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Centromere that plays a pivotal role in chromosome segregation is composed of repetitive elements in many eukaryotes. Although chromosomal regions containing repeats are the hotspots of rearrangements, little is known about the stability of centromere repeats. Here, by using a minichromosome that has a complete set of centromere sequences, we have developed a fission yeast system to detect gross chromosomal rearrangements (GCRs) that occur spontaneously. Southern and comprehensive genome hybridization analyses of rearranged chromosomes show two types of GCRs: translocation between homologous chromosomes and formation of isochromosomes in which a chromosome arm is replaced by a copy of the other. Remarkably, all the examined isochromosomes contain the breakpoint in centromere repeats, showing that isochromosomes are produced by centromere rearrangement. Mutations in the Rad3 checkpoint kinase increase both types of GCRs. In contrast, the deletion of Rad51 recombinase preferentially elevates isochromosome formation. Chromatin immunoprecipitation analysis shows that Rad51 localizes at centromere around S phase. These data suggest that Rad51 suppresses rearrangements of centromere repeats that result in isochromosome formation.

Critically shortened, dysfunctional telomeres may play a role in the genetic instabilities commonly found in cancer. We analyzed 30 surgical specimens of invasive breast carcinoma from women aged 34 to 91 years and estimated telomere lengths as telomere-to-centromere ratio values in the 5 different cell types comprising breast tissue in order to clarify telomere length variations within and between individuals using our tissue quantitative fluorescence in situ hybridization method. We obtained 3 novel findings. (1) In corresponding normal tissues, telomere length decreased in the order myoepithelial cells > normal-appearing fibroblasts > luminal epithelial cells, and telomere lengths were characteristic in these 3 cell types within each individual. (2) As expected, cancer cells had significantly shorter telomeres than myoepithelial cells (P < .0001) and normal-appearing fibroblasts (P = .0161), but there was no significant difference in telomere length between luminal cells and cancer cells (P = .6270). (3) Fibroblasts adjacent to cancer had longer telomeres than normal-appearing fibroblasts distant from cancer (P < .0001). This study, which represents the first reported assessment of telomere length variations in the 5 cell types comprising breast tissue within and between individuals, revealed that normal luminal epithelial cells and cancer cells had the shortest telomeres. Our new findings indicate that telomeres of background luminal cells are as short as those of cancer cells. Tissue quantitative fluorescence in situ hybridization, applicable to analysis of individual cells in tissue sections, is considered to be a powerful technique with considerable promise for studies in oncology.

Previous studies of telomeres and telomerase have focused mostly on mammals, and data for other vertebrates are limited. We analyzed both telomere length (terminal restriction fragment length) and telomerase activity in a small freshwater teleost fish, the medaka (Oryzias latipes), and found that the telomeres shorten during ageing despite the fact that a considerable amount of telomerase activity is ubiquitously detectable throughout the life of the fish. Since the telomere attrition rate during development was greater than that in adulthood, telomere length is inversely correlated with the increase in body length. The difference in telomere length among medaka individuals was similar to that in humans, and the individual specific differences were evident even at the earliest embryonic stage. Telomerase activity was ubiquitously detectable not only in the body of the embryo but also in the systemic organs of mature individuals throughout their entire life span. These data suggest that telomere attrition during ageing in medaka, which is similar to that in humans, may be a major factor determining their mortality, and that telomere maintenance through strong telomerase activity may be required for the characteristic lifelong continuous growth of this fish.

Many studies have demonstrated the association between telomere length in mitotic cells and carcinogenesis and mortality, but little attention has been focused on post-mitotic cells and human life expectancy. We assessed the relationship between telomere length in cerebral gray and white matter and longevity in 72 autopsied Japanese patients aged 0-100 years using Southern blot hybridization. The mean telomere lengths in the gray and white matter were 12.3+/-2.5 kilobase pairs and 11.4+/-2.1 kilobase pairs, respectively. The mean telomere lengths in 60-69 year decadal group were less than those of neonates, and declined further in the 70-79-year age group, but those in groups of further advanced age were longer than in the 70-79 year group (70-79<80-89<90-100 years of age). Thus, the 90-100-year age group possessed significantly longer telomeres than the 70s (p=0.029). Autopsy protocols showed a decrease in the rate of cancer death in individuals in their 80s (p=0.041) and 90s (p=0.017) versus those in their 60s, and in their 80s the mean telomere length in the gray matter from cancer death patients was significantly shorter than that of patients who died of other diseases (p=0.04). These data suggest that innate telomere lengths are maintained very well in the cerebrum, and are associated with longevity. Our study lends indispensable support to the hypothesis that longer telomeres protect the genome from instability (a major cause of carcinogenesis) and are beneficial for longevity.

