Does restriction of mandibular movements during sleep influence jaw-muscle activity?

To investigate the effect of restriction of mandibular movements during sleep on jaw-muscle electromyographic (EMG) activity. Eleven healthy subjects (four men and seven women; age, 25·9 ± 3·1 years) with self-reports of sleep bruxism participated in three randomised sessions with three different types of oral appliances: (i) full-arch maxillary and mandibular appliances which did not allow any mandibular movement, that is, restrictive oral appliance (restrict-MMOA), (ii) full-arch maxillary and mandibular oral appliances (free-MMOA) with no restrictions of mandibular movements and (iii) conventional full-arch flat stabilisation appliance, that is, maxillary oral appliance (free-MOA). Baseline recordings (1st EMG recording) of jaw-muscle activity during sleep without any oral appliance were performed and followed by 1 week of nightly use of each oral appliance (three sessions). During the last night in each session, jaw-muscle activity was recorded (2nd, 3rd and 4th EMG recordings) and compared to baseline values. All EMG data were analysed in accordance with the gold-standard diagnostic method. The average jaw-muscle activity expressed as number of EMG episodes and bursts per hour sleep was significantly reduced during any combination of appliance compared to baseline values. The inhibitory effect of the appliances was specific to the number of phasic EMG episodes and bursts (P < 0·01), with no effects on tonic EMG bursts or episodes (P > 0·30). The results indicated that restriction of mandibular movements with oral appliances do not have any major influence on jaw-muscle activity during sleep but rather that the immediate effect of any combination of oral appliances lead to a suppression of phasic EMG bursts and episodes.

The association of DNA-dependent protein kinase activity of peripheral blood lymphocytes with prognosis of cancer.

BACKGROUND: Repair of various types of DNA damages is critical for genomic stability. DNA-dependent protein kinase (DNA-PK) has an important role in DNA double-strand break repair. We examined whether there may be a correlation between DNA-PK activity in peripheral blood lymphocytes (PBLs) and survival percentages in various cancer patients. We also investigated the changes of DNA-PK activity in PBLs after radiotherapy. METHODS: A total of 167 of untreated cancer patients participated in this study. Peripheral blood was collected, separated, and centrifuged. DNA-PK activity was measured by DNA-pull-down assay. Chromosomal aberrations were examined by cytogenetic methods. RESULTS: DNA-PK activity of PBLs in advanced cancer patients was significantly lower than that in early stage. The patients with lower DNA-PK activity in PBLs tended to have the lower disease-specific survivals and distant metastasis-free survivals than those with higher DNA-PK activity in advanced stages. There was also a tendency of inverse correlation between DNA-PK activity and excess fragments. The DNA-PK activity of PBLs in most patients decreased in response to radiation as the equivalent whole-body dose increased. CONCLUSION: Cancer patients in advanced stage, with lower DNA-PK activity of PBLs might have higher distant metastasis and exhibit poorer prognosis. Therefore, DNA-PK activity in PBLs could be used as a marker to predict the chromosomal instability and poorer prognosis.

Frequent epigenetic inactivation of SFRP genes and constitutive activation of Wnt signaling in gastric cancer.

Activation of Wnt signaling has been implicated in gastric tumorigenesis, although mutations in APC (adenomatous polyposis coli), CTNNB1 (beta-catenin) and AXIN are seen much less frequently in gastric cancer (GC) than in colorectal cancer. In the present study, we investigated the relationship between activation of Wnt signaling and changes in the expression of secreted frizzled-related protein (SFRP) family genes in GC. We frequently observed nuclear beta-catenin accumulation (13/15; 87%) and detected the active form of beta-catenin in most (12/16; 75%) GC cell lines. CpG methylation-dependent silencing of SFRP1, SFRP2 and SFRP5 was frequently seen among GC cell lines (SFRP1, 16/16, 100%; SFRP2, 16/16, 100%; SFRP5, 13/16, 81%) and primary GC specimens (SFRP1, 42/46, 91%; SFRP2, 44/46, 96%; SFRP5, 30/46, 65%), and treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine rapidly restored SFRP expression. Ectopic expression of SFRPs downregulated T-cell factor/lymphocyte enhancer factor transcriptional activity, suppressed cell growth and induced apoptosis in GC cells. Analysis of global expression revealed that overexpression of SFRP2 repressed Wnt target genes and induced changes in the expression of numerous genes related to proliferation, growth and apoptosis in GC cells. It thus appears that aberrant SFRP methylation is one of the major mechanisms by which Wnt signaling is activated in GC.

