[Meteorological factors and related lag effects on scrub typhus in southwestern Yunnan].

Objective: To analyze the influence and related lag effects of meteorological factors on scrub typhus (ST) in southwestern Yunnan, to provide a reference for the corresponding prevention and treatment measures. Methods: Data on ST and meteorology in Yunnan province from 2007 to 2018 were collected. A distributed lag nonlinear model was conducted to study the cumulative lag effects of temperature, relative humidity, and rainfall on ST. Results: From 2007-2018, a total of 19 975 ST cases were reported in southwestern Yunnan. Weekly mean temperature, mean relative humidity, and the risk of ST all showed J-shaped curves. The cumulative risk of ST increased with mean temperature >23 ℃, mean relative humidity >80%, and cumulative rainfall between 20 and 60 mm or over 100 mm, weekly. Taking the median value as the reference, higher temperature (22.27 ℃, 23.45 ℃), relative humidity (80.14%, 84.38%) and rainfall (37.17 mm, 74.42 mm) all increased the risk of disease while lower temperature (11.22 ℃,14.83 ℃), relative humidity (53.18%,65.36%) and rainfall (0.00 mm,0.55 mm) showed opposite effects. The temperature-lag effect lasted for 10 and 16 weeks, respectively, with ST's risk the highest during the week of exposure. Humidity-lag effects usually last for 10 and 17 weeks. The lag effect of rainfall lasted for 25 weeks, while the disease's risk was the highest in the 4th week. Conclusion: Factors as temperature, relative humidity, and precipitation showed nonlinear and lag effects on ST. High temperature, high relative humidity, and an appropriate amount of rainfalls increase the risk of ST. The authorities of public health should implement effective prevention and control measures according to meteorological conditions.

Circulating exosomal miRNAs as diagnostic biomarkers in Parkinson's disease.

OBJECTIVE: This research has showed that exosomal miRNAs from cerebrospinal fluid could act as biomarkers for Parkinson's disease (PD). However, no analysis has been conducted to explore the potential value of exosomal miRNAs from plasma. PATIENTS AND METHODS: 52 patients with PD were included in study group. 48 healthy adults were included in control group. Blood samples were collected from all those people and then exosomes were extracted from the plasma. RESULTS: Compared with controls, patients with PD showed a significantly higher expression of circulating exosomal miR-331-5p. ROC curve showed that the area values under the curve of miR-331-5p and miR-505 were 0.849 and 0.898, respectively. CONCLUSIONS: Exosomal miRNAs, including miR-331-5p and miR-505, could potentially act as biomarkers for PD.

[Report of incidence and mortality of gallbladder cancer in China, 2014].

Objective: The incidence and mortality of gallbladder cancer from Chinese cancer registries in 2014 were analyzed to describe the prevalence of gallbladder cancer in China. Methods: Incidence and mortality data of gallbladder cancer in 2014 derived from registration data in 2017, collected by the National Central Cancer Registry (NCCR). Qualified data from 339 cancer registries were calculated after evaluating. According to the national population data of 2014, the gallbladder cancer incidence and mortality of China in 2014 were stratified by the area, gender and age.The age composition of standard population of Chinese census in 2000 and Segi's population were used for age-standardizes incidence and mortality in China and worldwide. Results: 339 cancer registries cover a total of 288 243 347 population including 146 203 891 males and 142 039 456 females (144 061 915 in urban and 144 181 432 in rural areas). The mortality to incidence ratio of gallbladder cancer was 0.74. The morphologically verified cases (MV%) and death certificate-only cases (DCO%) were 48.38% and 2.66%, respectively. Unclear diagnosis cases (UB%) was 0.48%. The crude incidence of gallbladder cancer in China in 2014 was 3.82/100 000, which accounted for 1.37% of new cancer cases (4.48/100 000 in urban areas and 3.01/100 000 in rural areas, 3.59/100 000 for male and 4.05/100 000 for female). Age-standardized incidence rates by Chinese standard population (ASR China) and world standard population (ASR world) were 2.38/100 000 and 2.37/100 000, respectively, and the cumulative incidence rate (0-74 age years old) was 0.27%.Besides, the crude mortality of gallbladder cancer was 2.86/100 000 (3.47/100 000 in urban areas and 2.12/100 000 in rural areas, 2.59/100 000 for male and 3.14/100 000 for female). Age-standardized mortality rates by ASR China and ASR world were 1.72/100 000 and 1.71/100 000, with a cumulative mortality rate (0-74 age years old) of 0.19%. Conclusion: The incidence and mortality of gallbladder cancer were significantly different between the city and country, while not obviously different between the female and male.

