Hyperprogressive disease during PD-1/PD-L1 blockade in patients with non-small-cell lung cancer.

BACKGROUND: Immune checkpoint blockade with Programmed cell death 1 (PD-1)/PD-L1 inhibitors has been effective in various malignancies and is considered as a standard treatment modality for patients with non-small-cell lung cancer (NSCLC). However, emerging evidence show that PD-1/PD-L1 blockade can lead to hyperprogressive disease (HPD), a flair-up of tumor growth linked to dismal prognosis. This study aimed to evaluate the incidence of HPD and identify the determinants associated with HPD in patients with NSCLC treated with PD-1/PD-L1 blockade. PATIENTS AND METHODS: We enrolled patients with recurrent and/or metastatic NSCLC treated with PD-1/PD-L1 inhibitors between April 2014 and November 2018. Clinicopathologic variables, dynamics of tumor growth, and treatment outcomes were analyzed in patients with NSCLC who received PD-1/PD-L1 blockade. HPD was defined according to tumor growth kinetics (TGK), tumor growth rate (TGR), and time to treatment failure (TTF). Immunophenotyping of peripheral blood CD8+ T lymphocytes was conducted to explore the potential predictive biomarkers of HPD. RESULTS: A total of 263 patients were analyzed. HPD was observed in 55 (20.9%), 54 (20.5%), and 98 (37.3%) patients according to the TGK, TGR, and TTF. HPD meeting both TGK and TGR criteria was associated with worse progression-free survival [hazard ratio (HR) 4.619; 95% confidence interval (CI) 2.868-7.440] and overall survival (HR, 5.079; 95% CI, 3.136-8.226) than progressive disease without HPD. There were no clinicopathologic variables specific for HPD. In the exploratory biomarker analysis with peripheral blood CD8+ T lymphocytes, a lower frequency of effector/memory subsets (CCR7-CD45RA- T cells among the total CD8+ T cells) and a higher frequency of severely exhausted populations (TIGIT+ T cells among PD-1+CD8+ T cells) were associated with HPD and inferior survival rate. CONCLUSION: HPD is common in NSCLC patients treated with PD-1/PD-L1 inhibitors. Biomarkers derived from rationally designed analysis may successfully predict HPD and worse outcomes, meriting further investigation of HPD.

Three-gene predictor of clinical outcome for gastric cancer patients treated with chemotherapy.

To identify transcriptional profiles predictive of the clinical benefit of cisplatin and fluorouracil (CF) chemotherapy to gastric cancer patients, endoscopic biopsy samples from 96 CF-treated metastatic gastric cancer patients were prospectively collected before therapy and analyzed using high-throughput transcriptional profiling and array comparative genomic hybridization. Transcriptional profiling identified 917 genes that are correlated with poor patient survival after CF at P<0.05 (poor prognosis signature), in which protein synthesis and DNA replication/recombination/repair functional categories are enriched. A survival risk predictor was then constructed using genes, which are included in the poor prognosis signature and are contained within identified genomic amplicons. The combined expression of three genes-MYC, EGFR and FGFR2-was an independent predictor for overall survival of 27 CF-treated patients in the validation set (adjusted P=0.017), and also for survival of 40 chemotherapy-treated gastric cancer patients in a published data set (adjusted P=0.026). Thus, combined expression of MYC, EGFR and FGFR2 is predictive of poor survival in CF-treated metastatic gastric cancer patients.

Aspirin use and bleeding risk after endoscopic submucosal dissection in patients with gastric neoplasms.