Epigenetic inactivation of the candidate tumor suppressor gene HOXB13 in human renal cell carcinoma.

Epigenetic alterations like DNA methylation and the resulting inactivation of cancer-related genes often contribute to the development of various cancers. To identify the genes that are silenced by aberrant methylation in renal cell carcinoma (RCC), we subjected two RCC lines to methylated CpG island amplification/representational difference analysis. This identified 27 CpG islands. Combined bisulfite restriction analysis of these CpG islands in primary RCC cases revealed that four were methylated in a tumor-specific manner. One of these was identified as the human homeo-box gene B13 (HOXB13) gene, but the remaining three CpG islands were not associated with known genes. The methylation frequencies of HOXB13 in primary RCC samples and lines were 30 and 73%, respectively. The methylation status of HOXB13 correlated with the loss of its expression both in RCC lines and primary tumors, and methyltransferase inhibitor treatment induced the recovery of its expression. Exogenous expression of HOXB13 in RCC cells that lacked endogenous HOXB13 expression suppressed colony formation and induced apoptotic features. Furthermore, HOXB13 methylation correlated positively with tumor grade and microvessel invasion. These results suggest that HOXB13 is a novel candidate tumor suppressor gene in RCC and that its inactivation may play an important role in both RCC tumorigenesis and progression.

Inactivation of CACNA1G, a T-type calcium channel gene, by aberrant methylation of its 5' CpG island in human tumors.

Using a newly developed PCR-based technique called methylated CpG island amplification, we have identified several DNA fragments that are aberrantly methylated in a colon cancer cell line. One of the fragments, termed MINT31, mapped to human chromosome 17q21, where frequent loss of heterozygosity is detected in various human tumors. By characterizing the genomic sequence around this area, we identified a gene encoding a T-type calcium channel, CACNA1G, as a target for hypermethylation in human tumors. By reverse transcriptase-PCR we detected expression of CACNA1G in normal colon and bone marrow, but expression was absent in the five tumor cell lines in which methylation was found. After treatment with the methylation inhibitor 5-deoxyazacytidine, the expression of CACNA1G was restored in all five cell lines. Detailed methylation mapping of the 5' CpG island by bisulfite-PCR revealed that methylation of a region 300-800 bp upstream of the translation initiation site closely correlated with the inactivation of CACNA1G. This region contained the transcription start site, as determined by 5' rapid amplification of cDNA ends analysis. Aberrant methylation of CACNA1G was also examined in various human primary tumors and was detected in 17 of 49 (35%) colorectal cancers, 4 of 16 (25%) gastric cancers, and 3 of 23 (13%) acute myelogenous leukemia cases. Inactivation of CACNA1G may play a role in cancer development by modulating calcium signaling, which potentially affects cell proliferation and apoptosis.

Aberrant methylation of the Cyclooxygenase 2 CpG island in colorectal tumors.

Cyclooxygenases (COXs) are key enzymes that convert arachidonic acid to prostaglandins. Overexpression of one of the COX isozymes, COX2, has been shown to play an important role in colorectal cancer progression. Recently, however, low expression of COX2 has been reported in a subset of colorectal and gastric cancers. Aberrant CpG island methylation and associated transcriptional silencing are common in colorectal cancer, and we therefore investigated the potential role of methylation in the transcriptional silencing of COX2. We examined the methylation status of the COX2 5' CpG island in a series of tumor cell lines. Among the 33 cell lines examined, dense methylation (>70%) of COX2 was detected in 5 cell lines, and partial methylation was detected in 10 cell lines. Detailed methylation mapping using bisulfite genomic sequencing revealed that loss of expression of COX2 mRNA was closely correlated with methylation of a region upstream of exon 1, and expression could be restored by demethylation using the DNA methyltransferase inhibitor 5-aza-deoxycytidine. Aberrant methylation of COX2 was also detected in 12 of 92 (13%) unselected sporadic primary colorectal cancers and 7 of 50 (14%) colorectal adenomas. COX2 methylation was strongly associated with the presence of the CpG island methylator phenotype (P<0.01), inversely related to p53 gene mutation (P<0.01), and unrelated to microsatellite instability status. We propose that COX2 expression in colorectal tumors is modulated by functional factors that favor high expression and by the CpG island methylator phenotype that favors silencing in a subset of cases. These results raise the possibility that tumors with COX2 methylation may be less sensitive to treatment using specific COX2 inhibitors.

Complementary DNA cloning and characterization of truncated form of c-kit in human colon carcinoma cells.