We developed a novel method for evaluating telomere length in 6 cell types of noncancerous and cancerous mucosal tissues from 11 cases of gastric neoplasm using the quantitative fluorescence in situ hybridization method with telomere and centromere peptide nucleic acid probes. Our telomere length estimates were determined from the background-corrected telomere intensity divided by the background-corrected centromere intensity (telomere-to-centromere ratio). Our results indicated that telomere lengths in each of the cases studied were reduced in turn from fibroblasts to fundic gland cells, to glandular neck cells, and then to surface foveolar cells. However, the telomere lengths of intestinalized cells located among fundic glands were not always shorter than those of the other cell types, as reported previously by others. Helicobacter pylori infection was suggested to induce the telomere shortening seen in the fundic glands. Although the mean telomere lengths varied among the 8 gastric cancer cases, correlation of the telomere lengths with the Ki-67 labeling index was established after normalization with the fibroblast measurements. We conclude that our telomere-to-centromere ratio method can reliably estimate the telomere lengths of the 6 cell types in the gastric mucosa and clarifies the relationship between proliferative activity and the telomere length of cancer cells.

Faithful replication of chromosomes is crucial to genome integrity. In yeast, the ORC binds replication origins throughout the cell cycle. However, Cdc45 binds these before S-phase, and, during replication, it moves along the DNA with MCM helicase. When replication progression is inhibited, checkpoint regulation is believed to stabilize the replication fork; the detailed mechanism, however, remains unclear. To examine the relationship between replication initiation and elongation defects and the response to replication elongation block, we used fission yeast mutants of Orc1 and Cdc45--orp1-4 and sna41-928, respectively--at their respective semipermissive temperatures with regard to BrdU incorporation. Both orp1 and sna41 cells exhibited HU hypersensitivity in the absence of Chk1, a DNA damage checkpoint kinase, and were defective in full activation of Cds1, a replication checkpoint kinase, indicating that normal replication is required for Cds1 activation. Mrc1 is required to activate Cds1 and prevent the replication machinery from uncoupling from DNA synthesis. We observed that, while either the orp1 or the sna41 mutation partially suppressed HU sensitivity of cds1 cells, sna41 specifically suppressed that of mrc1 cells. Interestingly, sna41 alleviated the defect in recovery from HU arrest without increasing Cds1 activity. In addition to sna41, specific mutations of MCM suppressed the HU sensitivity of mrc1 cells. Thus, during elongation, Mrc1 may negatively regulate Cdc45 and MCM helicase to render stalled forks capable of resuming replication.

We have conducted systematic studies to measure telomere length in human tissues of all types. Progressive telomere shortening with aging was studied in specimens of normal pancreas obtained at autopsy from 69 subjects aged 0 to 100 yr, and age-related shortening of telomere length at a rate of 36 base pairs (bp) per year was detected. Mean telomere length (+/-SD) was 13.9+/-1.4 kilobase pairs (kbp) in 16 neonates, as opposed to 8.4 kbp in 2 centenarians. Mean telomere length (+/-SD) in four age groups, 0-24, 25-49, 50-74, and 75-100 yr, was 13.5+/-1.5, 12.3+/-0.7, 11.3+/-2.5, and 10.7+/-1.8, respectively.

