Increased risk of skin cancer in Japanese heterozygotes of xeroderma pigmentosum group A.

This study was designed to learn if asymptomatic heterozygotes with mutations in a DNA repair gene are at an increased risk for cancer. To examine this, we focused on carriers of an XPA founder mutation because the frequency of xeroderma pigmentosum (XP) patients is much greater among Japanese than Caucasians, more than half of Japanese XP patients are affected at the XPA gene, and the majority of XP-A patients carry the same founder mutation in the XPA gene. Here we show that the frequency of XPA heterozygote was 14/1698 (0.8%) in cancer-free controls, and the corresponding frequency in patients with nonmelanocytic skin cancer that developed in sun-exposed areas was 11/440 (2.5%, OR = 3.08, p = 0.0097) for basal cell carcinoma, and 3/272 (1.1%, OR = 1.34, p = 0.72) for squamous cell carcinoma. These results suggest a moderately elevated risk for skin cancer among XPA heterozygotes.

Unrepairable DNA double-strand breaks that are generated by ionising radiation determine the fate of normal human cells.

After an exposure to ionising radiation, cells can quickly repair damage to their genomes; however, a few unrepairable DNA double-strand breaks (DSBs) emerge in the nucleus in a prolonged culture and perpetuate as long as the culture continues. These DSBs may be retained forever in cells such as non-dividing ageing tissues, which are resistant to apoptosis. We show that such unrepairable DSBs, which had been advocated by the classical target theory as the 'radiation hit', could account for permanent growth arrest and premature senescence. The unrepairable DSBs build up with repeated irradiation, which accounts for an accumulated dose. Because these DSBs tend to be paired, we propose that the untethered and 'torn-off' molecular structures at the broken ends of the DNA result in an alteration of chromatin structure, which protects the ends of the DNA from genomic catastrophe. Such biochemical responses are important for cell survival but may cause gradual tissue malfunction, which could lead to the late effects of radiation exposure. Thus, understanding the biology of unrepairable damage will provide new insights into the long-term effects of radiation.

Photoresponsive two-component organogelators based on trisphenylisoxazolylbenzene.

Photochromic tris(phenylisoxazolyl)benzene 1 and bispyridine derivatives 2a­e were mixed in a certain ratio to generate stable gels in benzyl alcohol, 4-methoxybenzyl alcohol, and aniline. Supramolecular assembly of 1 in solution was confirmed by 1H NMR study. The Tgel value was saturated in a 2 : 3 ratio of 1 and 2c. The intermolecular hydrogen bonds OH···N and salt bridge O(−)···H(­)N(+) between 1 and 2c coexisted evidently, and these hydrogen bonds contributed to the stabilization of the gel networks. The lengths of alkyl chains of 2a­e governed the stabilities of the gels. The gel formations were driven by the morphological transition of 1 before and after the addition of 2a­e. Mixtures of 1 and 2a­e led to the well developed fibrillar networks, generating a lot of voids that are responsible for immobilizing solvent molecules. When the benzyl alcohol gel was irradiated at 360 nm, the gel turned to the sol. The sol was reversed to the gel by warming. This gel-to-sol phase transition was completely reversible.

Circadian Clock genes Per2 and clock regulate steroid production, cell proliferation, and luteinizing hormone receptor transcription in ovarian granulosa cells.

Circadian Clock genes are associated with the estrous cycle in female animals. Treatment with Per2 and Clock siRNAs decreased the number of granulosa cells and LHr expression in follicle-stimulating hormone FSH-treated granulosa cells. Per2 siRNA treatment did not stimulate the production of estradiol and expression of P450arom, whereas Clock siRNA treatment inhibited the production of estradiol and expression of P450arom mRNA. Per2 and Clock siRNA treatment increased and unchanged, respectively, progesterone production in FSH-treated granulosa cells. Similarly, expression of StAR mRNA was increased by Per2 siRNA and unchanged by Clock siRNA. Our data provide a new insight that Per2 and Clock have different action on ovarian granulosa cell functions.

Easy detection of GFP-positive mutants following forward mutations at specific gene locus in cultured human cells.

