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Background: In an extensive genomic analysis of lung adenocarcinomas (LUADs), driver mutations have been recognized as potential targets for molecular therapy. However, there remain cases where target genes are not identified. Super-enhancers and structural variants are frequently identified in several hundred loci per case. Despite this, most cancer research has approached the analysis of these data sets separately, without merging and comparing the data, and there are no examples of integrated analysis in LUAD., Methods: We performed an integrated analysis of super-enhancers and structural variants in a cohort of 174 LUAD cases that lacked clinically actionable genetic alterations. To achieve this, we conducted both WGS and H3K27Ac ChIP-seq analyses using samples with driver gene mutations and those without, allowing for a comprehensive investigation of the potential roles of super-enhancer in LUAD cases., Results: We demonstrate that most genes situated in these overlapped regions were associated with known and previously unknown driver genes and aberrant expression resulting from the formation of super-enhancers accompanied by genomic structural abnormalities. Hi-C and long-read sequencing data further corroborated this insight. When we employed CRISPR-Cas9 to induce structural abnormalities that mimicked cases with outlier ERBB2 gene expression, we observed an elevation in ERBB2 expression. These abnormalities are associated with a higher risk of recurrence after surgery, irrespective of the presence or absence of driver mutations., Conclusions: Our findings suggest that aberrant gene expression linked to structural polymorphisms can significantly impact personalized cancer treatment by facilitating the identification of driver mutations and prognostic factors, contributing to a more comprehensive understanding of LUAD pathogenesis., (© 2024. The Author(s).)

High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecological malignancy. To date, the profiles of gene mutations and copy number alterations in HGSOC have been well characterized. However, the patterns of epigenetic alterations and transcription factor dysregulation in HGSOC have not yet been fully elucidated. In this study, we performed integrative omics analyses of a series of stepwise HGSOC model cells originating from human fallopian tube secretory epithelial cells (HFTSECs) to investigate early epigenetic alterations in HGSOC tumorigenesis. Assay for transposase-accessible chromatin using sequencing (ATAC-seq), chromatin immunoprecipitation sequencing (ChIP-seq), and RNA sequencing (RNA-seq) methods were used to analyze HGSOC samples. Additionally, protein expression changes in target genes were confirmed using normal HFTSECs, serous tubal intraepithelial carcinomas (STICs), and HGSOC tissues. Transcription factor motif analysis revealed that the DNA-binding activity of the AP-1 complex and GATA family proteins was dysregulated during early tumorigenesis. The protein expression levels of JUN and FOSL2 were increased, and those of GATA6 and DAB2 were decreased in STIC lesions, which were associated with epithelial-mesenchymal transition (EMT) and proteasome downregulation. The genomic region around the FRA16D site, containing a cadherin cluster region, was epigenetically suppressed by oncogenic signaling. Proteasome inhibition caused the upregulation of chemokine genes, which may facilitate immune evasion during HGSOC tumorigenesis. Importantly, MEK inhibitor treatment reversed these oncogenic alterations, indicating its clinical effectiveness in a subgroup of patients with HGSOC. This result suggests that MEK inhibitor therapy may be an effective treatment option for chemotherapy-resistant HGSOC., (© 2023. The Author(s).)

Ovarian clear cell carcinoma (OCCC) has a poor prognosis, and its therapeutic strategy has not been established. PRELP is a leucine-rich repeat protein in the extracellular matrix of connective tissues. Although PRELP anchors the basement membrane to the connective tissue and is absent in most epithelial cancers, much remains unknown regarding its function as a regulator of ligand-mediated signaling pathways. Here, we obtained sets of differentially expressed genes by PRELP expression using OCCC cell lines. We found that more than 1000 genes were significantly altered by PRELP expression, particularly affecting the expression of a group of genes involved in the PI3K-AKT signaling pathway. Furthermore, we revealed the loss of active histone marks on the loci of the PRELP gene in patients with OCCC and how its forced expression inhibited cell proliferation. These findings suggest that PRELP is not only a molecule anchored in connective tissues but is also a signaling molecule acting in a tumor-suppressive manner. It can serve as the basis for early detection and novel therapeutic approaches for OCCC toward precision medicine.

High-grade serous ovarian carcinoma (HGSOC) is the most aggressive gynecological malignancy, resulting in approximately 70% of ovarian cancer deaths. However, it is still unclear how genetic dysregulations and biological processes generate the malignant subtype of HGSOC. Here we show that expression levels of microtubule affinity-regulating kinase 3 (MARK3) are downregulated in HGSOC, and that its downregulation significantly correlates with poor prognosis in HGSOC patients. MARK3 overexpression suppresses cell proliferation and angiogenesis of ovarian cancer cells. The LKB1-MARK3 axis is activated by metabolic stress, which leads to the phosphorylation of CDC25B and CDC25C, followed by induction of G2/M phase arrest. RNA-seq and ATAC-seq analyses indicate that MARK3 attenuates cell cycle progression and angiogenesis partly through downregulation of AP-1 and Hippo signaling target genes. The synthetic lethal therapy using metabolic stress inducers may be a promising therapeutic choice to treat the LKB1-MARK3 axis-dysregulated HGSOCs., (© 2022. The Author(s).)