[Significance of bacteria detection with filter paper method on diagnosis of diabetic foot wound infection].

Objective: To evaluate the significance of bacteria detection with filter paper method on diagnosis of diabetic foot wound infection. Methods: Eighteen patients with diabetic foot ulcer conforming to the study criteria were hospitalized in Liyuan Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology from July 2014 to July 2015. Diabetic foot ulcer wounds were classified according to the University of Texas diabetic foot classification (hereinafter referred to as Texas grade) system, and general condition of patients with wounds in different Texas grade was compared. Exudate and tissue of wounds were obtained, and filter paper method and biopsy method were adopted to detect the bacteria of wounds of patients respectively. Filter paper method was regarded as the evaluation method, and biopsy method was regarded as the control method. The relevance, difference, and consistency of the detection results of two methods were tested. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of filter paper method in bacteria detection were calculated. Receiver operating characteristic (ROC) curve was drawn based on the specificity and sensitivity of filter paper method in bacteria detection of 18 patients to predict the detection effect of the method. Data were processed with one-way analysis of variance and Fisher's exact test. In patients tested positive for bacteria by biopsy method, the correlation between bacteria number detected by biopsy method and that by filter paper method was analyzed with Pearson correlation analysis. Results: (1) There were no statistically significant differences among patients with wounds in Texas grade 1, 2, and 3 in age, duration of diabetes, duration of wound, wound area, ankle brachial index, glycosylated hemoglobin, fasting blood sugar, blood platelet count, erythrocyte sedimentation rate, C-reactive protein, aspartate aminotransferase, serum creatinine, and urea nitrogen (with F values from 0.029 to 2.916, P values above 0.05), while there were statistically significant differences among patients with wounds in Texas grade 1, 2, and 3 in white blood cell count and alanine aminotransferase (with F values 4.688 and 6.833 respectively, P<0.05 or P<0.01). (2) According to the results of biopsy method, 6 patients were tested negative for bacteria, and 12 patients were tested positive for bacteria, among which 10 patients were with bacterial number above 1×10(5)/g, and 2 patients with bacterial number below 1×10(5)/g. According to the results of filter paper method, 8 patients were tested negative for bacteria, and 10 patients were tested positive for bacteria, among which 7 patients were with bacterial number above 1×10(5)/g, and 3 patients with bacterial number below 1×10(5)/g. There were 7 patients tested positive for bacteria both by biopsy method and filter paper method, 8 patients tested negative for bacteria both by biopsy method and filter paper method, and 3 patients tested positive for bacteria by biopsy method but negative by filter paper method. Patients tested negative for bacteria by biopsy method did not tested positive for bacteria by filter paper method. There was directional association between the detection results of two methods (P=0.004), i. e. if result of biopsy method was positive, result of filter paper method could also be positive. There was no obvious difference in the detection results of two methods (P=0.250). The consistency between the detection results of two methods was ordinary (Kappa=0.68, P=0.002). (3) The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of filter paper method in bacteria detection were 70%, 100%, 1.00, 0.73, and 83.3%, respectively. Total area under ROC curve of bacteria detection by filter paper method in 18 patients was 0.919 (with 95% confidence interval 0-1.000, P=0.030). (4) There were 13 strains of bacteria detected by biopsy method, with 5 strains of Acinetobacter baumannii, 5 strains of Staphylococcus aureus, 1 strain of Pseudomonas aeruginosa, 1 strain of Streptococcus bovis, and 1 strain of bird Enterococcus. There were 11 strains of bacteria detected by filter paper method, with 5 strains of Acinetobacter baumannii, 3 strains of Staphylococcus aureus, 1 strain of Pseudomonas aeruginosa, 1 strain of Streptococcus bovis, and 1 strain of bird Enterococcus. Except for Staphylococcus aureus, the sensitivity and specificity of filter paper method in the detection of the other 4 bacteria were all 100%. The consistency between filter paper method and biopsy method in detecting Acinetobacter baumannii was good (Kappa=1.00, P<0.01), while that in detecting Staphylococcus aureus was ordinary (Kappa=0.68, P<0.05). (5) There was no obvious correlation between the bacteria number of wounds detected by filter paper method and that by biopsy method (r=0.257, P=0.419). There was obvious correlation between the bacteria numbers detected by two methods in wounds with Texas grade 1 and 2 (with r values as 0.999, P values as 0.001). There was no obvious correlation between the bacteria numbers detected by two methods in wounds with Texas grade 3 (r=-0.053, P=0.947). Conclusions: The detection result of filter paper method is in accordance with that of biopsy method in the determination of bacterial infection, and it is of great importance in the diagnosis of local infection of diabetic foot wound.