We have generated a new mutation assay system using HT1080 human fibrosarcoma cells, which consists of a combination of tetracycline-operator dependent GFP gene (TetO-EGFP) and tetracycline repressor (TetR) genes, where the expression of GFP gene is under strict control of TetR protein, and the TetR gene is located within the endogenous HPRT gene. In this system, any inactivating mutation at the TetR gene or large deletions including the gene itself results in high expression of GFP gene (>200-fold increase) in the cells, which can be readily scored not only by a flow cytometer but also under a fluorescent microscope. With this new cell line, we show that the spontaneous mutation rate at the TetR locus was 2.8-3.4×10(-6)/cell division, slightly lower than the rate at the endogenous HPRT gene of HT1080 cells, and has a dose response to X rays as a mutagen. We also isolated variant clones with elevated spontaneous mutation rate (i.e., genetically unstable cells) following X irradiation. Spontaneous GFP-positive mutants were predominantly base-change mutations at the TetR gene while those obtained after X irradiation often contained large deletions which spanned up to 6Mb. The results indicate that the bacterial TetR/TetO regulatory units work extremely well as a mutation detection system in human cells, and any part of the human genome may be tested for mutation sensitivity following targeted insertion of the TetR gene in a stably expressing gene.

Tooth enamel ESR doses and cytogenetic doses of Nagasaki atomic-bomb survivors in comparison with DS02R1 doses.

PURPOSE: Cancer risks for Nagasaki survivors once appeared to be lower than for Hiroshima survivors. The possibility that this was due to overestimation of the doses for the Nagasaki survivors was tested by measuring biological doses of Nagasaki survivors and comparing them with DS02R1 individual doses as previously done for Hiroshima survivors. MATERIALS AND METHODS: The electron spin resonance (ESR) method and cytogenetic method were used to estimate radiation doses for 24 Nagasaki survivors, and the results were compared to calculated DS02R1 doses. RESULTS: Six factory workers and 10 other survivors showed ESR or cytogenetically estimated doses that were in reasonably good agreement with their DS02R1 doses, while one factory worker was found to have an ESR dose estimate of nearly one half of the DS02R1 dose to the eye lens (a proxy organ for teeth). A few outliers were also observed. CONCLUSIONS: Although apparently lower cancer risks were observed in the past for Nagasaki survivors when compared to Hiroshima survivors, the present results do not indicate the existence of a trend that DS02R1 doses are overestimated when compared with biologically estimated tooth or cytogenetic doses. This observation is in line with the recent disappearance of the city difference in cancer risks.

Population structure and antimicrobial susceptibility of Paenibacillus larvae isolates from American foulbrood cases in Apis mellifera in Japan.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), the most destructive disease of the honey bee brood. In this study, we investigated the population structure and antimicrobial susceptibility of Japanese P. larvae using 100 isolates isolated between 1993 and 2017 in 17 prefectures. Using repetitive-element PCR and multilocus sequence typing, isolates from diverse origins were classified into six genotypes, including the novel genotype ERIC II-ST24. Among these genotypes, ERIC I-ST15 is the most common in Japan, while ERIC II-ST10 isolates were found to be increasing during the 2010s. Regardless of genotype or origin, all isolates were susceptible to the major antimicrobials used in the control of AFB, including mirosamicin and tylosin, which were approved for the prevention of AFB in Japan in 1999 and 2017 respectively. Despite nearly 20 years of use, mirosamicin is still effective against Japanese P. larvae in vitro; however, the development of AFB in honey bee colonies may not always be suppressed by this drug. The case information collected in this study provides insight into the conditions under which prophylactic medicine may not exert sufficient preventive effects in vivo.

Prognosis in patients with hepatocellular carcinoma correlates to mutations of p53 and/or hMSH2 genes.