RNA modifications have attracted increasing interest in recent years because they have been frequently implicated in various human diseases, including cancer, highlighting the importance of dynamic post‑transcriptional modifications. Methyltransferase‑like 6 (METTL6) is a member of the RNA methyltransferase family that has been identified in many cancers; however, little is known about its specific role or mechanism of action. In the present study, we aimed to study the expression levels and functional role of METTL6 in hepatocellular carcinoma (HCC), and further investigate the relevant pathways. To this end, we systematically conducted bioinformatics analysis of METTL6 in HCC using gene expression data and clinical information from a publicly available dataset. The mRNA expression levels of METTL6 were significantly upregulated in HCC tumor tissues compared to that in adjacent non‑tumor tissues and strongly associated with poorer survival outcomes in patients with HCC. CRISPR/Cas9‑mediated knockout of METTL6 in HCC cell lines remarkably inhibited colony formation, cell proliferation, cell migration, cell invasion and cell attachment ability. RNA sequencing analysis demonstrated that knockout of METTL6 significantly suppressed the expression of cell adhesion‑related genes. However, chromatin immunoprecipitation sequencing results revealed no significant differences in enhancer activities between cells, which suggests that METTL6 may regulate genes of interest post‑transcriptionally. In addition, it was demonstrated for the first time that METTL6 was localized in the cytosol as detected by immunofluorescence analysis, which indicates the plausible location of RNA modification mediated by METTL6. Our findings provide further insight into the function of RNA modifications in cancer and suggest a possible role of METTL6 as a therapeutic target in HCC.

Although chromatin immunoprecipitation and next-generation sequencing (ChIP-seq) using formalin-fixed paraffin-embedded tissue (FFPE) has been reported, it remained elusive whether they retained accurate transcription factor binding. Here, we developed a method to identify the binding sites of the insulator transcription factor CTCF and the genome-wide distribution of histone modifications involved in transcriptional activation. Importantly, we provide evidence that the ChIP-seq datasets obtained from FFPE samples are similar to or even better than the data for corresponding fresh-frozen samples, indicating that FFPE samples are compatible with ChIP-seq analysis. H3K27ac ChIP-seq analyses of 69 FFPE samples using a dual-arm robot revealed that driver mutations in EGFR were distinguishable from pan-negative cases and were relatively homogeneous as a group in lung adenocarcinomas. Thus, our results demonstrate that FFPE samples are an important source for epigenomic research, enabling the study of histone modifications, nuclear chromatin structure, and clinical data.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and is a leading cause of cancer-related death worldwide. Given that the standard-of-care for advanced liver cancer is limited, there is an urgent need to develop a novel molecular targeted therapy to improve therapeutic outcomes for HCC. In order to tackle this issue, we conducted functional analysis of the histone lysine-specific demethylase (LSD1) to explore the possibility that this enzyme acts as a therapeutic target in HCC. According to immunohistochemical analysis, 232 of 303 (77%) HCC cases showed positive staining of LSD1 protein, and its expression was correlated with several clinicopathological characteristics, such as female gender, AFP (alpha-fetoprotein) levels, and HCV (hepatitis C virus) infectious. The survival curves for HCC using the Kaplan-Meier method and the log-rank test indicate that positive LSD1 protein expression was significantly associated with decreased rates of overall survival (OS) and disease-free survival (DFS); the multivariate analysis indicates that LSD1 expression was an independent prognostic factor for both OS and DFS in patients with HCC. In addition, knockout of LSD1 using the CRISPR/Cas9 system showed a significantly lower number of colony formation units (CFUs) and growth rate in both SNU-423 and SNU-475 HCC cell lines compared to the corresponding control cells. Moreover, LSD1 knockout decreased cells in S phase of SNU-423 and SNU-475 cells with increased levels of H3K4me1/2 and H3K9me1/2. Finally, we identified the signaling pathways regulated by LSD1 in HCC, including the retinoic acid (RA) pathway. Our findings imply that deregulation of LSD1 can be involved in HCC; further studies may explore the usefulness of LSD1 as a therapeutic target of HCC., Competing Interests: The authors declare no conflicts of interest.