BACKGROUND AND STUDY AIM: The risk of bleeding after endoscopic submucosal dissection (ESD) in patients with early gastric neoplasms who do not discontinue aspirin for the procedure has not been established. We aimed to investigate whether post-ESD gastric bleeding is increased in patients who take aspirin. PATIENTS AND METHODS: Patients who underwent ESD for early gastric neoplasms at the National Cancer Center Hospital, Korea, between November 2008 and January 2011 were enrolled. The risk of post-ESD bleeding was evaluated using Poisson regression analysis. RESULTS: We categorized 514 patients into three groups according to aspirin intake at the time of the procedure: patients who never used aspirin (n=439), patients who interrupted aspirin use for 7 days or more (n=56), and patients who continuously used aspirin (n=19). Post-ESD bleeding occurred in 4.1% (21/514) overall, and was more frequent in continuous aspirin users (4/19 [21.1%]) than in those who never used aspirin (15/439 [3.4%]) (P=0.006) and those with interrupted aspirin use (2/56 [3.6%]) (P=0.033). Multivariate analysis showed that use of aspirin by itself was associated with post-ESD bleeding (relative risk [RR] 4.49; 95% confidence interval [95%CI] 1.09-18.38). The resumption of clopidogrel combined with aspirin use (RR 26.71, 95%CI 7.09-100.53), and increased iatrogenic ulcer size (RR 1.52, 95%CI 1.14-2.02), were significantly associated with post-ESD bleeding. CONCLUSIONS: Continuous aspirin use increases the risk of bleeding after gastric ESD. Aspirin use should be stopped in patients with a low risk for thromboembolic disease to minimize bleeding complications.

Effect of chemotherapy on the outcome of self-expandable metallic stents in gastric cancer patients with malignant outlet obstruction.

BACKGROUND AND STUDY AIM: Chemotherapy has been suggested to affect the outcome of pyloric stent placement. This study aimed to investigate the association between the response to chemotherapy and pyloric stent outcome. PATIENTS AND METHODS: Data from 113 patients with inoperable gastric cancer who received chemotherapy after pyloric stent placement at the National Cancer Center hospital were analyzed retrospectively. Chemotherapy response was assessed using the Response Evaluation Criteria in Solid Tumors. A Cox proportional hazards model was used to evaluate the effect of chemotherapy response on the complications of stents. RESULTS: The stent migration rate was 15.9% (18/113) and the re-stenosis rate was 30.1% (34/113). The response rates to chemotherapy were higher in the first-line group than in the salvage chemotherapy group (second-line or more) (44.8% [26/58] vs. 3.6% [2/55], respectively; P < 0.001). The proportion of patients with long time-to-progression (> 8 weeks) was also higher in the first-line than the salvage chemotherapy group (81.0% [47 /58] vs. 61.8% [34 /55], respectively; P = 0.036). Although, the response to chemotherapy was not associated with stent migration or re-stenosis, a long time-to-progression (adjusted hazard ratio [aHR] = 0.29, 95% confidence interval [CI] 0.13-0.67) and first-line chemotherapy (aHR = 0.45, 95%CI 0.22-0.93) were protective factors against re-stenosis in the multivariate analysis. In patients who received first-line chemotherapy, the median duration of patency of covered and uncovered stents was 20 weeks (95%CI 11-29) and 33 weeks (95 %CI 18-48), respectively (P = 0.317). CONCLUSIONS: A long time-to-progression and first-line chemotherapy were significant protective factors against re-stenosis. In chemotherapy-naïve gastric cancer patients with pyloric obstruction, placement of an uncovered stent followed by chemotherapy can be considered to increase stent patency.

CYP2A6 and ERCC1 polymorphisms correlate with efficacy of S-1 plus cisplatin in metastatic gastric cancer patients.

