Gastric cancer is the second leading cause of cancer death worldwide and in China. The mechanism of gastric carcinogenesis is not fully understood. Epigenetic studies indicated that inactivation of tumor suppressor genes by DNA hypermethylation plays a crucial role in the progression of gastric cancer. Epigenetic inactivation of secreted frizzled-related protein (SFRP 1) by methylation plays a pivotal role on the development of various cancers. However, the role of SFRP family genes in gastric cancer remains largely unknown. We aimed to characterize the epigenetic abnormalities and discover novel biomarkers for early detection of gastric cancer. We investigated the epigenetic alterations in gastric adenocarcinoma by microarray based analysis and gene promoter hypermethylation. Based of the microarray data, we determined the functional significance and frequency of SFRP family genes hypermethylation in human gastric cancer. We screened the mRNA expression and methylation status of the SFRP family members in human gastric cancer cell lines and primary gastric cancer samples. Demethylation study of SFRP family genes were done by treating gastric cancer cell lines with 5'Aza. The biological effects of SFRP were analyzed by flow cytometry, cell viability assay and tumor growth in nude mice. SFRP1, 2, 4 and 5 were undetectable in 100% (7/7), 100% (7/7), 42.8% (3/7) and 85.7% (6/7) of gastric cancer cell lines, respectively. However, only SFRP2 showed significant down-regulation in gastric cancer compared with adjacent non-cancer samples (P<0.01). Treatment with demethylation agent, 5'-Aza, restored the expression of SFRP2 in all 7 cancer cell lines. Promoter hypermethylation of SFRP2 was detected in 73.3% of primary gastric cancer samples and 20% of adjacent non-cancer tissue (P<0.01). Bisulfite sequencing confirmed the density of promoter methylation in cell line, primary gastric cancer tissue and their adjacent non-cancer tissue. Transfection of SFRP2 induced cell apoptosis, inhibited proliferation in vitro and suppressed tumor growth in vivo. Furthermore, SFRP2 methylation was detected in 37.5% of samples showing intestinal metaplasia. Methylated SFRP2 was also detected in 66.7% of serum samples from cancer patients but not in normal controls. Epigenetic inactivation of SFRP2, but not SFRP1, SFRP4 and SFRP5 is a common and early event of carcinogenesis. Hence, detection of SFRP2 methylation in serum may have diagnostic value in gastric cancer patients. / by Cheng, Yuen Yee. / Adviser: FKL Chan. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0803. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 165-179). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / School code: 1307.

ABC transporters consist of two transmembrane domains (TMDs) that form the transport channel and two cytosolic nucleotide-binding domains (NBDs) that energize transport via ATP binding and hydrolysis. Using site-directed mutagenesis, the role of highly conserved residues in the NBDs of Abcb1a was investigated. / In both NBDs of Abcb1a the A-loop aromatic residue is a tyrosine: Y397 in NBD1 and Y1040 in NBD2. Another tyrosine (618 in NBD1 and 1263 in NBD2) also appears to lie close to the ATP molecule. These four tyrosine residues were mutated to tryptophan and the effect of these substitutions on transport properties, ATP binding, and ATP hydrolysis was analyzed. Y618W and Y1263W enzymes had catalytic characteristics similar to wild-type (WT) Abcb1a. On the other hand, Y397W and Y1040W showed impaired transport and greatly reduced ATPase activity, including an ∼10-fold increase in KM(ATP). Thus, Y397 and Y1040 play an important role in Abcb1a catalysis. / Since it was speculated that ABC transporters utilize a catalytic base to hydrolyse the beta-gamma phosphodiester bond of ATP, a search for that residue was undertaken. Six pairs of highly conserved acidic residues in the NBDs of Abcb1a were investigated. Removal of the charge in D558N and D1203N as well as in E552Q and E1197Q produced enzymes with severely impaired transport. These mutants were purified and characterized with respect to ATPase activity. Mutants D558N and D1203N retained some drug-stimulated ATPase activity and vanadate (Vi) trapping of 8-azido-[alpha32P]nucleotide confirmed slower basal and drug-stimulated hydrolysis. The E552Q and E1197Q mutants showed absence of ATPase activity but Vi trapping of 8-azido-[alpha 32P]nucleotide was observed, at a level similar to that of WT Abcb1a. Photolabelling by 8-azido-[alpha32P]nucleotide, in the presence or absence of drug, was also detected in the absence of Vi. The ATPase activity, binding affinity, and trapping properties of these glutamate residues were further analyzed. In addition to the E→Q mutants, the glutamates were individually mutated to D, N, and A. The double mutants E552Q/E1197Q, E552Q/K1072R, and K429R/E552Q were also analyzed. The results obtained suggest that 1) the length of the side-chain is important for the catalytic activity, whereas the charge is critical for full turnover to occur, 2) formation of the catalytic transition state does occur in the mutant site in the single-site mutants, suggesting that E552 and E1197 are not classical catalytic carboxylates, 3) steps after formation of the transition state are severely impaired in these mutant enzymes, 4) NBD1 and NBD2 are functionally asymmetric, and 5) the glutamates are involved both in NBD-NBD communication and transition-state formation through orientation of the linchpin residue.

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Indiana University-Purdue University Indianapolis (IUPUI) / External apical root resorption (EARR) is a common sequela of orthodontic treatment, although it may also occur without orthodontic treatment. Despite rigorous investigation, no single factor or group of factors that directly causes root resorption has been identified. Experiment 1. A sample of 83 pairs of full siblings who had undergone orthodontic treatment was studied. Measurements were made of the longest maxillary central incisor, mandibular central incisor and mesial and distal roots of the mandibular first molars. Heritability estimates were generated by generalized liner models. Our results showed that the heritability estimate of the EARR was 64% on average. It was concluded that there was sufficient heritability for EARR to pursue genetic analysis. Experiment 2. Five polymorphic markers flanking or lying within the IL-IA , IL-JB, TNSALP, TNFA, and TNFRSFJ JA genes were used in a candidate gene approach to assess linkage and association with EARR in 38 pedigrees. Suggestive evidence for linkage between EARR and the polymorphic marker D18S64 was obtained with the analysis program MAPMAKER/SIBS (LOD score 2.51). The Q-TDT program showed highly significant (p = 0.0003) evidence of linkage disequilibrium of IL-1 B polymorphisms with EARR. Our analysis indicates that the JL -1 B polymorphism accounts for 15% of the total EARR variation. Experiment 3. Nine-week-old male mice were randomly selected as controls or for placement under anesthesia of an open coil spring ligated to the left maxillary first molar producing a force of approximately 25 g. The control (C) or treated (T) per strain were A/J (C=3,T=9), C57BL/6J (C=7,T=8), C3H/HeJ (C = 4,T=6), BALB/cJ (C=4,T=6), 129P3 /J (C=6,T=8), DBA/2J (C=8,T=9), SJL/J (C=8,T= 10), and AKR/J (C=9,T =8). Animals were sacrificed after nine days of treatment or control; maxillae were immediately removed, prepared, sectioned, mounted, stained with H&E, and observed microscopically at 1 OOX to determine root resorption. Mice were grouped into root resorption resistant (A/J, C57BL/6J and SJL/J); intermediate (C3H/HeJ and AKR/J); and susceptible (BALB/cJ, DBA/2J, and 129P3/J) strains. It was concluded that there were differential susceptibility or resistance to root resorption among inbred mouse strains, indicating that genotype is an influencing factor.