Introduction: We investigated the 2-year survival rate and incidence of spontaneous tumors in F344/DuCrlCrlj rats used in carcinogenicity studies of chemical substances. Records for animals used in the control groups of carcinogenicity studies which were conducted during the last 10 years were obtained from the database of the Japan Bioassay Research Center (JBRC). Six hundred ninety-nine males and 550 females were used in 14 and 11 inhalation studies, respectively, and 500 animals of each sex were used in 10 male and 10 female oral studies., Methods: In each study, SPF (specific pathogen free) animals were housed for 2 years (104 weeks) as control groups in the carcinogenicity studies. All animals underwent necropsy and histopathological examination. Each study was conducted in accordance with the Good Laboratory Practice., Results: The incidence of interstitial cell tumors was highest in both inhalation studies and oral studies (inhalation studies 86.1%, oral studies 68.6%). Tumors which had an incidence of 6% or higher were adenoma of the pituitary, C-cell adenoma of the thyroid, and mononuclear cell leukemia (LGL leukemia) of the spleen in male and female rats; fibroma of the subcutaneous tissue, adrenal pheochromocytoma, and islet cell adenoma of the pancreas in male rats; and endometrial stromal polyps and fibroadenoma of the mammary gland in female rats. Tumors other than the above had rare incidence rates. A clear difference in the incidence of spontaneous tumors was not observed between the inhalation and oral studies. The incidences of spontaneous tumors in control groups of previous oral studies are similar to our findings. There are no other reports of the spontaneous tumor incidence in the control groups of inhalation studies using F344/DuCrlCrlj rats. The 2-year survival rate was about 77% in both the inhalation and oral studies, and a gender difference was not observed. The F344/DuCrlCrlj rats used at JBRC had a higher 2-year survival rate than F344/N rats. This difference is possibly due to the low incidence of LGL leukemia in the F344/DuCrlCrlj rat., Conclusions: The incidences of spontaneous tumors in F344/DuCrlCrlj rats used in control groups of both inhalation and oral studies during the last 10 years at JBRC are similar to each other and similar to those reported in other studies. This is the first report on the incidence of spontaneous tumors in inhalation studies and contributes to the toxicological evaluation of studies using F344/DuCrlCrlj rats.

Accumulating evidence suggests that dysregulation of histone modification is involved in the pathogenesis and/or pathophysiology of psychiatric disorders. However, the abnormalities in histone modification in the animal model of schizophrenia and the efficacy of antipsychotics for such abnormalities remain unclear. Here, we investigated the involvement of histone modification in phencyclidine-induced behavioral abnormalities and the effects of antipsychotics on these abnormalities. After repeated phencyclidine (10 mg/kg) treatment for 14 consecutive days, mice were treated with antipsychotics (clozapine or haloperidol) or the histone deacetylase inhibitor sodium butyrate for 7 d. Repeated phencyclidine treatments induced memory impairment and social deficit in the mice. The acetylation of histone H3 at lysine 9 residues decreased in the prefrontal cortex with phencyclidine treatment, whereas the expression level of histone deacetylase 5 increased. In addition, the phosphorylation of Ca²⁺/calmodulin-dependent protein kinase II in the nucleus decreased in the prefrontal cortex of phencyclidine-treated mice. These behavioral and epigenetic changes in phencyclidine-treated mice were attenuated by clozapine and sodium butyrate but not by haloperidol. The dopamine D1 receptor antagonist SCH-23390 blocked the ameliorating effects of clozapine but not of sodium butyrate. Furthermore, clozapine and sodium butyrate attenuated the decrease in expression level of GABAergic system-related genes in the prefrontal cortex of phencyclidine-treated mice. These findings suggest that the antipsychotic effect of clozapine develops, at least in part, through epigenetic modification by activation of the dopamine D1 receptor in the prefrontal cortex.

Carcinogenicity of 1,1,1-trichloroethane (TCE) was examined by an inhalation exposure of F344 rats and BDF1 mice of both sexes to TCE at 0, 200, 800 or 3200 ppm for 6 h/d, 5 d/week for 104 weeks. In male rats, the incidences of bronchiolo-alveolar adenomas and peritoneal mesotheliomas were significantly increased in the 800 and 3200 ppm-exposed groups, respectively. The incidence of bronchiolo-alveolar adenomas in the 3200 ppm-exposed groups exceeded the range of historical control data in the Japan Bioassay Research Center. In female rats, the tumor incidences were not increased in any organs of the TCE-exposed groups. In male mice, a significant positive trend with dose was shown for incidences of bronchiolo-alveolar carcinomas, combined incidences of bronchiolo-alveolar adenomas/carcinomas and hepatocellular adenomas. The incidence of Harderian gland adenomas was significantly increased in the 3200 ppm-exposed group, and malignant lymphomas of spleen at this highest dose exceeded the range of historical control data. In female mice, the combined incidence of bronchiolo-alveolar adenomas/carcinomas was significantly increased in the 3200 ppm-exposed group, and the incidences of hepatocellular adenomas and combined incidences of hepatocellular adenomas/carcinomas were significantly increased in the 200, 800 and 3200 ppm-exposed groups with dose dependence except the combined incidence of hepatocellular adenomas/carcinomas in the 200 ppm-exposed group. The incidences of bronchiolo-alveolar adenomas in the 3200 ppm-exposed group and combined incidences of hepatocellular adenomas/carcinomas in the 200 ppm-exposed groups exceeded the ranges of historical control data. Thus, this study provided clear evidence of inhalation carcinogenicity for TCE in both rats and mice.