BACKGROUND: We evaluated the association between polymorphisms of cytochrome P450 2A6 (CYP2A6)/excision repair cross-complementation group 1 (ERCC1)/X-ray repair cross-complementing group 1(XRCC1) and treatment outcomes of metastatic gastric cancer (MGC) patients treated with S-1/cisplatin. METHODS: Among MGC patients (n=108), who received S-1 (40 mg m(-2) b.i.d., days 1-14) and cisplatin (60 mg m(-2), day 1) every 3 weeks, we analysed the wild-type allele (W) and variants (V) of CYP2A6 (*4, *7, *9, *10), and the polymorphisms of ERCC1 (rs11615, rs3212986) and XRCC1 (rs25487). RESULTS: Patients having fewer CYP2A6 variants had better response rates (W/W vs W/V other than *1/*4 vs V/V or *1/*4=66.7 vs 58.3 vs 32.3%; P=0.008), time to progression (TTP) (7.2 vs 6.1 vs 3.5 months, P=0.021), and overall survival (23.2 vs 15.4 vs 12.0 months, P=0.004). ERCC1 19442C>A (rs3212986) was also associated with response rate (C/C, 46.7% vs C/A, 55.3% vs A/A, 87.5%) (P=0.048) and TTP (4.4 vs 7.6 vs 7.9 months) (P=0.012). Patients carrying both risk genotypes of CYP2A6 (V/V or 1/*4) and ERCC1 19442C>A (C/C) vs those carrying none showed an adjusted odds ratio of 0.113 (P=0.004) for response, and adjusted hazard ratios of 3.748 (P=0.0001) for TTP and 2.961 (P=0.006) for death. CONCLUSION: Polymorphisms of CYP2A6 and ERCC1 19442C>A correlated with the efficacy of S-1/cisplatin.

Phase II study of a triplet regimen of S-1 combined with irinotecan and oxaliplatin in patients with metastatic gastric cancer: clinical and pharmacogenetic results.

BACKGROUND: The aim of this study was to investigate the efficacy and safety of S-1/irinotecan/oxaliplatin (TIROX) in metastatic gastric cancer (MGC) and the association between treatment outcome and uridine diphosphate-glucuronosyltransferase (UGT) 1A polymorphisms. PATIENTS AND METHODS: Patients with previously untreated MGC received S-1 40 mg/m(2) b.i.d. on days 1-14 and irinotecan 150 mg/m(2) plus oxaliplatin 85 mg/m(2) on day 1 every 3 weeks. RESULTS: Forty-four patients were enrolled. In intent-to-treat analysis, the objective response rate was 75%, including the complete response (CR) rate of 14%. The median time to progression and overall survival was 10.2 and 17.6 months, respectively. Ten (26%) of the 39 patients with primary gastric tumor showed biopsy-confirmed gastric CR. Grade 3/4 neutropenia developed in 66% of patients and grade 3 febrile neutropenia in 16%. The most common grade 3 nonhematologic toxic effects were abdominal pain (18%), anorexia (16%), and diarrhea (14%). UGT1A polymorphisms were associated with significantly higher incidence of grade 4 leukopenia (UGT1A1*6), neutropenia (UGT1A1*6, UGT1A6*2, and UGT1A7*3), grade 3/4 febrile neutropenia (UGT1A1*6), and grade 3 abdominal pain (UGT1A1*6). CONCLUSIONS: The TIROX regimen induced marked tumor reduction and promising survival with a manageable toxicity profile in MGC patients. UGT1A genotype may be predictive of TIROX toxicity.

Risk of high-grade dysplasia or carcinoma in gastric biopsy-proven low-grade dysplasia: an analysis using the Vienna classification.