Association of gene alterations and prognosis has not fully been elucidated in hepatocellular carcinoma (HCC). To clarify the relationship between p53 and hMSH2 mutations and prognosis, we analysed these mutations in 83 HCC cases and assessed their association with various clinicopathological factors. The 3-year disease-free survival (DFS) or overall survival (OS) rates in HCC patients with p53 mutation and p53 wild/hMSH2 mutation significantly decreased compared with those without these mutations (14.3% and 37.5% versus 67.5% for DFS; 35.7% and 50.0% versus 96.4% for OS, respectively). In the multivariate analysis, categories by p53 and hMSH2 mutation status, and liver cirrhosis demonstrated statistical significances for DFS and OS. Moreover, the frequency of patients with p53 and/or hMSH2 mutations in intrahepatic metastasis (75.0%) was significantly higher than that in multicentric occurrence (14.3%). Thus, p53 and hMSH2 mutations will be useful for identifying subsets of HCC patients with poor prognosis.

Cytogenetic Reconstruction of Gamma-Ray Doses Delivered to Atomic Bomb Survivors: Dealing with Wide Distributions of Photon Energies and Contributions from Hematopoietic Stem/Progenitor Cells.

Retrospective estimation of the doses received by atomic bomb (A-bomb) survivors by cytogenetic methods has been hindered by two factors: One is that the photon energies released from the bomb were widely distributed, and since the aberration yield varies depending on the energy, the use of monoenergetic 60Co gamma radiation to construct a calibration curve may bias the estimate. The second problem is the increasing proportion of newly formed lymphocytes entering into the lymphocyte pool with increasing time intervals since the exposures. These new cells are derived from irradiated precursor/stem cells whose radiosensitivity may differ from that of blood lymphocytes. To overcome these problems, radiation doses to tooth enamel were estimated using the electron spin resonance (ESR; or EPR, electron paramagnetic resonance) method and compared with the cytogenetically estimated doses from the same survivors. The ESR method is only weakly dependent on the photon energy and independent of the years elapsed since an exposure. Both ESR and cytogenetic doses were estimated from 107 survivors. The latter estimates were made by assuming that although a part of the cells examined could be lymphoid stem or precursor cells at the time of exposure, all the cells had the same radiosensitivity as blood lymphocytes, and that the A-bomb gamma-ray spectrum was the same as that of the 60Co gamma rays. Subsequently, ESR and cytogenetic endpoints were used to estimate the kerma doses using individual DS02R1 information on shielding conditions. The results showed that the two sets of kerma doses were in close agreement, indicating that perhaps no correction is needed in estimating atomic bomb gamma-ray doses from the cytogenetically estimated 60Co gamma-ray equivalent doses. The present results will make it possible to directly compare cytogenetic doses with the physically estimated doses of the survivors, which would pave the way for testing whether or not there are any systematic trends or factors affecting physically estimated doses.

Heterozygous individuals bearing a founder mutation in the XPA DNA repair gene comprise nearly 1% of the Japanese population.

Individuals who are homozygotes for mutations in DNA repair genes are at high risk for cancer. It is not well documented, however, if the heterozygous carriers of the mutation are also predisposed to cancer. To address the issue, xeroderma pigmentosum (XP) in Japan is an interesting candidate because of three major reasons: XP is an autosomal recessive disorder with an enormously elevated risk of skin cancer, the frequency of XP patients is higher in Japan than in other parts of the world, and more than half of Japanese XP patients are homozygous for the same founder mutation in the XPA gene. We screened archival blood samples from Japanese individuals who resided in Hiroshima or Nagasaki. A simple PCR-RFLP method was developed that is highly specific for detection of XPA heterozygotes carrying the founder mutation. We identified nine XPA heterozygotes among 1,020 individuals screened for a prevalence of 0.88%. This rate, if representative, implies that there are about 1 million carriers of the XPA founder mutation in the Japanese population. Thus, investigation of their cancer risk may be warranted.

Serial evaluation of acute cerebral hyperperfusion by transcranial color-coded sonography.

Using transcranial color-coded sonography (TCCS), we evaluated the acute changes in the hemodynamics of cerebral hyperperfusion in two cases. The mean flow velocity of the cerebral arteries increased at the onset of clinical symptoms, together with an increase in the regional cerebral blood flow (rCBF). In serial follow-up studies, the flow velocity gradually returned to normal in parallel with the normalization of the rCBF values. TCCS can be useful for evaluation of acute cerebral hyperperfusion.

Existence of Paenibacillus larvae genotypes ERIC I-ST2, ERIC I-ST15 and ERIC II-ST10 in the western region of Aichi prefecture, Japan.