A number of histone demethylases have been identified and biochemically characterized, yet their biological functions largely remain uncharacterized, particularly in the context of human diseases such as cancer. In this study, we describe important roles for the histone demethylase KDM3A, also known as JMJD1A, in human carcinogenesis. Expression levels of KDM3A were significantly elevated in human bladder carcinomas compared with nonneoplastic bladder tissues (p < 0.0001), when assessed by real-time PCR. We confirmed that some other cancers including lung cancer also overexpressed KDM3A, using cDNA microarray analysis. Treatment of cancer cell lines with small interfering RNA targeting KDM3A significantly knocked down its expression and resulted in the suppression of proliferation. Importantly, we found that KDM3A activates transcription of the HOXA1 gene through demethylating histone H3 at lysine 9 di-methylation by binding to its promoter region. Indeed, expression levels of KDM3A and HOXA1 in several types of cancer cell lines and bladder cancer samples were statistically correlated. We observed the down-regulation of HOXA1 as well as CCND1 after treatment with KDM3A siRNA, indicating G(1) arrest of cancer cells. Together, our results suggest that elevated expression of KDM3A plays a critical role in the growth of cancer cells, and further studies may reveal a cancer therapeutic potential in KDM3A inhibition., (Copyright © 2011 UICC.)

N-methyl-D-aspartate (NMDA) receptor is a glutamate receptor which has an important role on mammalian brain development. We have reported that prenatal treatment with phencyclidine (PCP), a NMDA receptor antagonist, induces long-lasting behavioral deficits and neurochemical changes. However, the mechanism by which the prenatal antagonism of NMDA receptor affects neurodevelopment, resulting in behavioral deficits, has remained unclear. Here, we report that prenatal NMDA receptor antagonism impaired the proliferation of neuronal progenitors, leading to a decrease in the progenitor pool in the ventricular and the subventricular zone. Furthermore, using a PCR array focused on neurogenesis and neuronal stem cells, we evaluated changes in gene expression causing the impairment of neuronal progenitor proliferation and found aberrant gene expression, such as Notch2 and Ntn1, in prenatal PCP-treated mice. Consequently, the density of glutamatergic neurons in the prefrontal cortex was decreased, probably resulting in glutamatergic hypofunction. Prenatal PCP-treated mice displayed behavioral deficits in cognitive memory and sensorimotor gating until adulthood. These findings suggest that NMDA receptors regulate the proliferation and maturation of progenitor cells for glutamatergic neuron during neurodevelopment, probably via the regulation of gene expression.

Although heat-shock protein 70 (HSP70), an evolutionarily highly conserved molecular chaperone, is known to be post-translationally modified in various ways such as phosphorylation, ubiquitination and glycosylation, physiological significance of lysine methylation has never been elucidated. Here we identify dimethylation of HSP70 at Lys-561 by SETD1A. Enhanced HSP70 methylation was detected in various types of human cancer by immunohistochemical analysis, although the methylation was barely detectable in corresponding non-neoplastic tissues. Interestingly, methylated HSP70 predominantly localizes to the nucleus of cancer cells, whereas most of the HSP70 protein locates to the cytoplasm. Nuclear HSP70 directly interacts with Aurora kinase B (AURKB) in a methylation-dependent manner and promotes AURKB activity in vitro and in vivo. We also find that methylated HSP70 has a growth-promoting effect in cancer cells. Our findings demonstrate a crucial role of HSP70 methylation in human carcinogenesis.

Objectives: Carcinogenicity and chronic toxicity of indium-tin oxide (ITO) were examined by inhalation exposure of rats and mice to ITO aerosol., Methods: Fifty mice of both sexes were exposed to ITO at 0 (control), 0.01, 0.03 or 0.1 mg/m(3) for 6 h/day, 5 day/wk for 104 wk, and 50 rats of both sexes were exposed to 0, 0.01 or 0.03 mg/m(3) ITO for the same time period. The repeated exposure of 50 rats of both sexes to 0.1 mg/m(3) ITO was discontinued at the 26th wk, followed by clean air exposure for the remaining 78 wk., Results: In rats, incidences of bronchiolo-alveolar adenomas and carcinomas, bronchiolo-alveolar hyperplasia, alveolar wall fibrosis and thickened pleural wall, alveolar proteinosis and infiltrations of alveolar macrophages and inflammatory cells were significantly increased. Combined incidences of malignant lung tumors in male rats and total lung tumors in male and female rats were significantly increased at exposure to 0.01 mg/m(3) ITO. In mice, no carcinogenic response occurred, but thickened pleural wall, alveolar proteinosis and alveolar macrophage infiltration were induced. Mice were less susceptible to ITO than rats. The lung content of indium was the greatest, followed by the spleen, kidney and liver. Blood indium levels increased dose-dependently., Conclusions: There was clear evidence of carcinogenicity of inhaled ITO in male and female rats but not clear evidence in mice, together with occurrence of the chronic pulmonary lesions in both rats and mice.