The intrapapillary capillary loop (IPCL) changes in superficial esophageal lesions.

The aims of this study were to investigate intrapapillary capillary loops (IPCLs) of superficial esophageal lesions changes in different types classified by the Japan Esophageal Society classification. The calibers, areas, and densities of IPCLs were detected in 34 cases of esophageal lesions using immunohistochemical analysis. Statistically significant differences in calibers, areas, and densities of IPCLs were observed between type A, type B1/B2, and type B3 area (P < 0.001). In conclusion, the results of this observation showed the Japan Esophageal Society classification of IPCL would help endoscopists to diagnose the type and the invasion depth of lesion in esophagus, and decide the treatment strategy.

Role of pelvic radiotherapy for locally advanced rectal cancer and synchronous unresectable distant metastases.

PURPOSE: To evaluate the efficacy and safety of pelvic irradiation combined systematic chemotherapy in patients with locally advanced (cT3-T4 and/or cN+) rectal cancer and synchronous unresectable distant metastases. PATIENTS AND METHODS: A total of 76 eligible patients who received pelvic radiotherapy and concurrent capecitabine-based chemotherapy were retrospectively reviewed. Patients survival curves were constructed using the Kaplan-Meier method, and a multivariate analysis was performed to identify independent prognostic factors. RESULTS: Most of the adverse events were mild during the period of combined chemoradiotherapy. Twenty-two patients experienced resection of primary tumour and 16 patients underwent radical surgery of all lesions. Only five patients had pelvic progression during the follow-up period. The median progression-free survival and median overall survival were 13 and 30 months, respectively. Radical surgery of all lesions following chemoradiotherapy was found to be an independent prognostic factor according to multivariate analysis. CONCLUSIONS: Pelvic irradiation combined with systematic chemotherapy in patients with locally advanced rectal cancer and synchronous unresectable distant metastases is effective and tolerable, both for pelvic and distant control. A curative resection following chemoradiotherapy was associated with prolonged survival.

[Validity, reliability, and acceptability of the brief version of the self-management knowledge, attitude, and behavior assessment scale for diabetes patients].

OBJECTIVE: To evaluate the validity, reliability, and acceptability of the brief version of the self-management knowledge, attitude, and behavior (KAB) assessment scale for diabetes patients. METHODS: Diabetes patients who were managed at the Xinkaipu Community Health Service Center of Tianxin in Changsha, Hunan Province were selected for survey by cluster sampling. A total of 350 diabetes patients were surveyed using the brief scale to collect data on knowledge, attitudes, and behaviors of self-management. Content validity was evaluated by Pearson correlation coefficient between the brief scale and subscales of knowledge, attitude, and behavior. Structure validity was evaluated by factor analysis, and discrimination validity was evaluated by an independent sample t-test between the high-score and low-score groups. Reliability was tested by internal consistency reliability and split-half reliability. The evaluation indexes of internal consistency reliability were Cronbach's α coefficients, θ coefficient, and Ω coefficient. Acceptability was evaluated by valid response rate and completion time of the brief scale. RESULTS: A total of 346(98.9%) valid questionnaires were returned, with average survey time of (11.43±3.4) minutes. Average score of the brief scale was 78.85 ± 11.22; scores of the knowledge, attitude, and behavior subscales were 16.45 ± 4.42, 21.33 ± 2.03, and 41.07 ± 8.34, respectively. Pearson correlation coefficients between the brief scale and the knowledge, attitude, and behavior subscales were 0.92, 0.42, and 0.60, respectively; P-values were all less than 0.01, indicating that the face validity and content validity of the brief scale were achieved to a good level. The common factor cumulative variance contribution rate of the brief scale and three subscales was from 53.66% to 61.75%, which achieved more than 50% of the approved standard. There were 11 common factors; 41 of the total 42 items had factor loadings above 0.40 in their relevant common factor, indicating that the brief scale and three subscales had good construct validity. Patients were divided into a high-score group and a low-score group, then scores of the brief scale and three subscales were compared between the groups using a t-test. The results were all significant, indicating that the brief scale and three subscales had good discriminate validity. Mean scores of the brief scale and three subscales of the high-score group were 91.55±6.81, 19.51±2.17, 22.74±1.88, and 49.30±6.20, respectively; these were higher than the low-score group (65.89±5.79, 12.29±4.76, 20.22±1.88, and 33.39±6.17, respectively) with t-values 27.76, 13.31, 9.20, and 17.56 (P-values were less than 0.001). The Cronbach's α coefficient, θ coefficient, Ω coefficient, and split-half reliability of the brief scale were 0.83, 0.87, 0.96, and 0.84, respectively. These values for the three subscales were all above 0.70, except for the θ coefficient of the attitude subscale with 0.64, indicating that the brief scale and three subscales had acceptable internal consistency reliability. CONCLUSION: The brief version of the diabetes self-management knowledge, attitude, and behavior assessment scale showed good acceptability, validity, and reliability, to responsibly evaluate self-management KAB among patients with diabetes.