We have obtained a novel c-kit complementary DNA (cDNA) from a colon carcinoma cell line, Colo201, and characterized its structure. The size of the transcript in Colo201 was approximately 3.5 kilobases and it hybridized to the c-kit cDNA fragments encompassing the kinase domain, but not to the cDNA fragments encoding extracellular and transmembrane domains. The predicted protein encoded by those cDNAs was composed of 257 amino acids containing the NH2-terminal 25 unique amino acids in frame by the COOH terminal of the KIT protein. Of interest, these 25 amino acids were encoded by intron 15 of the c-kit gene. The aberrant mRNA was also detected in another colon carcinoma cell line, BM314. The translation of this message in Colo201 was confirmed by flow cytometry and immunoblot analysis. This is the first report describing the aberrant transcript of c-kit in human tumor cells, and it is suggested that truncated form of c-kit might play a role in the onset and development of human colon carcinoma.

Inactivation of the 14-3-3 sigma gene is associated with 5' CpG island hypermethylation in human cancers.

The cell cycle checkpoint plays an important role in maintaining the integrity of cells. Recently, one of the 14-3-3 protein family members, 14-3-3sigma, was shown to be regulated by p53 and to play a role in the G2-M-phase checkpoint. To determine whether 14-3-3sigma is inactivated in human cancers, the methylation status of the 5' region of 14-3-3sigma was investigated in a series of gastric, colorectal, and hepatocellular cancer cell lines. Of 22 cell lines examined, 6 showed aberrant methylation. The methylation status of 14-3-3sigma was found to be correlated with loss of expression, which was restored by 5-aza-2'-deoxycytidine treatment. Furthermore, normal G2 arrest after DNA damage was not demonstrated in the cell lines with methylation. In primary gastric cancers, 14-3-3sigma hypermethylation was observed frequently in 26 of 60 (43%) cases and observed more frequently in poorly differentiated adenocarcinomas (P = 0.0017). Our findings suggest that 14-3-3sigma is inactivated by aberrant methylation of the 5' region in various human cancers and that it might play an important role in the development of undifferentiated gastric cancers.

Accelerated age-related CpG island methylation in ulcerative colitis.

CpG island hypermethylation is a mechanism of gene silencing that can be usurped by neoplastic cells to inactivate undesirable genes. In the colon, hypermethylation often starts in normal mucosa as a function of age and is markedly increased in cancer. To test the hypothesis that subjects at increased risk of colon cancer have higher levels of methylation in their nonneoplastic mucosa, we studied methylation patterns of five genes in the normal and dysplastic mucosa of patients with ulcerative colitis (UC), a condition associated with a marked increased risk of colon cancer. One gene (Mlh1) was unmethylated in all tissues examined. All four remaining genes had low but detectable levels of methylation in the epithelium of UC patients without evidence of dysplasia, and this methylation was not different from non-UC controls. By contrast, all four genes were highly methylated in dysplastic epithelium from high-grade dysplasia (HGD)/cancer patients with UC; methylation in HGD versus controls averaged 40.0% versus 7.4% (P = 0.00003) for ER, 44.0% versus 3.0% (P < 0.00003) for MYOD, 9.4% versus 2.4% (P = 0.03) for p16 exon 1, and 57.5% versus 30.6% (P = 0.01) for CSPG2. Importantly, three of the four genes were also highly methylated in the normal appearing (nondysplastic) epithelium from these same HGD/cancer patients, indicating that methylation precedes dysplasia and is widespread in these patients. Compared with controls, methylation averaged 20.1% versus 7.2% (P = 0.07) for ER, 18.4% versus 3.0% (P < 0.008) for MYOD, and 7.9% versus 2.4% (P = 0.007) for p16 exon 1. These results are consistent with the hypothesis that age-related methylation marks (and may lead to) the field defect that reflects acquired predisposition to colorectal neoplasia. Furthermore, the data suggest that chronic inflammation is associated with high levels of methylation, perhaps as a result of increased cell turnover, and that UC can be viewed as resulting in premature aging of colorectal epithelial cells.

Hypermethylation-associated inactivation of p14(ARF) is independent of p16(INK4a) methylation and p53 mutational status.