Objectives: Two- and 13-week inhalation toxicities of indium-tin oxide (ITO) and indium oxide (IO) were characterized for risk assessments of workers exposed to ITO., Methods: F344 rats of both sexes were exposed by inhalation to ITO or IO aerosol for 6 h/day, 5 day/wk for 2 wk at 0, 0.1, 1, 10 or 100 mg/m(3) or 13 wk at 0, 0.1 or 1 mg/m(3). An aerosol generator and inhalation exposure system was constructed., Results: Blood and lung contents of indium were elevated in a dose-related manner in the ITO- and IO-exposed rats. ITO and IO particles were deposited in the lung, mediastinal lymph node and nasal-associated lymphoid tissue. Exposures to ITO and IO induced alveolar proteinosis, infiltrations of alveolar macrophages and inflammatory cells and alveolar epithelial hyperplasia in addition to increased lung weight. ITO affected the lung more severely than IO did. Fibrosis of alveolar wall developed and some of these lesions worsened at the end of the 26-week post-exposure period., Conclusions: Persistent pulmonary lesions including alveolar proteinosis and macrophage infiltration occurred after 2- and 13-week inhalation exposures of rats to ITO and IO. Fibrosis of alveolar wall developed later. These lesions occurred after ITO exposure at the same concentration as the current occupational exposure limit in the USA and at blood indium levels below the biological exposure index in Japan for indium.

High-resolution scanning electron microscopy (HR-SEM) with backscattered electrons (BSE) under a low accelerating voltage provided images with compositional information about Pt nano-particles with a diameter of approximately 6 nm that were dispersed in mesoporous silica (SBA-15). The dispersion of Pt nano-particles in mesoporous silica was investigated using the BSE imaging method in combination with the broad ion beam (BIB) cross-section fabrication technique.

A 72-year-old woman presented with a metastatic brain tumor around the pituitary stalk compressing the chiasm anteriorly and upward. After tumor resection by a left pterional approach failed, a three-dimensional (3D) image of the area of interest was reconstructed using the Vecter Vision navigation system and iPlan Cranial planning software version 2.5. Preoperative 3D computed tomography and magnetic resonance imaging data were fused to demonstrate the anatomical relationship between vessels, nerves, and tumor. Clearer demonstration of the optic nerves utilized thin slice axial and sagittal views along the nerves. The 3D reconstructed image demonstrated the spatial relationship of the tumor and surrounding tissue, and suggested the necessity of a right orbitozygomatic approach to create adequate working space for tumor resection. Second surgery according to this preoperative planning was successful. High quality multimodal fusion images in a navigation system has distinct advantages in preoperative assessment of essential structural relationships allowing adequate exposure of certain lesions and surrounding structures in individual patients by defining specific surgical approaches.

Much concern has been raised over the health consequences of workers exposed to carbon nanotubes. In order to characterize multi-wall carbon nanotubes (MWCNT) suspended in a phosphate-buffered saline containing 0.1% Tween 80 for an intratracheal instillation study. Length and width distributions of the MWCNT fibers, dispersion of MWCNT in the suspension and in the lung tissue and the MWCNT contents of metal impurities were investigated. Arithmetic mean length and width of the MWCNT fibers as measured on scanning electron microscope (SEM) photographs were 5.0 microm and 88 nm, respectively, and fibers longer than 5.0 microm were 38.9% of all fibers measured. Dynamic light scattering size measurement revealed that 5-min ultrasonication, together with addition of Tween 80 into the suspension, decreased the hydrodynamic diameters of the agglomerated MWCNT to those of finer particles below 1.0 microm. SEM observation showed good dispersion of MWCNT in the suspension, and in the alveoli on Day 1 after instillation. Concentration of iron, chromium and nickel in the MWCNT were 4,400, 48 and 17 ppm (wt/wt), respectively, all of which were below levels that would elicit positive pulmonary toxic responses to these metals. The results suggest that well-dispersed, long and thin MWCNT fibers exhibit asbestos-like pathogenicity in the lung.

Carcinogenicity and chronic toxicity of 1,4-dioxane were examined by inhalation exposure of 50 male F344 rats to 1,4-dioxane vapor at 0 (clean air), 50, 250, or 1250 ppm (v/v) for 6 h/day, 5 days/wk, and 104 wk. Survival rates of 250 and 1250 ppm-exposed groups were decreased near the end of the 2-yr exposure period, due probably to the occurrence of malignant tumors. A statistically significant but marginal decrement of terminal body weight (<10%) was found in the 1250 ppm-exposed group, suggesting slight systemic toxicity. Significant changes in plasma levels of AST, ALT, ALP, and gamma-GTP and relative weight of the liver occurred in the 1250 ppm-exposed group. Dose-dependent and statistically significant increases in incidences of nasal squamous cell carcinomas, hepatocellular adenomas, and peritoneal mesotheliomas were found primarily in the 1250 ppm-exposed group. The incidences of renal cell carcinomas, fibroadenomas in the mammary gland, and adenomas in the Zymbal gland were also increased dose-dependently. Preneoplastic lesions occurred in the nasal cavity and liver of the 1,4-dioxane-exposed groups. As nonneoplastic lesions, the significantly increased incidences of nuclear enlargement, atrophy, and respiratory metaplasia in the nasal cavity were noted at 50 ppm and above. A LOAEL (lowest observed adverse effect level) was determined at 50 ppm for the nasal endpoint of general chronic toxicity. This study provides clear evidence of carcinogenicity for 1,4-dioxane in male rats. A cytotoxic-proliferative and in vivo genotoxic mode of action is suggested to operate in 1,4-dioxane-induced carcinogenesis.

