection (SSI). AIM: To evaluate the Korean Nosocomial Infection Surveillance System (KONIS) in order to assess its effects on SSI since it was introduced. METHODS: SSI data after gastrectomy, total hip arthroplasty (THA), and total knee arthroplasty (TKA) between 2008 and 2012 were analysed. The pooled incidence of SSI was calculated for each year; the same analyses were also conducted from hospitals that had participated in KONIS for at least three consecutive years. Standardized SSI rates for each year were calculated by adjusting for SSI risk factors. SSI trends were analysed using the Cochran-Armitage test. FINDINGS: The SSI rate following gastrectomy was 3.12% (522/16,918). There was a significant trend of decreased crude SSI rates over five years. This trend was also evident in analysis of hospitals that had participated for more than three years. The SSI rate for THA was 2.05% (157/7656), which decreased significantly from 2008 to 2012. The risk factors for SSI after THA included the National Nosocomial Infections Surveillance risk index, trauma, reoperation, and age (60-69 years). The SSI rate for TKA was 1.90% (152/7648), which also decreased significantly during a period of five years. However, the risk-adjusted analysis of SSI did not show a significant decrease for all surgical procedures. CONCLUSION: The SSI incidence of gastrectomy and prosthetic joint replacement declined over five years as a result of active surveillance by KONIS.

Trends in the distribution and antimicrobial susceptibility of causative pathogens of device-associated infection in Korean intensive care units from 2006 to 2013: results from the Korean Nosocomial Infections Surveillance System (KONIS).

BACKGROUND: For all countries, information on pathogens causing healthcare-associated infections is important in order to develop proper strategies for preventing and treating nosocomial infections. AIM: To assess the change in frequencies and antimicrobial resistance of pathogens causing device-associated infections (DAIs) in intensive care units (ICUs) in South Korea between July 2006 and June 2014. METHODS: Data from the Korean Nosocomial Infections Surveillance System (KONIS) were analysed, including three major DAI types in ICUs. FINDINGS: The frequency of Gram-negative bacteria gradually increased for central line-associated bloodstream infection (CLABSI) and ventilator-associated pneumonia (VAP) (from 24.6% to 32.6% and from 52.8% to 73.5%, respectively). By contrast, the frequency of Gram-positive bacteria decreased from 58.6% to 49.2% for CLABSI, and from 44.3% to 23.8% for VAP (P < 0.001). Staphylococcus aureus was the most frequent causative pathogen in CLABSI throughout the surveillance period, but for VAP was replaced as the most frequent pathogen by Acinetobacter baumannii as of 2010. Candida albicans was the most frequent pathogen for catheter-associated urinary tract infection. The meticillin resistance rate in S. aureus decreased from 95% to 90.2% (P < 0.001); amikacin resistance in Klebsiella pneumoniae and Escherichia coli decreased from 43.8% to 14.7% and from 15.0% to 1.8%, respectively (P < 0.001); imipenem resistance in A. baumannii increased from 52.9% to 89.8% (P < 0.001). CONCLUSION: The proportion of Gram-negative bacteria as nosocomial pathogens for CLABSI and VAP has increased. The prevalence of A. baumannii causing DAIs in Korean ICUs has increased rapidly, as has the rate of carbapenem resistance in these bacteria.

Remission of proteinuria indicates good prognosis in patients with diffuse proliferative lupus nephritis.

Proteinuria is a well-known risk factor for the progression of renal dysfunction in chronic kidney disease; however, its importance for estimating the prognosis of lupus nephritis requires verification. Korean adult patients with renal biopsy-diagnosed diffuse proliferative lupus nephritis who had undergone three or more consecutive urine protein to creatinine ratio or urine dipstick tests within six months after renal biopsy were enrolled. The cumulative risks, predictors, and outcomes of proteinuric remission and flare were evaluated. This study included 26 men and 167 women with a mean age at renal biopsy of 31.2 ± 9.8 years. Eighty-two (42.5%) patients experienced proteinuric remission during the follow-up period. During a mean follow-up of 157.9 ± 69.5 months, among patients who achieved proteinuric remission, one died, one developed end-stage renal disease (ESRD), and two had composite outcomes; among patients without remission, nine died, 24 developed ESRD, and 30 had composite outcomes. Patients who achieved proteinuric remission had a 0.089-fold risk (95% CI: 0.011-0.736) of mortality, 0.110-fold risk (95% CI: 0.013-0.904) of incident ESRD, and 0.210-fold risk (95% CI: 0.048-0.920) of a composite outcome compared to patients without remission. Among the 82 patients who achieved proteinuric remission, 59 (72.0%) experienced at least one proteinuria flare; however, relapse did not correlate with the incidence of outcomes. In conclusion, proteinuric remission is an independent predictive prognostic marker of good renal survival and mortality, regardless of the interval from biopsy to remission, recurrence of proteinuria after remission, renal function status at remission, or hematuria remission.

