Alcaligenes eutrophus H16 shows three distinct nitrate reductase activities (U. Warnecke-Eberz and B. Friedrich, Arch. Microbiol. 159:405-409, 1993). The periplasmic enzyme, designated NAP (nitrate reductase, periplasmic), has been isolated. The 80-fold-purified heterodimeric enzyme catalyzed nitrate reduction with reduced viologen dyes as electron donors. The nap genes were identified in a library of A. eutrophus H16 megaplasmid DNA by using oligonucleotide probes based on the amino-terminal polypeptide sequences of the two NAP subunits. The two structural genes, designated napA and napB, code for polypeptides of 93 and 18.9 kDa, respectively. Sequence comparisons indicate that the putative gene products are translated with signal peptides of 28 and 35 amino acids, respectively. This is compatible with the fact that NAP activity was found in the soluble fraction of cell extracts and suggests that the mature enzyme is located in the periplasm. The deduced sequence of the large subunit, NAPA, contained two conserved amino-terminal stretches of amino acids found in molybdenum-dependent proteins such as nitrate reductases and formate dehydrogenases, suggesting that NAPA contains the catalytic site. The predicted sequence of the small subunit, NAPB, revealed two potential heme c-binding sites, indicating its involvement in the transfer of electrons. An insertion in the napA gene led to a complete loss of NAP activity but did not abolish the ability ofA. eutrophus to use nitrate as a nitrogen source or as an electron acceptor in anaerobic respiration. Nevertheless, the NAP-deficient mutant showed delayed growth after transition from aerobic to anaerobic respiration, suggesting a role for NAP in the adaptation to anaerobic metabolism.Nitrate is a major nitrogen source for many bacteria. In the general assimilatory pathway, nitrate is converted via nitrite to ammonia, which is then assimilated into nitrogen metabolism. This metabolic route functions aerobically and anaerobically and involves assimilatory nitrate reductases which are repressed by ammonia. Nitrate can also serve as an electron acceptor for anaerobic respiration in the absence of oxygen. In this case nitrate is reduced by respiratory nitrate reductases to nitrite (reviewed in references 39 and 46). In a variety of bacteria, includingAlcaligenes eutrophus H16 (24), the end product of nitrate respiration is dinitrogen. This denitrification pathway involves, in addition to the respiratory nitrate reductase, further respiratory reductases for nitrite, nitric oxide, and nitrous oxide (reviewed in reference 46).A. eutrophus H16 is a gram-negative obligate respiratory bacterium which is able to grow heterotrophically on a broad range of substrates. Alternatively, it can grow autotrophically with hydrogen as an energy source, and in the absence of oxygen it can use nitrate or nitrite for anaerobic respiration (8). These alternative facultative metabolic pathways of A. eutrophus H16 are genetically linked to the megaplasmid pHG1 (13,31). Physiological and genetic ...

Pyridochromanones were identified by high throughput screening as potent inhibitors of NAD ؉ -dependent DNA ligase from Escherichia coli. Further characterization revealed that eubacterial DNA ligases from Gramnegative and Gram-positive sources were inhibited at nanomolar concentrations. In contrast, purified human DNA ligase I was not affected (IC 50 > 75 M), demonstrating remarkable specificity for the prokaryotic target. The binding mode is competitive with the eubacteriaspecific cofactor NAD ؉ , and no intercalation into DNA was detected. Accordingly, the compounds were bactericidal for the prominent human pathogen Staphylococcus aureus in the low g/ml range, whereas eukaryotic cells were not affected up to 60 g/ml. The hypothesis that inhibition of DNA ligase is the antibacterial principle was proven in studies with a temperature-sensitive ligase-deficient E. coli strain. This mutant was highly susceptible for pyridochromanones at elevated temperatures but was rescued by heterologous expression of human DNA ligase I. A physiological consequence of ligase inhibition in bacteria was massive DNA degradation, as visualized by fluorescence microscopy of labeled DNA. In summary, the pyridochromanones demonstrate that diverse eubacterial DNA ligases can be addressed by a single inhibitor without affecting eukaryotic ligases or other DNA-binding enzymes, which proves the value of DNA ligase as a novel target in antibacterial therapy.Multiple drug resistance among bacterial pathogens is spreading even in developed countries and has made many currently available antibiotics ineffective (1). As a consequence the number of reports on therapy failures increases and treatment costs rise, causing a growing public health problem. Thus, the search for novel antibacterial classes with innovative mechanisms of action is crucial to keep pace with the innate adaptability of the bacterial population. From the information revealed by sequencing more than 80 bacterial genomes, many novel target ideas have emerged in the last decade. However, even classical target areas such as cell wall, protein, or DNA synthesis contain many vital reactions not exploited in antibacterial therapy so far.DNA ligases are promising target candidates because they are indispensable for many fundamental processes in DNA metabolism including the linkage of Okazaki fragments during replication, recombination processes, and repair pathways requiring resynthesis of DNA (2, 3). Their crucial function is emphasized by the fact that eukaryotic cells contain several isoenzymes and that viruses encode their own ligases (3, 4). The reaction catalyzed by the DNA ligases, the joining of nicked DNA strands, proceeds in three sequential nucleotidyl transfer reactions (2). The first step is the nucleophilic attack by the active site lysine on the AMP moiety of a cofactor, resulting in a covalent enzyme-AMP intermediate. The AMP is then transferred to the 5Ј-phosphate end of the nicked duplex DNA and finally released when the ligase catalyzes the attack by the adjacent...