American foulbrood is the most destructive honeybee bacterial disease. The etiological agent, Paenibacillus larvae, has been classified into four genotypes by a repetitive-element PCR (ERIC I-IV) and 21 sequence types by multilocus sequence typing (ST1-21). In this study, we genotyped Japanese P. larvae isolates for the first time and revealed the presence of three genotypes (ERIC I-ST2, ERIC I-ST15 and ERIC II-ST10) in the western region of Aichi prefecture. ERIC I-ST15 and ERIC II-ST10 are globally distributed types, whereas the ERIC I-ST2 isolate was the first isolate of this genotype identified outside the native range of the European honeybee. The ERIC I and II isolates differed in phenotypes including cell morphology, and these may be useful for predicting ERIC types.

Photoresponsive Toroidal Nanostructure Formed by Self-Assembly of Azobenzene-Functionalized Tris(phenylisoxazolyl)benzene.

The self-assembly of tris(phenylisoxazolyl)benzene 1b with photochemically addressable azobenzene moieties produced toroidal nanostructures, the formation and dissociation of which were reversibly regulated upon photoirradiation. 1b displayed a mesogenic behavior. In the solution, the stacked assemblies along with their C3 axes were formed. In the mesophase, two molecules of 1b most likely adopted the antiparallel arrangement to stabilize the columnar organization. This assembling behavior most likely triggered the development of the supramolecular toroidal nanostructures.

Progerin, the protein responsible for the Hutchinson-Gilford progeria syndrome, increases the unrepaired DNA damages following exposure to ionizing radiation.

INTRODUCTION: Progerin, the protein responsible for the Hutchinson-Gilford Progeria Syndrome (HGPS), is a partially deleted form of nuclear lamin A, and its expression has been suggested as a cause for dysfunctional nuclear membrane and premature senescence. To examine the role of nuclear envelop architecture in regulating cellular aging and DNA repair, we used ionizing radiation to increase the number of DNA double strand breaks (DSBs) in normal and HGPS cells, and analyzed possible relationship between unrepaired DSBs and cellular aging. RESULTS: We found that HGPS cells are normal in repairing a major fraction of radiation-induced double strand breaks (M-DSBs)but abnormal to show increased amount of residual unrepaired DSBs (R-DSBs). Such unrepaired DSBs were 2.6 times (CI 95 %: 2.2-3.2) higher than that in normal cells one week after the irradiation, and 1.6 times (CI 95 %: 1.3-1.9) higher even one month after the irradiation. These damages tend to increase as the nuclear envelope become abnormal, a characteristic of both HGPS and normal human cells which undergo replicative senescence. The artificial, enforced over-expression of progerin further impaired the repair of M-DSBs, implying lamin A-associated nuclear membrane has an important role for DNA DSB repair. Introduction of telomerase gene function in HGPS cells reversed such aging phenotypes along with upregulation of lamin B1 and downregulation of progerin, which is a hallmark of young cells. CONCLUSION: We suggest that lamin A- or progerin-associated nuclear envelope is involved in cellular aging associated with DNA damage repair.

Creation of Mice Bearing a Partial Duplication of HPRT Gene Marked with a GFP Gene and Detection of Revertant Cells In Situ as GFP-Positive Somatic Cells.

It is becoming clear that apparently normal somatic cells accumulate mutations. Such accumulations or propagations of mutant cells are thought to be related to certain diseases such as cancer. To better understand the nature of somatic mutations, we developed a mouse model that enables in vivo detection of rare genetically altered cells via GFP positive cells. The mouse model carries a partial duplication of 3' portion of X-chromosomal HPRT gene and a GFP gene at the end of the last exon. In addition, although HPRT gene expression was thought ubiquitous, the expression level was found insufficient in vivo to make the revertant cells detectable by GFP positivity. To overcome the problem, we replaced the natural HPRT-gene promoter with a CAG promoter. In such animals, termed HPRT-dup-GFP mouse, losing one duplicated segment by crossover between the two sister chromatids or within a single molecule of DNA reactivates gene function, producing hybrid HPRT-GFP proteins which, in turn, cause the revertant cells to be detected as GFP-positive cells in various tissues. Frequencies of green mutant cells were measured using fixed and frozen sections (liver and pancreas), fixed whole mount (small intestine), or by means of flow cytometry (unfixed splenocytes). The results showed that the frequencies varied extensively among individuals as well as among tissues. X-ray exposure (3 Gy) increased the frequency moderately (~2 times) in the liver and small intestine. Further, in two animals out of 278 examined, some solid tissues showed too many GFP-positive cells to score (termed extreme jackpot mutation). Present results illustrated a complex nature of somatic mutations occurring in vivo. While the HPRT-dup-GFP mouse may have a potential for detecting tissue-specific environmental mutagens, large inter-individual variations of mutant cell frequency cause the results unstable and hence have to be reduced. This future challenge will likely involve lowering the background mutation frequency, thus reducing inter-individual variation.

