Targeting Base Excision Repair Glycosylases with DNA Containing Transition State Mimics Prepared via Click Chemistry.

DNA glycosylases of the base excision repair (BER) pathway are front-line defenders in removing compromising modifications of the DNA nucleobases. Aberrantly modified nucleobases mediate genomic mutations and inhibit DNA replication leading to adverse health consequences such as cancer, neurological diseases, and aging. In an effort to develop high-affinity transition state (TS) analogues as chemical biology probes for DNA glycosylases, oligonucleotides containing a propargyl-modified pyrrolidine TS mimic nucleotide were synthesized. A small library of TS mimic-containing oligonucleotides was generated using a structurally diverse set of five azides via copper(I)-catalyzed azide-alkyne cycloaddition "click" chemistry. The relative affinity ( Kd) was evaluated for BER glycosylases Escherichia coli MutY, bacterial formamidopyrimidine glycosylase (Fpg), and human OG glycosylase 1 (hOGG1) with the library of TS mimic DNA duplexes. All of the BER glycosylases were found to exhibit extremely high affinities (approximately picomolar Kd values) for the TS mimics. However, binding preferences, distinct for each glycosylase, for the TS mimic library members were observed, suggesting different modes of binding and transition state stabilization among the three glycosylases. Fpg bound all of the TS mimics with exceptionally high affinities, while the MutY binding affinity correlated inversely with the size of the appended moiety. Of note, we identified one member of the small TS mimic library that exhibited a particularly high affinity for hOGG1. These results strongly support the use of the propargyl-TS mimic oligonucleotides and elaboration via click chemistry in screening and identification of high-affinity ligands for BER glycosylases of interest.

Fe-S Clusters and MutY Base Excision Repair Glycosylases: Purification, Kinetics, and DNA Affinity Measurements.

A growing number of iron-sulfur (Fe-S) cluster cofactors have been identified in DNA repair proteins. MutY and its homologs are base excision repair (BER) glycosylases that prevent mutations associated with the common oxidation product of guanine (G), 8-oxo-7,8-dihydroguanine (OG) by catalyzing adenine (A) base excision from inappropriately formed OG:A mispairs. The finding of an [4Fe-4S]2+ cluster cofactor in MutY, Endonuclease III, and structurally similar BER enzymes was surprising and initially thought to represent an example of a purely structural role for the cofactor. However, in the two decades subsequent to the initial discovery, purification and in vitro analysis of bacterial MutYs and mammalian homologs, such as human MUTYH and mouse Mutyh, have demonstrated that proper Fe-S cluster coordination is required for OG:A substrate recognition and adenine excision. In addition, the Fe-S cluster in MutY has been shown to be capable of redox chemistry in the presence of DNA. The work in our laboratory aimed at addressing the importance of the MutY Fe-S cluster has involved a battery of approaches, with the overarching hypothesis that understanding the role(s) of the Fe-S cluster is intimately associated with understanding the biological and chemical properties of MutY and its unique damaged DNA substrate as a whole. In this chapter, we focus on methods of enzyme expression and purification, detailed enzyme kinetics, and DNA affinity assays. The methods described herein have not only been leveraged to provide insight into the roles of the MutY Fe-S cluster but have also been provided crucial information needed to delineate the impact of inherited variants of the human homolog MUTYH associated with a colorectal cancer syndrome known as MUTYH-associated polyposis or MAP. Notably, many MAP-associated variants have been found adjacent to the Fe-S cluster further underscoring the intimate relationship between the cofactor, MUTYH-mediated DNA repair, and disease.

Selective base excision repair of DNA damage by the non-base-flipping DNA glycosylase AlkC.

DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT-like repeat (HLR) fold. AlkD uses a unique non-base-flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3-methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non-base-flipping strategy distinct from that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin-like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA This active site can accommodate and excise N3-methylcytosine (3mC) and N1-methyladenine (1mA), which are also repaired by AlkB-catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.

Sonographic features of acute colonic diverticulitis: the "dome sign".