Diagnostic markers are needed for achieving a cure in esophageal cancer, detecting tumor cells earlier. Exosomes are bioactive vesicles secreted by cells into surrounding body fluids. Exosome formation, cargo content, and delivery have major impact in cancer development. This is the first isolation of exosomes from serum of patients with adenocarcinoma of the esophagus and comparison of exosomal miRNA profiles with matching primary tumor and normal tissues. RNA was extracted for miRNA profiling by real-time TaqMan miR arrays. The miR profiles of exosomal cargo, matching tumor, and normal tissue of a subgroup of adenocarcinoma patients have been compared. "Exosomal onco-miRs" such as miR-223-5p, miR-223-3p, miR-483-5p, miR-409-3p, miR-196b-5p, miR-192-5p, miR-146a-5p, and miR-126-5p have been identified as part of exosomal cargo being overexpressed in corresponding tumor compared to normal. Upregulation of miR-223-5p and miR-483-5p in adenocarcinoma (p = 0.034, p = 0.017) has been verified by an independent cohort of 43 patients with T2-3 adeno- and squamous cell carcinoma. In contrast, miR-224-5p, miR-452-5p, miR-23b-5p, miR-203-5p, miR-1201-5p, miR-149-5p, miR-671-3p, miR-944-5p, miR-27b-3p, and miR-22-3p have been identified to be significantly downregulated in adenocarcinoma versus normal and merely or not detectable in exosomes. "Exosomal onco-miRs" are a novel, stable, and noninvasive source for diagnosis and therapy monitoring of esophageal cancer. Oncogenic shuttle miRNAs present in exosomes may contribute to understanding how tumor cells spread their oncogenic potential to the environment. The "exosomal onco-miRs" identified seem to play a major role and may be applied for noninvasive diagnosis and therapy monitoring of adenocarcinoma of the esophagus.

Purpose: The excision repair cross-complementing 1 (ERCC1) gene is coding for a nucleotide excision repair protein involved in the repair of radiation-and chemotherapy-induced DNA damage. We examined the potential of quantitative ERCC1 mRNA expression to predict minor or major histopathological response to neoadjuvant radiochemotherapy (cisplatin, 5-fluorouracil, and 36 Gy of radiation) followed by transthoracic en bloc esophagectomy in patients with locally advanced esophageal cancer (cT 2-4 , N x , M 0 ).Experimental Design: Tissue samples were collected by endoscopic biopsy before treatment. RNA was isolated from biopsies, and quantitative real-time reverse transcriptase PCR assays were performed to determine ERCC1 mRNA expression. Relative mRNA levels (tumor/normal ratios) were calculated as (ERCC1/␤-actin in tumor)/(ERCC1/ ␤-actin in paired normal tissue). ERCC1 expression levels were correlated with the objective histopathological response in resected specimens. Histomorphological regression was defined as major response when resected specimens contained <10% of residual vital tumor cells or in case a pathologically complete response was achieved.Results: Twelve of 36 tumors showed a major histopathological response, and 24 of 36 showed a minor histopathological response. Relative expression levels of ERCC1 of >1.09 were not associated with a major histopathological response (sensitivity, 62.5%; specificity, 100%) and 15 of 24 patients with minor histopathological response to the delivered neoadjuvant radiochemotherapy could be unequivocally identified. This association of dichotomized relative ERCC1 mRNA expression and histopathological response was statistically significant (P < 0.001).Conclusions: Relative expression levels of ERCC1 mRNA determined by quantitative real-time reverse transcriptase-PCR appear highly specific to predict minor response to our neoadjuvant radiochemotherapy protocol in patients with locally advanced esophageal cancer and could be applied to prevent expensive, noneffective, and potentially harmful therapies in a substantial number (42%) of patients.

Background Due to proliferation and increased metabolism, cancer cells have high glucose requirements. The glucose uptake of cells is influenced by a group of membrane proteins denoted the glucose transporter family (Glut-1 to -12). Whereas increased expression and a negative correlation with survival have been described for Glut-1 in several types of cancer, the impact of other glucose transporters on tumor biology is widely unknown. Methods In this retrospective study, gastric cancer specimens of 150 patients who underwent total gastrectomy between 2005 and 2010 were stained for Glut-1, -3, -6, and -10 by immunohistochemistry. Expression of Glut-1, -3, -6, and 10 was correlated to prognosis as well as clinical and pathological parameters. Results Glut-1, Glut-3, Glut-6, and Glut-10 were expressed in 22.0, 66.0, 38.0, and 43.3 % of the analyzed samples. Whereas Glut-1, -6, and -10 did not show a correlation with prognosis, positive staining for Glut-3 was associated with higher UICC stage and inferior prognosis. The mean overall survival was 38.6 months for Glut-3 positive patients, as compared to 51.2 months for Glut-3 negative patients (p \ 0.05). Coexpression of two or more of the analyzed glucose transporters was correlated to inferior prognosis. Glut-3 and UICC stage were significant prognostic factors in multivariate analysis. Conclusions All of the analyzed glucose transporters were expressed in a significant proportion of the gastric cancer samples. Glut-3 was associated with higher UICC stage and inferior prognosis. These findings are relevant to therapeutic approaches that target glucose metabolism as well as to imaging using radioactively labeled glucose.