Trends in the incidence rate of device-associated infections in intensive care units after the establishment of the Korean Nosocomial Infections Surveillance System.

BACKGROUND: The effectiveness of continuous nationwide surveillance on healthcare-associated infections should be investigated in each country. AIM: To assess the rate of device-associated infections (DAIs) in intensive care units (ICUs) since the establishment of the Korean Nosocomial Infections Surveillance System (KONIS). METHODS: Nationwide data were obtained on the incidence rate of DAI in ICUs reported to KONIS by all participating hospitals. The three major DAIs were studied: ventilator-associated pneumonia (VAP), central line-associated bloodstream infection (CABSI), and catheter-associated urinary tract infection (CAUTI). The pooled and year-wise incidence rates (cases per 1000 device-days) of these DAIs were determined for the period 2006 and 2012. In addition, data from institutions that had participated in KONIS for at least three consecutive years were analysed separately. FINDINGS: The number of ICUs participating in KONIS gradually increased from 76 in 2006 to 162 in 2012. Between 2006 and 2012, the incidence rate per 1000 device-days for VAP decreased significantly from 3.48 to 1.64 (F = 11, P < 0.01), for CAUTI the rate decreased non-significantly from 1.85 to 1.26 (F = 2.02, P = 0.07), and for CABSI the rate also decreased non-significantly from 3.4 to 2.57 (F = 1.73, P = 0.12). In the 132 ICUs that had participated in KONIS for at least three consecutive years, the VAP rate significantly decreased from the first year to third year (F = 20.57, P < 0.01), but the rates of CAUTI (F = 1.06, P = 0.35) and CABSI (F = 1.39, P = 0.25) did not change significantly. CONCLUSION: The decreased incidence rate of VAP in ICUs in Korea might be associated with the continuous prospective surveillance provided by KONIS.

Long-term efficacy of biofeedback therapy in patients with dyssynergic defecation: results of a median 44 months follow-up.

BACKGROUND: The beneficial effect of biofeedback therapy (BFT) over a period of more than 2 years has not been studied in a large group of patients. The aim of this study was to evaluate the long-term efficacy of BFT for dyssynergic defecation (DD). METHODS: We evaluated the results for 347 consecutive constipated patients with DD who underwent BFT for a median of five sessions between 2004 and 2009. Initial responses were assessed immediately after the completion of BFT. A responder was defined as a subject with at least a three-point improvement from before to after BFT on an 11-point global bowel satisfaction (GBS) scale, or a two-point improvement if the baseline GBS was more than six points. The probability of remaining a responder was estimated by non-parametric maximum likelihood estimation. KEY RESULTS: The initial response rate to BFT was 72.3% (n = 251), Parkinson's disease and higher baseline GBS scores were associated with initial non-response. The long-term efficacy of BFT was analyzed in 103 patients who were followed up for more than 6 months; the initial effects of BFT were maintained in 85 of the patients (82.5%) during a median of 44 months of follow-up (IQR = 12-68). The probability of remaining a responder was 60% at 2 years, and 58% at 5 years. CONCLUSIONS & INFERENCES: The efficacy of BFT is maintained for more than 2 years after BFT in a considerable proportion of constipated patients with DD. BFT is effective and durable treatment for managing DD.

Normal values for high-resolution anorectal manometry/topography in a healthy Korean population and the effects of gender and body mass index.