BACKGROUND AND AIMS: Therapeutic guidelines have not yet been established for low-grade gastric adenomas/dysplasias (LGD), which have a low risk of progression to high-grade adenomas/dysplasias (HGD) or to invasive carcinomas. This study aimed to evaluate risk factors for HGD/carcinoma that indicate a need for resection in biopsy-proven LGD lesions. PATIENTS AND METHODS: In total, 236 LGD lesions from 208 consecutive patients treated with endoscopic resection (ER) were retrospectively studied between 2004 and 2008. The Vienna classification was used for histological diagnosis. A generalized estimating equation (GEE) logistic regression model was used for multivariate analysis. RESULTS: Among the 236 LGD lesions, the final pathology diagnosed 9 (3.8 %) as invasive carcinoma (category 5), 71 (30.1 %) as HGD (category 4), 148 (62.7 %) as LGD (category 3), and 8 (3.4 %) as negative/indefinite for dysplasia (category 1/2). Lesions ≥ 1 cm were classified as HGD/carcinoma in 39.4 % of patients (65/165). Multivariate analysis indicated that size of ≥ 1 cm (OR 1.93 [95 % CI, 1.06 - 3.52]), depressed morphology (OR 3.81 [95 % CI, 1.22 - 11.9]), and erythema (OR 2.49 [95 % CI, 1.31 - 4.72]) were significantly associated with HGD/carcinoma. The OR increased to 47.6 (95 % CI, 4.27 - 530.65) when the risk factors were all positive. The sensitivity and negative predictive value for ≥ 1 risk factors were 93.8 % and 90.9 %, respectively. As the number of risk factors of a lesion increased, the specificity and positive predictive value also increased. CONCLUSIONS: Endoscopic resection can be recommended if a low-grade dysplastic lesion has at least one of the following risk factors: depressed morphology, surface erythema, or a size of 1 cm or greater. For lesions that have none of the three risk factors, follow-up endoscopy is recommended.

Risk of hemorrhagic gastropathy associated with colonoscopy bowel preparation using oral sodium phosphate solution.

BACKGROUND AND STUDY AIMS: Oral sodium phosphate (NaP) solution is widely used for colonoscopy bowel preparation and it may cause aphthous ulcers in the colon. Our aim was to evaluate whether oral NaP solution is associated with gastric mucosal lesions. METHODS: A total of 20 070 individuals underwent esophagogastroduodenoscopy (EGD) with colonoscopy, and 4271 individuals underwent EGD without colonoscopy, for cancer screening. Oral NaP solutions were used for bowel preparation prior to colonoscopy. Hemorrhagic gastropathy was graded using a five-point scale for erosive mucosal injury. The effect of NaP bowel preparation on hemorrhagic gastropathy was estimated using multiple logistic regression analysis with odds ratios (ORs) and 95 % confidence intervals (CIs). RESULTS: The incidence of hemorrhagic gastropathy was 1.6 % (70/4271) in the EGD only group and 4.0 % (809/20 070) in the EGD with colonoscopy group ( P < 0.001, unadjusted OR 2.55, 95 %CI 1.99 - 3.27). The ORs for mild (grade 1 - 2), moderate (grade 3), and severe (grade 4) hemorrhagic gastropathy according to NaP use were 1.92 (95 %CI 1.45 - 2.54), 4.72 (95 %CI 2.65 - 8.47), and 5.99 (95 %CI 1.46 - 24.63), respectively. After adjustment for confounding factors, NaP solution was a significant risk factor for acute hemorrhagic gastropathy in the multivariate analysis (OR 1.92, 95 %CI 1.34-2.74). In addition, male sex, a body mass index (kg/m (2)) of less than 20, concurrent use of antihypertensive or nonsteroidal anti-inflammatory drugs, and duodenal ulcers were independently associated with the development of hemorrhagic gastropathy. HELICOBACTER PYLORI infection and atrophic gastritis were negatively associated with hemorrhagic gastropathy. CONCLUSION: Oral NaP bowel preparation for colonoscopy was associated with hemorrhagic gastropathy.

DNA sequence and comparative analysis of chimpanzee chromosome 22.

Human-chimpanzee comparative genome research is essential for narrowing down genetic changes involved in the acquisition of unique human features, such as highly developed cognitive functions, bipedalism or the use of complex language. Here, we report the high-quality DNA sequence of 33.3 megabases of chimpanzee chromosome 22. By comparing the whole sequence with the human counterpart, chromosome 21, we found that 1.44% of the chromosome consists of single-base substitutions in addition to nearly 68,000 insertions or deletions. These differences are sufficient to generate changes in most of the proteins. Indeed, 83% of the 231 coding sequences, including functionally important genes, show differences at the amino acid sequence level. Furthermore, we demonstrate different expansion of particular subfamilies of retrotransposons between the lineages, suggesting different impacts of retrotranspositions on human and chimpanzee evolution. The genomic changes after speciation and their biological consequences seem more complex than originally hypothesized.