The INK4a/ARF locus encodes two cell cycle-regulatory proteins, p16INK4a andp14ARF, which share an exon using different reading frames. p14ARF antagonizes MDM2-dependent p53 degradation. However, no point mutations in p14ARF not altering p16INK4a have been described in primary tumors. We report that p14ARF is epigenetically inactivated in several colorectal cell lines, and its expression is restored by treatment with demethylating agents. In primary colorectal carcinomas, p14ARF promoter hypermethylation was found in 31 of 110 (28%) of the tumors and observed in 13 of 41 (32%) colorectal adenomas but was not present in any normal tissues. p14ARF methylation appears in the context of an adjacent unmethylated p16INK4a promoter in 16 of 31 (52%) of the carcinomas methylated at p14ARF. Although p14ARF hypermethylation was slightly overrepresented in tumors with wild-type p53 compared to tumors harboring p53 mutations [19 of 55 (34%) versus 12 of 55 (22%)], this difference did not reach statistical significance. p14ARF aberrant methylation was not related to the presence of K-ras mutations. Our results demonstrate that p14ARF promoter hypermethylation is frequent in colorectal cancer and occurs independently of the p16INK4a methylation status and only marginally in relation to the p53 mutational status.

Hypermethylation of multiple genes in pancreatic adenocarcinoma.

Hypermethylation of CpG islands is a common mechanism by which tumor suppressor genes are inactivated. We studied 45 pancreatic carcinomas and 14 normal pancreata for aberrant DNA methylation of CpG islands of multiple genes and clones using methylation-specific PCR (MSP) and bisulfite-modified sequencing. Using MSP, we detected aberrant methylation of at least one locus in 60% of carcinomas. The genes analyzed included RARbeta (methylated in 20%), p16 (18%), CACNA1G (16%), TIMP-3 (11%), E-cad (7%), THBS1 (7%), hMLH1 (4%), DAP kinase (2%), and MGMT (0%). In addition, aberrant methylation was found in three CpG islands (MINT31, -1, and -2) in 38, 38, and 14% of carcinomas, respectively. Hypermethylation was largely confined to the carcinomas with only three loci (E-cad, DAP kinase, and MINT2) harboring methylation in some normal pancreata (36, 21, and 14%, respectively). Simultaneous methylation of at least four loci was observed in 5 of 36 (14%) pancreatic adenocarcinomas. We defined this subgroup of pancreatic adenocarcinomas as "CpG island-methylator-phenotype positive (CIMP+)." Two of four carcinomas with microsatellite instability harbored promoter hypermethylation of hMLH1, and both cases were CIMP+. Thus, we conclude that many pancreatic carcinomas hypermethylate a small percentage of genes, whereas a subset displays a CIMP+ phenotype.

Identification of differentially methylated sequences in colorectal cancer by methylated CpG island amplification.

CpG island methylation has been linked to tumor suppressor gene inactivation in neoplasia and may serve as a useful marker to clone novel cancer-related genes. We have developed a novel PCR-based method, methylated CpG island amplification (MCA), which is useful for both methylation analysis and cloning differentially methylated genes. Using restriction enzymes that have differential sensitivity to 5-methyl-cytosine, followed by adaptor ligation and PCR amplification, methylated CpG rich sequences can be preferentially amplified. In a model experiment using a probe from exon 1 of the p16 gene, signal was detected from MCA products of a colorectal cancer cell line but not in normal colon mucosa. To identify novel CpG islands differentially methylated in colorectal cancer, we have applied MCA coupled with representational difference analysis to the colon cancer cell line Caco2 as a tester and normal colon mucosa as a driver. Using this strategy, we isolated 33 differentially methylated DNA sequences, including fragments identical to several known genes (PAX6, Versican, alpha-tubulin, CSX, OPT, and rRNA gene). The association of hypermethylation of the clones obtained and transcriptional suppression in colorectal cancer was confirmed by examining the Versican gene, which we found to be silenced in methylated cell lines and reactivated by the methylation inhibitor 5-aza-2'-deoxycytidine. We therefore propose that MCA is a useful technique to study methylation and to isolate CpG islands differentially methylated in cancer.

Aberrant methylation in gastric cancer associated with the CpG island methylator phenotype.