PURPOSE: This study was performed to clarify the sonographic features of acute colonic diverticulitis to enable its differentiation from appendicitis. METHODS: Of 119 patients who were referred to our hospitals for lower abdominal pain between June 1997 and December 1998 and underwent sonography, 12 patients had a definitive diagnosis of acute colonic diverticulitis and 4 patients a tentative diagnosis. Seventy-eight patients were diagnosed as having acute appendicitis, confirmed by appendectomy. In the 16 patients with diagnoses of diverticulitis, the sonographic and clinical features of acute colonic diverticulitis were studied. RESULTS: Among the 12 patients with definitive diagnoses of acute colonic diverticulitis, sonographic findings included localized thickening of the colonic wall (100%) and a hemispheric mass (the "dome sign") protruding at the thickened colonic wall (100%) and consisting of a hypoechoic wall (100%) and a central echogenic area (66%). The presence of diverticula was confirmed by barium-enema x-ray study in all 12 patients. The 4 patients with tentative diagnoses of acute colonic diverticulitis all had colonic wall thickening but no dome sign. Colonoscopy revealed colitis in 3 of these patients. All 16 patients recovered with conservative treatment, without laparotomy. CONCLUSIONS: Sonography was useful for differentiating acute colonic diverticulitis from appendicitis. The sonographic finding of the dome sign seems to be specific for acute colonic diverticulitis.

[Surgical treatment of N2 involved non-small cell lung cancer--the systematic extended lymph node dissection based on the regional lymphatic drainage].

From the study on regional lymphtic drainage, we have decided the extent of lymphadenectomy as follows; a) For the left lung cancer and the right upper lobe primary, systematic bilateral mediastinal dissection (R3 alpha) through a median sternotomy, b) For the cases with the highest mediastinal node involvement, lower half of radical neck dissection (R3 gamma) through a cervical collar incision in addition to a). The cN diagnosis by CT interpretation and pN diagnosis were compared. The under estimated rates of N were 32% of 137 patients with the left lung primary. 46 patients with pN2(+) included 14 patients (31%) with pN3 disease. As for the right upper lobe primary, 17 patients with pN2(+) included 13 patients (76%) with pN3 disease. Postoperative survival rates calculated with Kaplan-Meiermethod; 1) The five-year survival rates were 43% of 46 patients with pT1-3 N2-3 of the left lung primary. 2) As for the right upper lobe primary, the two-year survival rates were 51% of 17 patients with pT1-4 N2-3. 3) The three-year survival rates of 26 patients with pN3 gamma diagnosed as cN0-3 alpha preoperatively were 41%. These systematic extended dissection (R3 alpha, R3 gamma) would bring better prognosis in the patients with pN2-3 disease.

[Basic and clinical studies on cefmenoxime in pediatric field].

Basic and clinical studies were made on cefmenoxime (CMX) in pediatric field, and the following results were obtained. 1. The antibacterial activity of CMX against clinically isolated and maintained strains was examined. CMX had stronger antibacterial activity than CEZ against Escherichia coli, Salmonella, Klebsiella pneumoniae, Proteus mirabilis, Serratia marcescens and Pseudomonas aeruginosa, but CEZ had stronger antibacterial activity against Staphylococcus aureus. 2. The blood concentrations of CMX, 0.5, 1, 2, 4 and 6 hours after a one-shot intravenous injection of 20 mg/kg of CMX were 33.6, 15.1, 4.5, 2.5 and 0.6 mcg/ml, respectively, with the half-life of 1.04 hours. 3. The blood concentrations of CMX, 0.5, 1, 2, 4 and 6 hours after a 1-hour intravenous drip infusion of 20 mg/kg of CMX were 32.0, 55.2, 8.4, 4.2 and 1.0 mcg/ml, respectively, with the half-lite of 0.96 hour. 4. A complete or partial clinical response to therapy with CMX was obtained in all 10 children with infectious diseases. 5. Bacteriological examination made on 3 patients showed that all bacteria had been eradicated, and that therapy was effective. The bacteria were E. coli in 2 patients and Proteus mirabilis in 1 patient. 6. The side effects produced were neutropenia, eosinophilia and skin eruption in 1 patient, and diarrhea in 1 patient.

[Clinical investigation of cefadroxil in pediatric field (author's transl)].

(1) Cefadroxil powder for syrup was administered in 24 cases of respiratory tract infection and urinary tract infection, and the efficacy was obtained in 21 cases, effective ratio being 87.5%. (2) Clinical effect could be obtained satisfactorily at a daily dose of 10-15 mg/kg divided into 3 times after each meal. (3) As to the side effect, GOT and GPT rose in 1 case, and stomatitis in 1 case, though the patients returned to normal after discontinuation of the drug. (4) Haemophilus appeared by pharyngeal culture after administration of the drug, and attention should be paid on an alteration of pharyngeal flora.