Two-dimensional complementary deoxyribonucleic acid electrophoresis revealing up-regulated human epididymal protein-1 and down-regulated CL-100 in thyroid papillary carcinoma.

Two-dimensional cDNA electrophoresis was used to analyze gene expressions in papillary carcinoma and normal tissue of thyroid glands. Pooled thyroid tissues were used to extract mRNA. Complementary DNAs, synthesized with NotI anchor primers, were digested with three restriction enzymes, NotI, EcoRV, and PvuII. The protruding NotI ends were filled in with (32)P deoxynucleotide triphosphates, and the radiolabeled cDNA fragments were separated in two dimensions. Approximately 500 cDNA fragments were visualized as discrete spots without probes. A total of 20 spots, 9 up-regulated and 11 down-regulated cDNAs in papillary carcinoma, were selected and cloned for sequencing. This experiment lent itself to a novel discovery of up-regulated human epididymal protein 1 (HE-1) and down-regulated CL-100 genes in thyroid papillary carcinomas confirmed by Northern blot analysis. Immunohistochemical stains showed abundant HE-1 protein in the papillary carcinoma, whereas little or no HE-1 protein was detected in other types of thyroid cancers and normal thyroid tissues. The restricted localization of HE-1 protein to the portions of papillary projections suggests an involvement of HE-1 protein for forming papillary shape. Our study showed that two-dimensional cDNA electrophoresis is a useful method of detecting differentially expressed genes in human diseases as demonstrated for HE-1 and CL-100 in papillary carcinoma.

Diagnostic impact of transcranial color-coded real-time sonography with echo contrast agents for hyperperfusion syndrome after carotid endarterectomy.

BACKGROUND AND PURPOSE: The purpose of the present study was to evaluate availability of transcranial color-coded real-time sonography (TCCS) to detect hyperperfusion after carotid endarterectomy (CEA). METHODS: This prospective study included 105 consecutive patients who underwent CEA for severe carotid stenosis. TCCS with echo contrast agents was performed serially to evaluate flow velocity of the middle cerebral artery (MCA). Regional cerebral blood flow (rCBF) and vasodilatory capacity of the MCA territory were evaluated using single-photon emission computed tomography. We compared the changes in MCA flow velocity with rCBF. RESULTS: Using echo contrast agents, we could evaluate the MCA flow in 95 (90%) of 105 patients. Twelve patients showed hyperperfusion syndrome. Changes in the MCA mean flow velocity (MFV) before and 4 days after CEA were significantly correlated with those in rCBF (r=0.48; P<0.0001). An increase of >50% in MCA MFV was observed within 4 days after CEA in all 12 patients with hyperperfusion syndrome. Multivariate analysis revealed that reduced vasodilatory capacity (odds ratio, 0.14; 95% CI, 0.04 to 0.46) was an independent risk factor for a 1.5-fold increase in the MFV of MCA ipsilateral to CEA. CONCLUSIONS: Findings of a 1.5-fold increase in the MCA MFV can accurately identify those patients with high risk of developing post-CEA hyperperfusion syndrome. TCCS with echo contrast agents is available for the evaluation of hyperperfusion after CEA.

Uneven distributions of naïve and memory T cells in the CD4 and CD8 T-cell populations derived from a single stem cell in an atomic bomb survivor: implications for the origins of the memory T-cell pools in adulthood.