Lynch syndrome (hereditary non-polyposis colorectal cancer) is an inherited disease caused by germ-line mutation in mismatch repair genes such as MLH1, MSH2, and MSH6. The mutations include missense and nonsense mutations, small insertions and deletions, and gross genetic alterations including large deletions and duplications. In addition to these genetic changes, mutations in introns are also involved in the pathogenesis. However, it is sometimes difficult to interpret correctly the pathogenicity of variants in exons as well as introns. To evaluate the effect of splice-site mutations in two Lynch syndrome patients, we carried out a functional splicing assay using minigenes. Consequently, this assay showed that the mutation of c.1731+5G>A in MLH1 led to exon15 skipping, and that the mutation of c.211+1G>C in MSH2 created an activated cryptic splice-site 17-nucleotides upstream in exon1. These aberrant splicing patterns were not observed when wild type sequence was used for the assay. We also obtained concordant results by RT-PCR experiments with transcripts from the patients. Furthermore, additional functional splicing assays using two different intronic mutations described in earlier studies revealed splicing alterations that were in complete agreement with the reports. Therefore, functional splicing assay is helpful for evaluating the effects of genetic variants on splicing.

A lamellar material composed of alternating layers of magnesium phosphate sheets and n-hexadecylammonium (n-C16H33NH3+) ions was synthesized by the reaction between magnesium acetate tetrahydrate and phosphoric acid. The chemical formula of the composite material was determined to be (n-C16H33NH3+)Mg2+(HPO4(2-))(H2PO4-) by ICP, TG, and 31P MAS NMR measurements. The lamellar composite was obtained under basic conditions adjusted with tetramethylammonium (TMAOH) and tetraethylammonium hydroxides (TEAOH), while ammonium ions did not act as an alkali source useful for obtaining the composite material. The formation of crystalline byproducts such as hydrated newberyite (MgHPO4.3H2O) and ammonium-type struvite (MgNH4PO4. 6H2O) was suppressed by using the EtOH/H2O mixed solution and by adding TMAOH and TEAOH to adjust the synthetic condition, respectively. Including the data on calcium, barium and strontium phosphates, we can totally understand the formation of the mesostructured materials composed of ionic frameworks in the presence of n-alkylamines in detail. It is quite important to suppress the formation of crystalline alkaline earth metal phosphates and control the solubilities of reactants and products for synthesizing pure mesostructured materials with ionically crosslinked frameworks.

Mesostructured calcium phosphates constructed by ionic frameworks were synthesized using carboxylic acid- and amine-type surfactants in mixed solvent systems of ethanol and water. A lamellar mesostructured calcium phosphate was prepared using palmitic acid as an anionic surfactant, as in the case using n-alkylamines. A wormhole-like mesostructured calcium phosphate can be obtained using dicarboxyl N-lauroyl- l-glutamic acid, whose headgroup is larger than that of palmitic acid. Similar mesostructured product was obtained using 4-dodecyldiethylenetriamine with a large headgroup containing two primary amine groups. Interactions of carboxyl and primary amino groups in the surfactant molecules with inorganic species are quite important for the formation of mesostructured calcium phosphates. The Ca/P molar ratio of mesostructured calcium phosphates was strongly affected by the molecular structure of surfactants containing carboxyl and primary amino groups. Ca-rich materials can be obtained using carboxylic acid-type surfactants (Ca/P approximately 1.7) rather than amine-type surfactants (Ca/P approximately 1.0).

Carcinogenicity and chronic toxicity of carbon tetrachloride were examined by inhalation exposure of 50 F344 rats and 50 BDF1 mice of both sexes to carbon tetrachloride at 0 (clean air), 5, 25, or 125 ppm (v/v) for 6 h/day, 5 days/wk, for 104 wk. Incidences of hepatocellular adenomas and carcinomas in rats and mice of both sexes and of adrenal pheochromocytomas in mice of both sexes were significantly increased dose-dependently. Hepatocellular carcinomas and cirrhosis significantly occurred in the 125-ppm-exposed rats of both sexes, and 3 cases of hepatocellular carcinomas and increased incidences of hepatic altered cell foci were noted in the 25-ppm-exposed female rats. Hepatocellular carcinomas were induced in mice of both sexes at 25 and 125 ppm, and hepatocellular adenomas occurred in females at 5 ppm without any degenerative or necrotic change in hepatocytes. Hepatocellular carcinomas metastasized to the lung. The chronic hepatotoxicity was characterized by cirrhosis, fibrosis, and fatty change in rats, and ceroid deposition, bile-duct proliferation, and hydropic change in mice. Survival rates were decreased in the 125-ppm-exposed rats and mice of both sexes and in the 25-ppm-exposed female mice, in association with decreased body weights. The decreased survival rates were considered to be causally related to both various tumors including hepatocellular carcinomas and severe chronic progressive nephropathy in rats and to hepatocellular carcinomas in mice. This study provided clear evidence of carcinogenicity for carbon tetrachloride in rats and mice. A cytotoxic-proliferative and genotoxic mode of action for carbon tetrachloride-induced hepatocarcinogenesis was suggested.