Aberrant methylation of 5' CpG islands is thought to play an important role in the inactivation of tumor suppressor genes in cancer. In colorectal cancer, a group of tumors is characterized by a hypermethylator phenotype termed CpG island methylator phenotype (CIMP), which includes methylation of such genes as p16 and hMLH1. To study whether CIMP is present in gastric cancer, the methylation status of five newly cloned CpG islands was examined in 56 gastric cancers using bisulfite-PCR. Simultaneous methylation of three loci or more was observed in 23 (41%) of 56 cancers, which suggests that these tumors have the hypermethylator phenotype CIMP. There was a significant concordance between CIMP and the methylation of known genes including p16, and hMLH1; methylation of p16 was detected in 16 (70%) of 23 CIMP+ tumors, 1 (8%) of 12 CIMP intermediate tumors, and 1 (5%) of 21 CIMP- tumors (P<0.0001). Methylation of the hMLH1 gene was detected in three of five tumors that showed microsatellite instability, and all three of the cases were CIMP+. The CIMP phenotype is an early event in gastric cancer, being present in the normal tissue adjacent to cancer in 5 of 56 cases. These results suggest that CIMP may be one of the major pathways that contribute to tumorigenesis in gastric cancers.

Promoter hypermethylation of the DNA repair gene O(6)-methylguanine-DNA methyltransferase is associated with the presence of G:C to A:T transition mutations in p53 in human colorectal tumorigenesis.

Defects in DNA repair may be responsible for the genesis of mutations in key genes in cancer cells. The tumor suppressor gene p53 is commonly mutated in human cancer by missense point mutations, most of them G:C to A:T transitions. A recognized cause for this type of change is spontaneous deamination of the methylcytosine. However, the persistence of a premutagenic O(6)-methylguanine can also be invoked. This last lesion is removed in the normal cell by the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT). In many tumor types, epigenetic silencing of MGMT by promoter hypermethylation has been demonstrated and linked to the appearance of G to A mutations in the K-ras oncogene in colorectal tumors. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of p53 mutations, we studied 314 colorectal tumors for MGMT promoter hypermethylation and p53 mutational spectrum. Inactivation of MGMT by aberrant methylation was associated with the appearance of G:C to A:T transition mutations at p53 (Fischer's exact test, two-tailed; P = 0.01). Overall, MGMT methylated tumors displayed p53 transition mutations in 43 of 126 (34%) cases, whereas MGMT unmethylated tumors only showed G:C to A:T changes in 37 of 188 (19%) tumors. A more striking association was found in G:C to A:T transitions in non-CpG dinucleotides; 71% (12 of 17) of the total non-CpG transition mutations in p53 were observed in MGMT aberrantly methylated tumors (Fischer's exact test, two-tailed; P = 0.008). Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to G:C to A:T transition mutations in p53.

Identification and characterization of differentially methylated CpG islands in pancreatic carcinoma.

To identify CpG islands differentially methylated in pancreatic adenocarcinoma, we used methylated CpG island amplification (MCA) coupled with representational difference analysis. Of 42 CpG islands identified by MCA/representational difference analysis, 7 CpG islands [methylated in carcinoma of the pancreas (MICP)] were differentially methylated in a panel of eight pancreatic cancer cell lines compared with normal pancreas. In a larger panel of 75 pancreatic adenocarcinomas, these 7 MICPs (ppENK, Cyclin G, ZBP, MICP25, 27, 36, and 38) were methylated in 93, 3, 9, 15, 48, 19, and 41% of cancers, respectively, by methylation-specific PCR but not in any of 15 normal pancreata. In pancreatic cancer cell lines, methylation of ppENK, a gene with known growth suppressive properties, was associated with transcriptional silencing that was reversible with 5-aza-2'-deoxycytidine treatment. Relationships between the methylation patterns of pancreatic adenocarcinomas and their clinicopathological features were also determined. Larger pancreatic cancers and those from older patients (P = 0.017) harbored more methylated loci than smaller tumors and those from younger patients (P = 0.017). ppENK, MICP25, and 27 were variably methylated in normal gastric, duodenal, and colonic mucosae. These data indicate that aberrant methylation of ppENK and its transcriptional repression is a common event in pancreatic carcinogenesis.

Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis.

O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair protein that removes mutagenic and cytotoxic adducts from the O6 position of guanine. O6-methylguanine mispairs with thymine during replication, and if the adduct is not removed, this results in conversion from a guanine-cytosine pair to an adenine-thymine pair. In vitro assays show that MGMT expression avoids G to A mutations and MGMT transgenic mice are protected against G to A transitions at ras genes. We have recently demonstrated that the MGMT gene is silenced by promoter methylation in many human tumors, including colorectal carcinomas. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of K-ras mutations, we studied 244 colorectal tumor samples for MGMT promoter hypermethylation and K-ras mutational status. Our results show a clear association between the inactivation of MGMT by promoter hypermethylation and the appearance of G to A mutations at K-ras: 71% (36 of 51) of the tumors displaying this particular type of mutation had abnormal MGMT methylation, whereas only 32% (12 of 37) of those with other K-ras mutations not involving G to A transitions and 35% (55 of 156) of the tumors without K-ras mutations demonstrated MGMT methylation (P = 0.002). In addition, MGMT loss associated with hypermethylation was observed in the small adenomas, including those that do not yet contain K-ras mutations. Hypermethylation of other genes such as p16INK4a and p14ARF was not associated with either MGMT hypermethylation or K-ras mutation. Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to a particular genetic change in human cancer, specifically G to A transitions in the K-ras oncogene.