Polyaniline-carboxymethyl cellulose nanocomposite for cholesterol detection.

Cholesterol oxidase (ChOx) has been covalently immobilized onto polyaniline-carboxymethyl cellulose (PANI-CMC) nanocomposite film deposited onto indium-tin-oxide (ITO) coated glass plate using glutaraldehyde as a cross-linker. Fourier transform infrared (FTIR) spectroscopic and electrochemical studies have been used to characterize the PANI-CMC/ITO nanocomposite electrode and ChOx/PANI-CMC/ITO bioelectrode. Scanning electron microscopy (SEM) studies reveal the formation of PANI-CMC nanocomposite fibers of size approximately 150 nm in diameter. The ChOx/PANI-CMC/ITO bioelectrode exhibits linearity as 0.5-22 mM, detection limit as 1.31 mM, sensitivity as 0.14 mA/mM cm2, response time as 10 s and shelf-life of about 10 weeks when bioelectrode is stored at 4 degrees C. The low value of Michaelis-Menten constant (K(m)) obtained as 2.71 mM reveals high affinity of immobilized ChOx for PANI-CMC/ITO nanocomposite electrode.

Learning curve for identification of sentinel lymph node based on a cumulative sum analysis in gastric cancer.

BACKGROUND/AIMS: Lymph node metastasis is the most important point to consider when deciding on the modality of resection in patients with early gastric cancer. This study was conducted to evaluate the learning curve for identification of sentinel lymph nodes in patients with gastric cancer. METHODS: The investigators included the results from 2 prospective series of sentinel lymph node mapping. Cumulative sum (CUSUM) analysis was performed to assess the learning curves for identification of sentinel lymph nodes at CUSUM target success rates of 95%. RESULTS: One surgeon performed 135 sentinel lymph node mappings for 2 prospective series. The success rate exceeded 90%. The learning period for gastric cancer sentinel node mapping was calculated to be 26 cases for achieving a 95% success rate. Multiple logistic regression analysis for successful detection of sentinel nodes showed that surgical experience of sentinel lymph node mapping was an independent factor for successful detection of sentinel nodes. CONCLUSIONS: This study suggests that the learning period for identification of sentinel lymph nodes in gastric cancer would be 26 cases. In clinical trials for gastric cancer with sentinel lymph node mapping, the learning curve should be considered to minimize bias due to surgical factors.

Comparative assessment of gene quantification using real-time PCR and water quality parameters in unsanitary landfill.

Because of increasing demanding for development of direct ecological landfill monitoring methods, there is a requirement for the condition of landfills and their influence on the environment to be characterized by the behavior of enzymes and bacteria mainly concerned with biochemical reaction in the landfills. This study was thus conducted to understand the fates of contaminants in association with groundwater quality parameters. For the study, groundwater was seasonally sampled from four closed unsanitary landfills in which microbial diversity was simultaneously obtained by 16S rDNA methods. Subsequently, a number of the specific genes of representative bacteria and encoding enzymes were quantified by real-time PCR. The relationship between water quality parameters and gene quantification were compared based on correlation factors. Correlation between DSR gene and BOD was greater than 0.8 while NSR gene and nitrate were related more than 0.9. For MTOT, it was at the highest related at 100% over BOD/COD and Dde genes were correlated over 0.8. In addition, anaerobic genes and DO were also related more than 0.8, showing anaerobic reactions generally dependent upon DO. As demonstrated in the study, molecular biological investigation and water quality parameters are highly co-linked, so that quantitative real-time PCR could be cooperatively used for assessing landfill stabilization in association with the conventional monitoring parameters.

Microbiological monitoring of acid mine drainage treatment systems and aquatic surroundings using real-time PCR.