BACKGROUND: High-resolution manometry (HRM) based on spatiotemporal plots is increasingly being used. The aim this study was to evaluate, for the first time, the influence of gender, with adjustment for age, body mass index (BMI), and vaginal delivery, on anorectal functions in asymptomatic adults. METHODS: Fifty-four asymptomatic healthy subjects (M : F = 27 : 27; age = 20-67 years) who were matched by age and gender were enrolled prospectively. We evaluated anorectal pressures, rectal sensation using a HRM probe, and balloon expulsion time. Multivariate linear regression analysis was performed to identify the independent effects of each factor. KEY RESULTS: Anal resting pressure (median [IQR]; 32 [18] vs 46 [17] mmHg, p < 0.001), anal squeeze pressure (75 [28] vs 178 [72] mmHg, p < 0.001), rectal pressure (33 [16] vs 53 [46] mmHg, p = 0.009) and anal pressure (16 [17] vs 30 [36] mmHg, p = 0.019) during simulated evacuation with rectal distention, and the threshold for the desire to defecate (60 [20] vs 80 [60] mL, p = 0.020) were significantly lower in women than in men. BMI was positively correlated with anal resting pressure (95% CI: 0.598-2.947) and negatively correlated with the threshold for first sensation (95% CI: -0.099 to -0.015). Vaginal delivery did not affect any of the anorectal HRM parameters. CONCLUSIONS & INFERENCES: HRM parameters may be associated with gender and BMI. Therefore, gender and BMI should be taken into consideration when interpreting HRM results.

Characteristics of testosterone deficiency syndrome in men with chronic kidney disease and male renal transplant recipients: a cross-sectional study.

OBJECTIVES: Testosterone deficiency syndrome (TDS) is common among male patients with chronic kidney disease (CKD). We compared the characteristics of TDS in men with CKD versus renal transplantation (RT) with those of age-matched normal controls. MATERIALS AND METHODS: The 129 patients were: RT recipients (n = 25) group I, CKD patients (n = 37) group II, and controls (n = 67). We performed estimates of testosterone, hemoglobin (Hgb), hematocrit (Hct), glucose, creatinine, and lipid profile. Self-assessment questionnaires-International Index of Erectile Function (IIEF), Aging Males' Symptoms (AMS), Center for Epidemiologic Studies Depression Scale-were used to evaluate erectile function, testosterone deficiency, and depression, respectively. We also investigated morning erection as well as the presence and duration of erectile dysfunction (ED). RESULTS: Group I (RT) showed significantly higher serum testosterone levels than group II (CKD), who displayed significantly worse erectile function, more severe testosterone deficiency symptoms, and a greater trend toward depression. Similarly, the prevalences of ED and TDS were significantly greater in group II than group I. Group I and controls differed significantly only in the results of serologic tests, such as serum creatinine, Hgb, and glucose and lipid profiles, but not in serum testosterone levels, scores of self-assessment questionnaires, or prevalence of ED or TDS. Serum testosterone levels correlated significantly with scores on the IIEF and AMS questionnaires in both group II and controls, but not group I. CONCLUSIONS: RT recipients showed higher serum testosterone levels and a lower prevalence of TDS with milder symptom severity than CKD patients. RT recipients beyond the early acute posttransplant period, displayed serum testosterone levels and TDS prevalence similar to those of healthy controls. Unlike CKD patients and normal controls, serum testosterone did not significantly influence TDS symptoms in RT recipients.

The effects of cyclophosphamide and mycophenolate on end-stage renal disease and death of lupus nephritis.

Debate continues about the optimal treatment modality of lupus nephritis (LN). We compared the efficacy and safety of intravenous cyclophosphamide (CYC) and mycophenolate mofetil (MMF) for LN treatment in Korea. After searching for systemic lupus erythematosus (SLE) patients diagnosed between 1998 and 2007 with the diagnostic code of ICD10, we selected the 71 patients who were treated with CYC or MMF without any other immunosuppressant except systemic steroid. Composite outcome was defined as progression to end-stage renal disease (ESRD) and/or all-cause mortality. The initial manifestations of the CYC group were more severe than those of the MMF group. The mean daily MMF dose was 980 ± 100 mg for 21.67 ± 18.25 months. The mean monthly dose per CYC pulse therapy was 850 ± 30 mg for 17.04 ± 13.15 months. The incidence of composite outcome was 5/20 (25%) in the MMF group and 4/51 (7.8%) in the CYC group. The relative risk (RR) for composite outcome in the CYC group was 0.249 (95% CI for RR: 0.067-0.934, p = 0.039) compared with the MMF group with Cox's hazard proportional analysis. In Kaplan-Meier analysis, the probability of composite outcome was lower in the CYC group than in the MMF group (Log rank test p-value = 0.026). The results of this retrospective study suggest that intravenous CYC therapy may be more efficacious in averting ESRD and death than MMF. These results need to be confirmed in a larger randomized controlled trial.