Comparative evaluation of the therapeutic effect of metformin monotherapy with metformin and acupuncture combined therapy on weight loss and insulin sensitivity in diabetic patients.

OBJECTIVE: Obesity induces insulin resistance (IR), the key etiologic defect of type 2 diabetes mellitus (T2DM). Therefore, an incidence of obesity-induced diabetes is expected to decrease if obesity is controlled. Although Metformin is currently one of the main treatment options for T2DM in obese patients, resulting in an average of 5% weight loss, adequate weight control in all patients cannot be achieved with Metformin alone. Thus, additional therapies with a weight loss effect, such as acupuncture, may improve the effectiveness of Metformin.Subjective:We designed this randomized clinical trial (RCT) to compare the effects of Metformin monotherapy with that of Metformin and acupuncture combined therapy on weight loss and insulin sensitivity among overweight/obese T2DM patients, to understand whether acupuncture plus Metformin is a better approach than Metformin only on treating diabetes. To understand whether acupuncture can be an insulin sensitizer and, if so, its therapeutic mechanism. RESULTS: Our results show that Metformin and acupuncture combined therapy significantly improves body weight, body mass index (BMI), fasting blood sugar (FBS), fasting insulin (FINS), homeostasis model assessment (HOMA) index, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), leptin, adiponectin, glucagon-like peptide-1 (GLP-1), resistin, serotonin, free fatty acids (FFAs), triglyceride (TG), low-density lipoprotein cholesterol (LDLc), high-density lipoprotein cholesterol (HDLc) and ceramides. CONCLUSIONS: Consequently, Metformin and acupuncture combined therapy is more effective than Metformin only, proving that acupuncture is an insulin sensitizer and is able to improve insulin sensitivity possibly by reducing body weight and inflammation, while improving lipid metabolism and adipokines. As a result, electro-acupuncture (EA) might be useful in controlling the ongoing epidemics in obesity and T2DM.

A triple suicide gene strategy that improves therapeutic effects and incorporates multimodality molecular imaging for monitoring gene functions.

Gene-directed enzyme prodrug therapy (GDEPT), or suicide gene therapy, has shown promise in clinical trials. In this preclinical study using stable cell lines and xenograft tumor models, we show that a triple-suicide-gene GDEPT approach produce enhanced therapeutic efficacy over previous methods. Importantly, all the three genes (thymidine kinase, cytosine deaminase and uracil phosphoribosyltransferase) function simultaneously as effectors for GDEPT and markers for multimodality molecular imaging (MMI), using positron emission tomography, magnetic resonance spectroscopy and optical (fluorescent and bioluminescent) techniques. It was demonstrated that MMI can evaluate the distribution and function/activity of the triple suicide gene. The concomitant expression of these genes significantly enhances prodrug cytotoxicity and radiosensitivity in vitro and in vivo.

Systematic review and meta-analysis of randomized controlled trials comparing stapled haemorrhoidopexy with conventional haemorrhoidectomy.