Analysis of adenomatous polyposis coli promoter hypermethylation in human cancer.

Germ-line mutations in the tumor suppressor gene APC are associated with hereditary familial adenomatous polyposis (FAP), and somatic mutations are common in sporadic colorectal tumors. We now report that methylation in the promoter region of this gene constitutes an alternative mechanism for gene inactivation in colon and other tumors of the gastrointestinal tract. The APC promoter is hypermethylated in 18% of primary sporadic colorectal carcinomas (n = 108) and adenoma (n = 48), and neoplasia with APC methylation fails to express the APC transcript. Methylation affects only wild-type APC in 95% of cases and is not observed in tumors from FAP patients who have germ-line APC mutations. As with APC mutation, aberrant APC methylation occurs early in colorectal carcinogenesis. When other tumor types are analyzed (n = 208), methylation of the APC promoter is not restricted to the colon but is present in tumors originating elsewhere in the gastrointestinal tract but rarely in other tumors. Our data suggest that hypermethylation of APC provides an important mechanism for impairing APC function and further underscores the importance of the APC pathway in gastrointestinal tumorigenesis.

Expression and identification of aberrant c-kit transcripts in human cancer cells.

We have previously cloned and sequenced a novel 3.5 kb c-kit mRNA expressed in a colon carcinoma cell line Colo201. Here we examined the expression of this truncated form of c-kit in 14 gastrointestinal cancer cells and 16 hematopoieic cancer cells by RT-PCR. Expression of the aberrant c-kit transcript was observed in various cancer cell lines. Furthermore, a new transcript which is 78 bp shorter than the transcript previously described was identified and characterized. These results indicate that two kinds of aberrant c-kit transcript produced by alternative promoter in intron 15 are expressed in human cancer cells.

Human exposure to carcinogenic heterocyclic amines and their mutational fingerprints in experimental animals.

Heterocyclic amines (HCAs) are mutagens/carcinogens to which humans are exposed on almost a daily basis. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP) is the most abundant of the various carcinogenic HCAs (present at a level of 0.56 to 69.2 ng/g of cooked meat or fish), with 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx) following it at 0.64 to 6.44 ng/g. HCAs have been found in the urine of healthy people who consume ordinary diets, while patients receiving parenteral alimentation lack, for example, PhlP and MelQx in their urine. Based on the concentrations of PhlP and MelQx in urine samples from 10 healthy volunteers, daily intake of MelQx in Japanese was calculated to be 0.3 to 3.9 micrograms/person, while that of PhlP was 0.005 to 0 micrograms. The Japanese consume more MelQx than Americans, whereas Japanese intake of PhlP was about one-third that of Americans. MelQx-DNA adducts have also detected in Japanese Kidney, colon, and rectum samples using the 32P-postlabeling method followed by identification using high-performance liquid chromatography (HPLC) analysis; the levels were 0.18, 1.8, and 1.4 per 10(9) nucleotides, respectively. In addition, we elucidated the mutational fingerprints of Phlp by analyzing Apc mutations in rat colon cancers induced by this carcinogen. Four of eight tumors had a total of five mutations in the Apc gene, four of which featured a guanine deletion from 5'-GTGGGAT-3' sequences. This specific mutation spectrum may be used as a fingerprint of PhlP in evaluating its risk potential for human colon carcinogenesis. Mutations were not found in similar 2-amino-3-methylimidazo[4,5-f]quinoline-induced colon lesions. Microsatellite instability was detected in both colon and mammary tumors induced by PhlP. The mechanisms involved in this development of microsatellite instability in PhlP. The mechanisms involved in this development of microsatellite instability in PhlP-induced cancers remain to be elucidated.

A stereoselective synthesis of the C-10 to C-18 (right-half) fragment of mycalamides employing lewis acid promoted intermolecular aldol reaction.

[reaction: see text] Beginning with D-mannitol, a stereoselective synthesis of the right-half segment of the mycalamides has been accomplished by employing Lewis acid catalyzed intermolecular aldol reaction and oxypalladation as the key steps.