A deubiquitinating enzyme, UCH/CeUBP130, has an essential role in the formation of a functional microtubule-organizing centre (MTOC) during early cleavage in C. elegans.

BACKGROUND: Deubiquitinating enzymes generate monomeric ubiquitin in protein degradation pathways and are known to be important for the early development in many organisms. RESULTS: RNA interference experiments targeted for a UBP homologue, UCH/CeUBP130, in C. elegans resulted in cell division defective embryos. Immunostaining localized UCH/CeUBP130 in the sperm and at the microtubule-organizing centre (MTOC) during early cleavage. Furthermore, the embryonic lethal phenotype was rescued by mating with wild-type males. CONCLUSIONS: Since it is known that the MTOC in the fertilized embryo is contributed by sperm asters in C. elegans, we suggest that UCH/CeUBP130 and ubiquitin protein degradation pathways may be involved in microtubule-based sperm aster formation. Therefore UCH/CeUBP130 is necessary for the formation of a functional MTOC in the fertilized embryo of C. elegans.

Regulation of gene expression, cellular localization, and in vivo function of Caenorhabditis elegans DNA topoisomerase I.

BACKGROUND: DNA topoisomerase I is dispensable in yeast, but is essential during the embryogenesis of Drosophila and mouse. In order to determine functions of the enzyme in the development of Caenorhabditis elegans, phenotypes resulting from the deficiency were observed and correlated with the expression of the gene. RESULTS: The transcriptional regulation of the C. elegans DNA topoisomerase I gene was investigated by mRNA localization and reporter gene expression in C. elegans. The mRNA was expressed in the gonad and in the early embryos, followed by a rapid decrease in its level during the late embryonic stage. A reporter gene expression induced by the 5'-upstream DNA sequence appeared at the comma stage of embryos, continued through the L1 larval stage, and began to decrease gradually afterwards. The DNA topoisomerase I protein was immuno-localized in the nuclei of meiotic gonad cells and interphase embryonic cells, and unexpectedly in centrosomes of mitotic embryonic cells. Double-stranded RNA interference of DNA topoisomerase I gene expression resulted in pleiotropic phenotypes showing abnormal gonadogenesis, oocyte development and embryogenesis. CONCLUSION: These phenotypes, along with expressional regulations, demonstrate that DNA topoisomerase I plays important roles in rapidly growing germ cells and embryonic cells.

The endonuclease activity of the yeast Dna2 enzyme is essential in vivo.

Dna2 is a multifunctional enzyme in yeast that possesses endonuclease activity well suited to remove RNA-DNA primers of Okazaki fragments, raising the question of whether endonuclease activity is essential for in vivo Dna2 function. Systematic site-directed mutations of amino acid residues in Saccharomyces cerevisiae DNA2 conserved in the central region of many eukaryotic DNA2 homologs allowed us to identify mutant dna2 alleles that were divided into three groups based on the viability of the mutant cells: (i) viable; (ii) inviable only when expression was repressed; (iii) inviable. Biochemical analyses of recombinant mutant Dna2 proteins isolated from the latter two groups revealed that they possessed normal ATPase/helicase activity, but were impaired in their endonuclease activity. Cells expressing mutant Dna2 enzymes partially impaired in endonuclease activity were viable, but were unable to grow when expression of their mutant Dna2 enzymes was further reduced. Their growth was restored when the mutant Dna2 proteins decreased in nuclease activity were induced to overexpress. In contrast, mutant Dna2 proteins lacking endonuclease activity did not allow cells to grow under any conditions tested. These in vivo and in vitro results demonstrate that the endonuclease activity of Dna2 is essential for Okazaki fragment processing.

Functional characterization of Caenorhabditis elegans DNA topoisomerase IIIalpha.