BACKGROUND: This paper compares stapled haemorrhoidopexy with conventional haemorrhoidectomy for the treatment of haemorrhoids. METHODS: An electronic literature search was undertaken to identify primary studies and systematic reviews. Results on efficacy and safety were analysed. A meta-analysis was conducted to examine long-term outcomes. RESULTS: Twenty-nine randomized clinical trials recruiting 2056 patients were identified. Meta-analysis showed that stapled haemorrhoidopexy was less painful than conventional haemorrhoidectomy. Stapled haemorrhoidopexy required a shorter inpatient stay (weighted mean difference (WMD) -0.95 (95 per cent confidence interval (c.i.) -1.32 to -0.59) days; P < 0.001) and operating time (WMD -11.42 (95 per cent c.i. -18.26 to -4.59) min; P = 0.001). It was also associated with a faster return to normal activities (WMD -11.75 (95 per cent c.i. -21.42 to -2.08) days; P = 0.017). No significant difference was noted between the two techniques in terms of the total incidence of complications. Stapled haemorrhoidopexy was associated with a higher rate of recurrent disease (relative risk 2.29 (95 per cent c.i. 1.57 to 3.33); P < 0.001). CONCLUSION: Stapled haemorrhoidopexy offers some short-term benefits over conventional operation but the total complication rates are similar for both techniques. Stapled haemorrhoidopexy is associated with a higher rate of recurrent disease.

A preliminary assessment of consumer's exposure to pesticide residues in fisheries products.

The fisheries products in this study comprise fish, bivalve, crustacean and cephalopod collected from different types of markets in Taiwan between the years 2001 and 2003. A total of 91 pesticide residues belonging to four major pesticide groups were tested and analyzed. The test results show that 65.40% of fish, 93.55% of shellfish, 84.92% of crustacean and 98.33% of cephalopod samples contain no detectable residues. There are only two kinds (organochlorine and organophosphate) of totally six pesticides (DDTs, dieldrin, chlorpyrifos, fenitrotion, fenthion and prothion) that have been detected from the fisheries products in this study. For there were pesticides present in the fish products, consumption of fisheries product there was no zero risk. But the exposure of consumer did not exceed the acceptable daily intakes (ADI). For male, there was the highest risk in exposure to dieldrin, which the percentage of ADI was 93.56%. This study also suggests that a yearly monitoring program for organophosphate pesticide residues in fish is necessity.

Allotype imbalance or microsatellite mutation in low-grade soft tissue sarcomas of the extremities in adults.

The ability to repair DNA double-strand breaks is essential to maintain chromosomal stability. Virtually all soft tissue sarcomas contain chromosomal instabilities, including clonal aberrations and cytogenetic aberrations. However, the relevance of DNA-dependent protein kinase (DNA-PK) in the pathogenesis of soft tissue sarcoma has not been clarified. The main aim of this work is to compare the prognostic impact of genotypic imbalance in low-grade soft tissue sarcomas of the extremities, and to correlate this with the translational level of DNA-PK. This study investigated 28 adult low-grade malignant spindle cell tumours of the extremities, predominantly fibrosarcomas, for loss of heterozygosity (LOH) and microsatellite mutation on flanking regions of each DNA-PK subunit, with identical immunophenotypes. Twelve different polymorphic markers flanking the specific loci of three subunits comprise the genetic map of DNA-PK, at 22q13, 2q35, and 8q11. Translational activity was also analysed by western blot and conventional immunohistochemistry. The overall sarcoma 5-year survival rate was 61.7%. LOH was identified in the specific coding region of DNA-PK in 39.29% for the DNA-PK catalytic subunit (cs), 17.86% for Ku70, and only 7.14% for Ku80. A positive LOH for DNA-PKcs was shown to be a significant factor for poor survival (log rank test p = 0.0160). Immunoreactivity and immunoblot results correlated with the loss of DNA-PKcs allotype in soft tissue sarcoma (Fisher's exact test p = 0.0037). Ku70 and DNA-PKcs were almost identical in terms of immunoreactivity. In conclusion, whereas microsatellite mutation seems an uncommon event during the evolution of low-grade fibrosarcoma of the extremities in adults, the loss of DNA-PKcs defines a biologically more aggressive subset.

DNA-PKcs is critical for telomere capping.