In general, acid mine drainage (AMD) causes low pH and high metal concentrations in mining areas and surroundings. The aim of this research was to achieve microbiological monitoring for AMD and to assess whether mine water outflows have any ecological effects on the aqueous ecosystem receiving effluents from different types of treatment system. The water quality of aquatic sample was analyzed and the molecular biological diversity of the samples was assessed using 16S rRNA methods, which were implemented to determine which bacteria existed throughout various unit processes for different AMD treatment systems and their receiving water environments. Acidiphilium cryptum, a heterotrophic acidophile, was found at the AMD sites, and Rhodoferax ferrireducens, which can reduce iron using insoluble Fe(III) as an electron acceptor, was detected at many AMD treatment facilities and downstream of the treatment processes. Subsequently, quantitative real-time PCR was conducted on specific genes of selected bacteria. Surprisingly, obvious trends were observed in the relative abundance of the various bacteria that corresponded to the water quality analytical results. The copy number of Desulfosporosinus orientus, a sulfate reducing bacteria, was also observed to decrease in response to decreases in metals according to the downstream flow of the AMD treatment system.

Optimization of biological wastewater treatment conditions for 1,4-dioxane decomposition in polyester manufacturing processes.

The solvent stabilizer 1,4-dioxane could have harmful effects on an ecosystem. The discharge limit of 1,4-dioxane in a body of water will be regulated at 5 mg/L in Republic of Korea. Thus, the currently operating activated sludge used in the manufacture of polyester should be properly treated to meet the regulations. Accordingly, the removal rate of 1,4-dioxane and its microbial properties was assessed at K, H and T corporations. The highest removal efficiencies were recorded at H. However, the concentration of 1,4-dioxane in the effluent of T exceeded the criterion. In addition, a microbial degradation test was conducted on 100 mg/L of 1,4-dioxane inoculated with the activated sludge from each of the three corporations. After 7 days, the 1,4-dioxane was completely removed with the H sludge and efficiencies were 67% in the T sludge and 52% in the K sludge. These results confirm that the biodegradability of 1,4-dioxane may vary in relation to the microbial properties. The microbial diversity of activated sludge of each company was therefore investigated by 16S rDNA cloning methods. In conclusion, the activated sludge of H is the most effective for the biodegradation of 1,4-dioxane. This fact is of significant concern for the industrial sector.

Decomposition of 1,4-dioxane by photo-Fenton oxidation coupled with activated sludge in a polyester manufacturing process.

The cyclic ether 1,4-dioxane is a synthetic industrial chemical that is used as a solvent in producing paints and lacquers. The EPA and the International Agency for Research on Cancer(IARC) classified 1,4-dioxane as a GROUP B2(probable human) carcinogen. 1,4-dioxane is also produced as a by-product during the manufacture of polyester. In this research, a polyester manufacturing company (i.e. K Co.) in Gumi, Korea was investigated regarding the release of high concentrations of 1,4-dioxane (about 600 mg/L) and whether treatment prior to release should occur to meet with the level of the regulation standard (e.g., 5 mg/L in 2010). A 10 ton/day pilot-scale treatment system using photo-Fenton oxidation was able to remove approximately 90% of 1,4-dioxane under the conditions that concentrations of 2800 ppm H(2)O(2) and 1,400 ppm FeSO(4) were maintained along with 10 UV-C lamps (240 microW/cm(2)) installed and operated continuously during aeration. However, the effluent concentration of 1,4-dioxane was still high at about 60 mg/L where TOC concentration in the effluent had been moreover increased due to decomposed products such as aldehydes and organic acids. Thus, further investigation is needed to see whether the bench scale (reactor volume, 8.9 L) of activated sludge could facilitate the decomposition of 1,4-dioxane and their by-products (i.e., TOC). As a result, 1,4-dioxane in the effluent has been decreased as low as 0.5 mg/L. The optimal conditions for the activated sludge process that were obtained are as follows: DO, 3-3.5 mg/L; HRT, 24 h; SRT 15 d; MLSS, 3,000 mg/L. Consequently, photo-Fenton oxidation coupled with activated sludge can make it possible to efficiently decompose 1,4-dioxane to keep up with that of the regulation standard.