The processes that lead to the establishment and maintenance of memory T-cell pools in humans are not well understood. In this study, we examined the emergence of naïve and memory T cells in an adult male who was exposed to an atomic bomb radiation dose of approximately 2 Gy in 1945 at the age of 17. The analysis presented here was made possible by our earlier observation that this particular individual carries a hematopoietic stem cell mutation at the hypoxanthine phosphoribosyltransferase (HPRT) locus that is almost certainly a result of his exposure to A-bomb radiation. Our key finding is that we detected a very much higher HPRT mutant frequency in the naive (CD45RA(+)) cell component of this individual's CD4 and CD8 T-cell populations than in the memory (CD45RA(-)) cell component of his CD4 and CD8 T-cell populations. This stands in marked contrast to our finding that HPRT mutant frequencies are fairly similar in the naïve CD45RA(+) and memory CD45RA(-) components of the CD4 and CD8 T-cell populations of three unexposed individuals examined concurrently. In addition we found that the HPRT mutant frequencies were about 30-fold higher in the naïve (CD45RA(+)) CD4 T cells of the exposed individual than in his memory (CD45RA(-)) cell populations, but that the effect was a little less striking in his CD8 cell populations, where the HPRT mutant frequencies were only about 15-fold higher in his naïve T-cell pools than in his memory T-cell pools. We further found that 100% of the HPRT mutant cells in both his CD4 and CD8 naïve cell subsets appeared to have originated from repeated divisions of the initial HPRT mutant stem cell, whereas only 4 of 24 and 5 of 6 mutant cells in his CD4 and CD8 memory cell subsets appeared to have originated from that same stem cell. The most straightforward conclusion may be that the great majority of the T cells produced by this individual since he was 17 years old have remained as naïve-type T cells, rather than having become memory-type T cells. Thus the T cells that have been produced from the hematopoietic stem cells of this particular A-bomb-exposed individual seldom seem to enter and/or to remain in the memory T-cell pool for long periods. We speculate that this constraint on entry into memory T-cell pools may also apply to unirradiated individuals, but in the absence of genetic markers to assist us in obtaining evidential support, we must await clarifying information from radically different experimental approaches.

Reversible posterior leukoencephalopathy syndrome in a postpartum woman without eclampsia.

We report a patient who developed reversible posterior leukoencephalopathy syndrome (RPLS) in puerperium without preeclampsia-eclampsia or chronic hypertension. The woman suddenly complained of visual loss and headache 10 days after delivery caused by edematous lesions mainly distributed in the bilateral occipital lobe. Apparent diffusion coefficient map was useful for distinction of this vasogenic edema from cytotoxic edema due to brain infarction. Under the diagnosis of RPLS, we successfully treated her disease using a trinitroglycerin as an antihypertensive, a hyperosmolar agent, methylprednisolone, and a free radical scavenger. Postpartum women may have the risk of development of RPLS even without preeclampsia-eclampsia. Vascular endothelial dysfunction may trigger RPLS, in addition to acute and modest increase in systemic pressure.

Gamma-ray and neutron dosimetry by EPR and AMS, using tooth enamel from atomic-bomb survivors: a mini review.

The electron paramagnetic resonance (EPR, or electron spin resonance) method was used to measure CO2⁻· radicals recorded in tooth enamel by exposure to atomic-bomb gamma rays. The EPR-estimated doses (i.e. 6°Co gamma-ray equivalent dose) were generally in good correlation with cytogenetic data of the same survivors, whereas plots of EPR-estimated dose or cytogenetically estimated dose against DS02 doses turned out to scatter more widely. Because those survivors whose EPR doses were higher (or lower) than DS02 doses tended to show also higher (or lower) responses for cytogenetic responses, the apparent variation appears primarily due to problems in individual DS02 doses rather than the measurement errors associated with the EPR or cytogenetic technique. A part of the enamel samples were also used for evaluation of neutron doses by measuring 4¹Ca/4°Ca ratios using the accelerator mass spectrometry technique. The results for the measured ratios were on average ~85 % of the calculated ratios by DS02 (but within the 95 % confidence bounds of the simulated results), which lends support to DS02-derived neutron doses to the survivors.