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is critical for DNA repair via the nonhomologous end joining pathway. Previously, it was reported that bone marrow cells and spontaneously transformed fibroblasts from SCID (severe combined immunodeficiency) mice have defects in telomere maintenance. The genetically defective SCID mouse arose spontaneously from its parental strain CB17. One known genomic alteration in SCID mice is a truncation of the extreme carboxyl terminus of DNA-PKcs, but other as yet unidentified alterations may also exist. We have used a defined system, the DNA-PKcs knockout mouse, to investigate specifically the role DNA-PKcs specifically plays in telomere maintenance. We report that primary mouse embryonic fibroblasts (MEFs) and primary cultured kidney cells from 6-8 month-old DNA-PKcs-deficient mice accumulate a large number of telomere fusions, yet still retain wild-type telomere length. Thus, the phenotype of this defect separates the two-telomere related phenotypes, capping, and length maintenance. DNA-PKcs-deficient MEFs also exhibit elevated levels of chromosome fragments and breaks, which correlate with increased telomere fusions. Based on the high levels of telomere fusions observed in DNA-PKcs deficient cells, we conclude that DNA-PKcs plays an important capping role at the mammalian telomere.

Dissipation of epoxiconazole in the paddy field under subtropical conditions of Taiwan.

The environmental fate and distribution of fungicide epoxiconazole were studied by a rice paddy field model ecosystem. One week before the head-sprouting stage, rice plant was treated separately once with OPUS (tradename of epoxiconazole) 12% SC 2.1 kg ha(-1) and 1.4 kg ha(-1), respectively. Soil, water and rice plant were sampled seven days intervals nine times after application. The bioconcentration factor of epoxiconazole on mosquito fish in the ecosystem was also determined, based on the amounts of epoxiconazole content both in fish and water. This was initiated one day after the fungicide treatment, and continued for four days. In addition, the residue of epoxiconazole in rice grains was analyzed after harvest. After harvest, both planted water spinach (Ipomoea aquatica Forsk) and edible amaranth (Amaranthus mangostanüs L.) were analyzed. The results showed that epoxiconazole degraded in the local environment under the experimental conditions described. The degradation equations were in accordance with the first order kinetics. The DT50 of soil, field water and rice plant were 20-69 days, 11-20 days and 14-39 days, respectively. The bioconcentration factors of epoxiconazole on mosquito fish were 12.9 and 10.6 from 2.1 kg ha(-1) and 1.4 kg ha(-1) treatment, respectively. Residues of epoxiconazole in both rice and harvest vegetables were non-detectable. This indicates that epoxiconazole applied to rice at the recommended rates and application frequencies will not accumulate on rice grain and successive cropping vegetables.

Rb-associated protein 46 (RbAp46) suppresses the tumorigenicity of adenovirus-transformed human embryonic kidney 293 cells.

The retinoblastoma suppressor (Rb)-associated protein 46 (RbAp46) is a nuclear protein of the WD-repeat protein family and a component of the histone deacetylase complex that physically interacts with Rb. We demonstrated that RbAp46 is a gene up-regulated by the Wilms' tumor suppressor (WT1) and functions as a negative regulator of cell growth. Here we have investigated the ability of RbAp46 to inhibit malignant phenotype of adenovirus-transformed human embryonic kidney (HEK) 293 cells in tumorigenesis assays. We have found that expression of RbAp46 suppressed clonal growth of HEK 293 cells in soft agar and inhibited tumor growth of these cells in nude mice. Furthermore, expression of RbAp46 resulted in an increase of cells in the G2/M fraction of cell cycle and augmented apoptosis in serum-starved cells. The results suggest that high levels of RbAp46 expression inhibit the transformation of tumor cells through interfering with normal cell cycle and/or enhancing apoptotic cell death.

Rb-associated protein 46 (RbAp46) inhibits transcriptional transactivation mediated by BRCA1.