To clarify the significance of hMLH1 promoter hypermethylation in the development of medullary-type poorly differentiated colorectal adenocarcinoma, we studied the status of promoter methylation and hMLH1 expression in 23 medullary-type and 12 pleomorphic-type carcinomas, as well as the pathology and microsatellite status. In medullary-type carcinomas, the percentages of cases with promoter methylation (83%) and an absence of hMLH1 expression (91%) were significantly higher than in pleomorphic-type carcinomas (14 and 17%), respectively. The rate of microsatellite instability in the medullary type was significantly higher than that of the pleomorphic type (87 vs 40%, P<0.01). Compared with pleomorphic-type carcinomas, medullary-type carcinomas were significantly associated with hMLH1 promoter methylation, absent expression of hMLH1 protein, microsatellite instability, as well as a proximal location, a Crohn's-like lymphoid reaction, a low incidence of lymph node metastasis, and a favorable outcome. Medullary-type carcinomas accumulated with advancing age, especially in the female. These results indicated that hMLH1 hypermethylation, concurrent with a lack of its protein expression, may play an important role in the development of medullary-type poorly differentiated colorectal adenocarcinomas in the elderly.

One major protein was selectively solubilized when phosphate analogues, such as inorganic vanadate (Vi), beryllium fluoride (BeFx) or aluminum fluoride (AlFx), were added to ciliary axonemes of Tetrahymena ssp. (T. pyriformis or T. thermophila) in the presence of ATP. This protein contains three high molecular weight polypeptides, characteristic of an outer arm dynein. Electron microscopic observation of the axonemes after solubilization using ATP and Vi revealed axonemes partially lacking outer arm dyneins. These results suggest that the solubilized protein is an outer arm dynein and also that a dynein-ADP-phosphate complex decreases its affinity with the adjacent microtubules within axonemes. Limited digestion with chymotrypsin revealed that each solubilized dynein has a similar conformation, but it is markedly different from that of dynein in the absence of ATP or a phosphate analogue. The solubilized dynein obtained by the addition of Vi and ATP to axonemes was digested by UV irradiation to yield at least five new polypeptides (240, 230, 225, 180 and 160 kDa) but the dyneins solubilized by BeFx (or AlFx) in the presence of ATP did not produce any photocleavage products under the same conditions.

Background and Objective: Telomerase activity is present in most malignant tumours and might provide a mechanism for unlimited replication of neoplastic cells. Expression of the gene encoding human telomerase reverse transcriptase (hTERT), the telomerase catalytic subunit gene, is associated with telomerase activity, and it is overexpressed in most parathyroid carcinomas. We have therefore studied hTERT expression in parathyroid tumours., Methods: In the present study, we assayed telomerase activity by the telomeric repeat amplification protocol (TRAP) and used in situ hybridization (ISH) to study hTERT mRNA expression in four parathyroid metastatic cancers, six adenomas and two parathyroid hyperplasia tissue specimens., Results: Telomerase activity was detected by the TRAP assay in all of the parathyroid cancers (100%) but in none of the eight parathyroid benign lesions. hTERT gene expression was detected in the nuclei of the parathyroid cancer cells in all of the lesions (100%) but in none of the eight parathyroid benign lesions., Conclusions: Our results demonstrate a correlation between telomerase activity and hTERT gene expression measured by ISH (P < 0.001) in parathyroid tumours. We succeeded in very clearly and sensitively demonstrating hTERT mRNA in parathyroid tissues by using a new probe.

Detailed studies of the phosphatase activity in the tidelands were examined including the localization of the phosphatase activities and the spatial distributions of the activity in the tidelands around Hiroshima Bay, the Aki-Bingo Open Sea and Isahaya Bay of Kyushu on the western side of Japan. The results indicate that the phosphatase activity of the sediment fraction accounts for 98% of the tideland activity. Furthermore, we classified 19 tidelands into four groups by a cluster analysis using two parameters: phosphatase activities and ATP (adenosine 5(')-triphosphate) biomass. The first group is a member of the tidelands located in areas remote from the cities. They have a low phosphatase activity and low ATP biomass and the seawater in the tideland is very clean. The second is a group with a low phosphatase activity and high ATP biomass. In the tidelands belonging to this group, large numbers of benthos and shellfish can be observed. The third group has a high phosphatase activity and a relatively low ATP biomass. The estuary tidelands located near Hiroshima City belong to the third group. The last one is tidelands with a high phosphatase activity and high ATP biomass. In this group, the ratio of the phosphatase activity to ATP biomass is relatively low. In the summer, we observed deterioration in the water quality and accumulation of sludge on the sea bottom in the tideland of the last group.Moreover, we employed a multiple regression analysis and proposed an equation to obtain the group number of the cluster analysis using the assayed values of the phosphatase activity and the ATP biomass.