To investigate the function of a DNA topoisomerase III enzyme in Caenorhabditis elegans, the full-length cDNA of C.elegans DNA topoisomerase IIIalpha was cloned. The deduced amino acid sequence exhibited identities of 48 and 39% with those of human DNA topoisomerase IIIalpha and Saccharomyces cerevisiae DNA topoisomerase III, respectively. The overexpressed polypeptide showed an optimal activity for removing negative DNA supercoils at a relatively high temperature of 52-57 degrees C, which is similar to the optimum temperatures of other eukaryotic DNA topoisomerase III enzymes. When topoisomerase IIIalpha expression was interfered with by a cognate double-stranded RNA injection, pleiotropic phenotypes with abnormalities in germ cell proliferation, oogenesis and embryo-genesis appeared. These phenotypes were well correlated with mRNA expression localized in the meiotic cells of gonad and early embryonic cells.

Helical periodicity of (PNA)2.(DNA) triplexes in strand displacement complexes.

To study the helical structure in a P-loop formed by an invasion of oligopyrimidine peptide nucleic acid (PNA) into DNA duplex, bent DNA fragments containing a homopurine.homopyrimidine sequence between two bent DNA loci were prepared. As the spacer DNA length between the two bent loci varied by 1 bp over one helical turn, the electrophoretic mobility, reflecting the overall extent of DNA bending, was modulated sinusoidally in non-denaturing 5% polyacrylamide gel. When the bent DNA fragments differing in the spacer DNA length were preincubated with an oligopyrimidine PNA, the gel mobilities were changed due to a P-loop formation. By analyzing the gel mobility data with variations of the P-loop size, average helical parameters at the P-loop structure were determined. (PNA)2. (DNA) triplex within a P-loop had the helical periodicities of 15. 6(0.2) bp per turn at 20 degrees C and 17.4(0.7) bp per turn at 10 degrees C. In addition, the results indicate that a helical unwinding by 57(7) degrees at 20 degrees C and 37(13) degrees at 10 degrees C is present at the two junctions between a P-loop and its adjacent DNA duplex.

Cloning and characterization of the 5'-flanking region for the human topoisomerase III gene.

The human DNA topoisomerase III (hTOP3) gene encodes a topoisomerase homologous to the Escherichia coli DNA topoisomerase I subfamily. To understand the mechanisms responsible for regulating hTOP3 expression, we have cloned the 5'-flanking region of the gene coding for the hTOP3 and analyzed its promoter activity. The presence of a single transcription initiation site was suggested by primer extension analysis. The hTOP3 gene promoter is moderately high in GC content and lacks a canonical TATA box, suggesting that hTOP3 promoter has overall similarity to promoters of a number of housekeeping genes. Examination of the promoter sequence indicated the presence of four Sp-1 consensus binding sequences and a putative initiator element surrounding the transcription initiation site. Transient expression of a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the 52-base pair region from -326 to -275 upstream of the transcription initiation site includes a positive cis-acting element(s) for the efficient expression of hTOP3 gene. On the basis of gel mobility shift and supershift assays, we demonstrated that both YY1 and USF1 transcription factors can bind to the 52-base pair region. When HeLa cells were transiently transfected with a mutant construct which had disabled both YY1- and USF1-binding sites, the luciferase activity was greatly reduced, suggesting that these binding elements play a functional role in the basal activation of the hTOP3 promoter. Transfection studies with mutations that selectively impaired YY1 or USF1 binding suggested that both YY1 and USF1 function as activators in the hTOP3 promoter.

Alternative splicing in the Caenorhabditis elegans DNA topoisomerase I gene.

5'-end cDNA fragments of the Caenorhabditis elegans DNA topoisomerase I gene were obtained by rapid amplification of the cDNA ends from C. elegans mRNAs. The presence of a SL1 sequence at the 5'-terminus of the cDNA sequence suggested trans-splicing of the pre-mRNA. By comparing the complete cDNA sequence with the genomic lambda DNA clones, the gene structure composed of five exons was established. Alternative splicing deleting the second exon was observed in the cDNA fragments obtained by a gene-specific reverse transcription followed by polymerase chain reactions. The shorter mRNA missing the second exon was expressed at all the developmental stages, while the full-length mRNA was present only in embryos.