The retinoblastoma suppressor (Rb)-associated protein 46 (RbAp46) is a member of the WD-repeat protein family and a component of the histone modifying and remodeling complexes. Previously, we demonstrated that RbAp46 is a potent growth inhibitor that can suppress the transformed phenotype of tumor cells. To explore the molecular mechanisms of RbAp46 function, we used RbAp46 as a bait in a yeast two-hybrid screening and found that RbAp46 interacts specifically with the C-terminal region of BRCA1 (the BRCT domain), a domain involved in the t transactivation activity of BRCA1. Coimmunoprecipitation assays demonstrated that the interaction of RbAp46 with BRCA1 requires the first two of the four Trp-Asp (WD)-repeats of RbAp46. We also showed that expression of RbAp46 represses the transactivation activity mediated by the BRCT/Gal4 fusion protein and inhibits the transactivation of the p21 promoter mediated by the full-length BRCA1. Interestingly, the association of BRCA1 and RbAp46 is disrupted in cells treated with DNA-damaging agents. These results suggest that RbAp46 may specifically interact with BRCA1 and modulate its transactivation activity in response to DNA damage.

Cytotoxic mechanism of XK469: resistance of topoisomerase IIbeta knockout cells and inhibition of topoisomerase I.

Topoisomerase IIbeta knockout mouse cells (beta-/-) were found to have only slight resistance to m-AMSA, a dual topoisomerase IIalpha-IIbeta poison, as compared to wild-type cells (beta+/+) during 1 h or 3 day exposures to the drug. In contrast, the beta-/- cells were greater than threefold resistant to XK469, a selective topoisomerase IIbeta poison during three day drug exposures (beta+/+ IC(50) = 175 microM, beta-/- IC(50) = 581 microM). Short term (1 h) exposure to XK469 was not cytotoxic to either beta-/- or beta+/+ cells, suggesting that anticancer therapy with XK469 may be more efficacious if systemic levels can be prolonged. During studies on topoisomerase activity in nuclear extracts of the beta+/+ and beta-/- cells, we found evidence that XK469 is a weak topoisomerase I catalytic inhibitor. The high IC(50) for topoisomerase I inhibition (2 mM) suggests that topoisomerase I is not a significant target for XK469 cytotoxicity.

ATM is required for IkappaB kinase (IKKk) activation in response to DNA double strand breaks.

Following challenge with proinflammatory stimuli or generation of DNA double strand breaks (DSBs), transcription factor NF-kappaB translocates from the cytoplasm to the nucleus to activate expression of target genes. In addition, NF-kappaB plays a key role in protecting cells from proapoptotic stimuli, including DSBs. Patients suffering from the genetic disorder ataxia-telangiectasia, caused by mutations in the ATM gene, are highly sensitive to inducers of DSBs, such as ionizing radiation. Similar hypersensitivity is displayed by cell lines derived from ataxia-telangiectasia patients or Atm knockout mice. The ATM protein, a member of the phosphatidylinositol 3-kinase (PI3K)-like family, is a multifunctional protein kinase whose activity is stimulated by DSBs. As both ATM and NF-kappaB deficiencies result in increased sensitivity to DSBs, we examined the role of ATM in NF-kappaB activation. We report that ATM is essential for NF-kappaB activation in response to DSBs but not proinflammatory stimuli, and this activity is mediated via the IkappaB kinase complex. DNA-dependent protein kinase, another member of the PI3K-like family, PI3K itself, and c-Abl, a nuclear tyrosine kinase, are not required for this response.

[Cloning and expression of human anti-colonic cancer single-chain Fv fragment].

We report a new strategy for the generation of human anticolon cancer monoclonal antibodies based on the molecular cloning and expression of immunoglobulin variable region cDNAs derived from peripheral blood mononuclear cells (PBMC) that were transformed by EBV. The immortalized B cells secreting tumor-specific antibodies were identified by HRT-18 cell ELISA and cloned by limiting dilution. Heavy- and light-chain VH-CH1 (gamma) and V kappa-C kappa cDNAs were rescued from ELISA-positive cells wells by RT-PCR. VH and V kappa were amplified by 2nd PCR and linked them together by 3rd PCR assembly with the use of a (Gly4Ser)3 linker. The ScFv cDNAs were then cloned into the fUSE 5 vector and displayed on phage. Phage clones were selected on HRT-18 cells and normal human PBMC. ELISA tested phage clones randomly picked after each panning step. > 80% of the clones showed a strong ELISA reaction, demonstrating the effectiveness of the panning procedure for selecting anticolon cancer antibodies. This approach offers an effective method by combining in vitro immunization, B-cell expansion for enrichment of specific B-lymphocytes, PCR gene cloning and phage display to make high-affinity human anticolon cancer monoclonal antibody molecules.