Many epidemiological studies have examined the associations of nutrients and foods with risk of stomach cancer. Few studies have addressed the dietary patterns that reflect various components of food consumption and their interactions. We identified major four dietary patterns; "vegetable and fruit", "Western breakfast", "meat", and "rice/snack" with principal component analysis using food consumption questionnaire in a prospective study of 5,765 middle-aged male workers in Tokyo. After 10 years of follow-up between September 1988 and August 1998, 84 incident cases of stomach cancer were documented. Using proportional hazards regression to estimate risk ratios, we found no clear association between each of dietary pattern and stomach cancer risk. After adjustment for age and other potential confounding factors and after exclusion of the cases diagnosed in first follow-up year, the risk ratio (RR) associated with high tertile compared to low tertile was 0.78(95%CI 0.42-1.44) for "vegetable and fruit" pattern and 0.71(95%CI 0.40-1.24) for "Western breakfast" pattern. The V-shaped associations between dietary patterns and stomach cancer risk were appeared in the "meat" (RR=1.00, 0.55, and 1.10) and the "rice/snack" (RR=1.00, 0.52, and 1.19) patterns, while the linear trend of these associations was statistically on borderline. The roll of overall dietary patterns in predicting stomach cancer risk requires further investigation.

We investigated progressive telomere shortening in normal human epidermis and lingual epithelium during aging, and attempted, in particular, to ascertain whether the telomere shortening that accompanies aging occurs at the same rate in different tissues. We studied telomeric DNA integrity, and estimated annual telomere loss, in 52 specimens of epidermis and 48 specimens of lingual epithelium collected at autopsy from subjects who had died at ages between 0 and 101 y. Most of the DNA samples were measured twice by southern blot hybridization. In addition, the correlation between telomere lengths in the two types of tissues was examined. The telomere reduction rates in epidermis and lingual epithelium were 36 bp and 30 bp per y, respectively, and these were significantly different. The rates obtained by the second measurements in epidermis and lingual epithelium were 39 and 32 bp per y, respectively, and these were also significantly different. The mean telomere lengths in the epidermis of eight neonates and the lingual epithelium of seven neonates were 13.2+/-1.0 and 13.8+/-1.0 kb, respectively. Comparison of telomere lengths in the two tissues for 41 paired samples showed that the mean telomere length in the epidermis (10.7+/-2.3 kb) was less than that in the lingual epithelium (12.4+/-2.5 kb); however, statistical analysis revealed a very significant relationship between epidermal and lingual epithelial telomere length (r=0.842, p<0.0001). These results indicate that the telomeres in epidermis and lingual epithelium are characterized by tissue-specific loss rates.

The hepatitis B virus (HBV) infection is a major health problem in China. This study examined liver function in relation to HBV infection, and the occupational and lifestyle factors among workers in Shanghai. The study included 690 male workers aged 20-59 employed at a steel manufacturing company. The occupational and lifestyle factors were evaluated by self-administered questionnaire addressing worksite, exposure to dust or chemicals, history of cigarette smoking and habitual alcohol consumption. The prevalence of hepatitis B surface antigen(HBsAg) seropositivity was 21.4%. Elevated values of aspartate aminotransferase (AST, >30IU/liter) appeared in HBsAg-positive and current alcohol drinking groups but statistically on the borderline. There was a positive linear trend in the odds ratios(ORs) among age groups and ethanol consumption levels for elevated values of g-glutamyl transferase (GGT, >50IU/liter). There was no clear association between occupational exposure and liver functions. When the effects of HBsAg and the current alcohol drinking status on the elevated value of AST were examined simultaneously, OR for cases with HBsAg-positive and current alcohol drinking rose to 2.85(95%CI.98-8.28) against reference cases with HBsAg-negative and non-alcohol drinking, although this association was statistically on the borderline. The results indicated that some interventional attempts including educational strategy for alcohol drinking would be important among the HBsAg-positive cases to reduce the risk of liver dysfunction and further, hepatocellular carcinoma.