Subchronic toxicity of carbon tetrachloride (CCl4) was examined by inhalation exposure of F344 rats and BDF1 mice of both sexes to 0, 10, 30, 90, 270 or 810 ppm (v/v) CCl4 vapor for 13 wk (6 h/d and 5 d/wk). In the high exposure levels at 270 and 810 ppm, altered cell foci in the livers of both rats and mice, and fibrosis and cirrhosis in the rat liver were observed. Hematoxylin and eosin-stained altered cell foci of rats were recognized as glutathione-S-transferase placental form (GST-P) positive foci, which are preneoplastic lesions of hepatocarcinogenesis. The most sensitive endpoint of CCl4-induced toxicity was fatty change with large droplets in rats of both sexes and male mice, and cytoplasmic globules in male mice, as well as increased relative liver weight in male rats. Those endpoints were manifested at 10 ppm and the LOAEL was determined as 10 ppm for the hepatic endpoints in rats and mice. Enhanced cytolytic release of liver transaminases into plasma in rats and mice and its close association with hepatic collapse in mice were observed at medium and high levels of inhalation exposure. Both CCl4-induced hematotoxicity and nephrotoxicity were observed in both rats and mice, but those toxicities were manifested at higher exposure concentrations than hepatotoxicity. The LOAEL for the hepatic endpoint and the GST-P-stained altered cell foci provide relevant animal data for reconsidering the occupational exposure limit val1ue of 5 ppm for CCl4 and strengthen the evidence of CCl4-induced hepatocarcinogenicity which is used in its carcinogenicity classification.

A 64 amino acid residue sialylated phosphorylated glycomacropeptide (GMP) from bovine sweet whey can be detected as a Coomassie blue-staining peptide by electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels. There is, however, limited information available concerning detection of GMP as a sialylated phosphorylated compound. Samples of GMP were electrophoresed on SDS-polyacrylamide gels or cellulose acetate strips (CAS). Immediately following electrophoresis, fractions obtained by cutting gels or strips were subjected to sialic acid determination by the thiobarbituric acid reaction and phosphorus determination by the malachite green dye reaction. Both determinations were found to be sensitive enough to detect approximately 20 and 40 microg of GMP in CAS and SDS gels, respectively. Further studies demonstrated that sialylated phosphorylated GMP can be detected on either SDS gels or CAS loaded with whey products or whey-added margarine residues.

Background: The primary malignant dural lymphoma of B-cell type is rare. Our review of the literature (24 cases) indicated that patients with this tumor had female predominance, immunocompetency, and longer survival compared with those with primary malignant intracerebral lymphomas. Based on its clinicopathological features, this dural lymphoma may be classified differently from other types of malignant lymphomas in the central nervous system., Case Description: The authors report an example of a patient who had a favorable course of malignant dural lymphoma. A 59-year-old woman presented with primary malignant dural lymphoma in the frontal area. Histological diagnosis was a diffuse large B-cell-type lymphoma of intermediate malignancy, but MIB-1 index was extremely high. Subtotal tumor removal was followed by 4 courses of chemotherapy. Thirty months after surgery, the patient is asymptomatic with no MRI evidence of recurrence. A combination of subtotal resection and chemotherapy for a primary malignant dural lymphoma is an effective means to attempt cure of this tumor., Conclusion: These rare tumors may be managed successfully by subtotal resection and postoperative treatment. We believe that chemotherapy is the treatment of choice after subtotal resection of malignant dural lymphomas, as experienced in our case.

We derived microglia from mouse embryonic stem cells (ES cells) at very high density. Using the markers Mac1(+)/CD45(low) and Mac1(+)/CD45(high) to define microglia and macrophages, respectively, we show that Mac1(+) cells are induced by GM-CSF stimulation following neuronal differentiation of mouse ES cells using a five-step method. CD45(low) expression was high and CD45(high) expression was low on induced cells. We used a density gradient method to obtain a large amount of microglia-like cells, approximately 90% of Mac1(+) cells. Microglia-like cells expressed MHC class I, class II, CD40, CD80, CD86, and IFN-gammaR. The expression level of these molecules on microglia-like cells was barely enhanced by IFN-gamma. Intravenously transferred GFP(+) microglia derived from GFP(+) ES cells selectively accumulated in brain but not in peripheral tissues such as spleen and lymph node. GFP(+) cells were detected mainly in corpus callosum and hippocampus but were rarely seen in cerebral cortex, where Iba1, another marker of microglia, is primarily expressed. Furthermore, both GFP(+) and Iba1(+) cells exhibited a ramified morphology characteristic of mature microglia. These studies suggest that ES cell-derived microglia-like cells obtained using our protocol are functional and migrate selectively into the brain but not into peripheral tissues after intravenous transplantation.