Phase I/II study of S-1 combined with weekly docetaxel in patients with metastatic gastric carcinoma.

We designed a phase I/II trial of S-1 combined with weekly docetaxel to determine the maximum tolerated dose (MTD) and recommended dose (RD) and to evaluate the efficacy and toxicity in metastatic gastric carcinoma (MGC). Patients with measurable disease received S-1 orally b.i.d. on days 1-14 and docetaxel intravenously on days 1 and 8 every 3 weeks. In phase I (n=30), each cohort received escalating doses of S-1 (30-45 mg m(-2) b.i.d.) and docetaxel (25-40 mg m(-2)); MTD was 45 mg m(-2) b.i.d. S-1/35 mg m(-2) docetaxel and RD was 40 mg m(-2) b.i.d. S-1/35 mg m(-2) docetaxel. Dose-limiting toxicities included grade 3 elevated liver enzymes, gastric perforation, grade 3 diarrhoea/fatigue, febrile neutropenia with grade 3 anorexia/fatigue, and neutropenic infection with grade 3 stomatitis/anorexia. In phase II (n=52), the overall response rate was 66.7% (95% confidence interval (CI): 53.8-79.6%) and the median time to progression and overall survival were 6.5 months (95% CI: 4.9-8.1) and 13.7 months (95% CI: 9.9-17.5), respectively. The most common grade 3/4 toxicity was neutropenia (29.4%), and febrile neutropenia/neutropenic infection occurred in 19.6% of patients. Non-haematological toxicities were generally mild. There was one treatment-related death due to pneumonitis. S-1 combined with weekly docetaxel is active in MGC with moderate toxicities.

Comparision of nirS, cnorB, MCR genes against water quality parameters to monitor uncontrolled landfills.

This study was conducted to investigate the possible relationship between a molecular biological investigation and water quality parameters in monitoring groundwater pollution at the immediate boundary of uncontrolled landfills and their downgradient aquifers, which may consequently facilitate unbiased monitoring for the sites. Two closed landfills, Jicksan and Taejang in Korea, were chosen for this study, where the diversity of the microbial community was characterized and three specific genes, i.e. nirS (nitrite reductase coding gene), cnorB (nitric oxide reductase coding gene) and MCR (methyl coenzyme M reductase coding gene), were quantified. The quantified genes were then compared with conventional water quality parameters. From the analyzed DNA sequences, Proteobacteria phylum was most dominantly observed. A quantitative analysis revealed that the copy numbers (gene abundance) of denitrification enzyme coding genes, i.e. nirS gene and cnorB gene in Jicksan (J) site, are seven and four times, respectively, higher than Taejang (T) site. This simply implied that denitrification was possibly higher in J site than T site. In addition, a methane production enzyme coding gene, i.e. MCR, in a J1 bore immediately bordering the sources in the J site showed the greatest concentration, but it was precipitously decreased in the downgradient direction toward the outer boundary of landfill. A comparative investigation between the copy numbers of three genes, i.e. nirS, cnorB, and MCR, and conventional monitoring parameters, i.e. Cl-, alkalinity, TOC, NH3-N, and NO2-N, showed that they had overall correlation as given by more than 0.99 of the squared correlation coefficient (R2) for almost all of the concerned bores. It was concluded that the comparison between the molecular biological investigation and the conventional groundwater monitoring parameters showed good relationship between them, so that both tools could be more efficiently used for assessing the levels of contamination and prediction of the fate of pollutants, rather than being applied separately.

Partial purification of a nuclear protein that binds to the CCAAT box of the mouse alpha 1-globin gene.

We enriched a fraction from nuclear extracts of murine erythroleukemia cells which contains a protein able to form stable complexes with the promoter region of the alpha 1-globin gene. Binding activity, which is present in mouse brain and a variety of cultured mouse and human cell lines, is not erythroid cell specific. Binding studies with alpha-globin gene promoter deletion mutants as well as DNase I footprinting and dimethyl sulfate protection studies showed that the factor bound specifically to the CCAAT box of the alpha 1 promoter. Enriched factor preparations exhibited weak binding to the promoter region of the beta maj-globin gene. This suggests that this protein could bind differentially to these two promoters in vivo. The enriched factor may be a ubiquitous nuclear protein involved in the differential regulation of the alpha 1- and beta maj-globin genes.

Purification of multiple erythroid cell proteins that bind the promoter of the alpha-globin gene.

Three erythroid cell factors that bind the murine alpha-globin promoter were enriched more than 1,000-fold by conventional and DNA sequence affinity chromatography. Visualization of enriched polypeptides revealed simple patterns suggesting that each binding activity was purified. Two of the purified proteins, alpha-CP1 and alpha-CP2, have been shown previously to interact with distinct binding sites that overlap in the alpha-globin CCAAT box. Affinity purification of alpha-CP1 revealed seven polypeptides with Mrs raging from 27,000 to 38,000. In contrast, purified alpha-CP2 was made up of a polypeptide doublet with Mrs of 64,000 and 66,000. The third purified binding activity, alpha-IRP, interacted with sequences that formed an inverted repeat (IR) between the alpha-globin CCAAT and TATAA boxes. Affinity-purified alpha-IRP was made up of a single polypeptide with an Mr of 85,000. We confirmed that the purified polypeptides corresponded to alpha-CP1-, alpha-CP2-, and alpha-IRP-binding activities by UV cross-linking experiments (alpha-CP2 and alpha-IRP) or by renaturation of binding activity after elution of polypeptides from sodium dodecyl sulfate-polyacrylamide gels (alpha-CP1 and alpha-CP2). The apparent complexity of the polypeptides accounting for alpha-CP1 binding activity prompted a further physical characterization of this factor. Sedimentation of affinity-purified alpha-CP1 in glycerol gradients containing 100 mM KCl showed that all seven polypeptides migrated as a complex that cosedimented with alpha-CP1-binding activity. In contrast, when sedimented in glycerol gradients containing 500 mM KCl, alpha-CP1 dissociated into at least two components. Under these conditions, alpha-CP1-binding activity was reduced or lost. Activity was reconstituted, however, by combining fractions that were enriched in the two components. These results were confirmed by experiments in which we showed that alpha-CP1-binding activity can be recovered only by combining distinct sets of polypeptides that were isolated and renatured from sodium dodecyl sulfate-polyacrylamide gels. Our results suggest that the seven polypeptides visualized after affinity purification of alpha-CP1 interact to form a heterotypic complex (or set of complexes) required for alpha-CP1-binding activity.

Purification and characterization of an erythroid cell-specific factor that binds the murine alpha- and beta-globin genes.

An erythroid cell-specific nuclear factor that binds tightly to a sequence motif (5'-GATAAGGA-3') shared by many erythroid cell-specific promoters was purified to homogeneity by DNA sequence affinity chromatography. Visualization of the purified factor, which we term EF-1, showed a simple pattern comprising a polypeptide doublet with Mrs of 18,000 and 19,000. We confirmed that these species account for EF-1-binding activity by eluting the polypeptides from sodium dodecyl sulfate-polyacrylamide gels and renaturing the appropriate binding activity. Using the purified polypeptides, we mapped seven factor-binding sites that are dispersed across the murine alpha- and beta-globin genes. The murine alpha-globin gene is flanked by at least two EF-1-binding sites. One site is centered at nucleotide (nt) -180 (with respect to the alpha-globin cap site). A fivefold-weaker site is located downstream of the alpha-globin poly(A) addition site, at nt +1049. We mapped five EF-1-binding sites near the murine beta-globin gene. The strongest site was centered at nt -210. Four additional sites were centered at nt -266 (adjacent to the binding site of a factor present in both murine erythroleukemia and Raji cells), -75 (overlapping the beta-globin CCAAT box), +543 (within the second intervening sequence), and -111.