This study was undertaken to develop a method to isolate glycomacropeptide (GMP), a bioactive compound, from sweet whey by using chitosan resins as anion exchangers. Shrimp shells were used to prepare two chitosan (polyglucosamine) resins, one with the primary amine (-NH(2)) (resin A) and the other with the secondary amine (-NH-) (resin B) as the major functional group. These resins were tested as adsorbents for the isolation of GMP from sweet whey, and the results obtained were compared with those obtained with commercial anion exchangers. The most important finding in this experiment was that the GMP binding capacity of resin A was much higher than that of resin B. Resin A may be the anion exchanger to be tested for industrial scale production of GMP. Amino acid analysis of the GMP-depleted whey fraction suggests that this product can replace sweet whey as an ingredient in various food products including infant formulas, bakery products, and beverages.

This case report describes an 11-year-old boy with primitive neuroectodermal tumor (PNET), which remains the pediatric brain tumor with the worst prognosis despite combination treatment with surgery, irradiation and anti-cancer drugs. The boy was successfully treated with intratumoral administration of interferon-beta (IFN-beta) following surgical resection and irradiation. Two million units of IFN-beta were locally administered into the post-operative cavity through the Ommaya's reservoir every two weeks after discharge. He was managed as an outpatient without serious side-effects to IFN. The patient has shown no tumor recurrence, mental retardation, or neuroendocrine impairment for over three years and has lived a normal school life with a full Karnofsky Performance Status score. The local administration of IFN-beta may be warranted for maintenance therapy in children with PNET.

Glycomacropeptide (GMP) found in sweet whey is a biologically active compound released from kappa-casein by the action of chymosin during cheese making. This study was undertaken to purify GMP from sweet whey as a research chemical on a laboratory scale. Glycomacropeptide was isolated from proteins and other non-GMP compounds by deproteinization with trichloroacetic acid and gel chromatography on Sephacryl S-200. The purified GMP accounted for 0.12% of dry sweet whey powder and contained 107.0, 50.9, 61.2 and 4.3 microg, respectively, of sialic acid, galactose, galactosamine and phosphorus per mg dry weight. The GMP was of high purity, with its amino acid composition showing undetectable levels of phenylalanine, tyrosine and arginine, the amino acids that do not occur in bovine GMP. On gel electrophoresis, the GMP showed a single broad band with an average mobility faster than that of carbonic anhydrase (molecular weight = 31 kDa). The purified GMP may be used as a standard glycopeptide in chromatography and electrophoresis and may also be used to test various known or unknown properties and biological activities of this compound.

Few reports exist comparing virological studies on hepatitis viruses with histopathological studies of autopsy cases other than those of liver clinics. Relations between hepatitis virus-related markers and hepatic histopathology were studied in 1044 autopsy cases (779 men and 265 women) at the Medical Examiner's Office, Tokyo. Heart blood was obtained at the autopsy, and the sera were submitted for virus-marker detection of HBV, HCV, and HGV/GBV-C. Hematoxylin and eosin-stained paraffin sections were used for histological assessment. Histopathologically, 463 cases were determined as so-called normal liver; among them 440 cases (95.0%) were negative for all hepatitis virus-related markers, but HBV-DNA was positive in 13 cases, three cases were positive for HCV-RNA (indicating a healthy carrier rate of HCV-RNA of 4.1%), and seven cases were positive for HGV/GBV-C RNA. The incidence of these three virus-related markers was low in cases with fatty liver and micronodular cirrhosis, but in cases with chronic hepatitis, macronodular cirrhosis and hepatocellular carcinoma, the incidence of HBV-DNA and HCV-RNA increased with advancing disease. A positive rate of anti-HBs or anti-HBc (HBV-Ab) or both was found between 30 and 50% in all histopathological groups, and no noticeable relations between the positive rate and microscopical changes were detected. The presence of HGV/GBV-C RNA seemed to be unrelated to hepatic inflammation or generalized inflammatory changes or both occurring together. The decadal age incidence of the virus-related markers and their incidence in various hepatic diseases are also reported.

The objectives of this study were to analyze glycosaminoglycan (GAG) and mineral composition in the chicken eggshell. Eggshells were decalcified with acetic acid, and GAG was extracted from the decalcified shell by digestion with papain. The eggshell contained an average of 0.024% of its dry weight as uronic acid, a carbohydrate moiety of GAG. The eggshell GAG consisted of approximately 48% hyaluronic acid and and 52% galactosaminoglycan. In the latter, chondroitin sulfate-dermatan sulfate copolymers were the major galactosaminoglycans with dermatan sulfate disaccharide as a relatively minor component. The inorganic material recovered after decalcification accounted for approximately 140% of dry weight of the eggshell and contained 24.11% calcium, 0.04% phosphorous, and 0.23% magnesium, with an undetectable amount of nitrogen.

Cases of 445 adult Japanese autopsies of the Tokyo Metropolitan Medical Examiner's Office were used in this study. They were either negative for all hepatitis virus-related markers examined or had little or no histopathological hepatic changes. The maximum liver weight was observed in the fifth decade in both sexes, and after the fifth decade the liver weight decreased markedly with increasing age. The sexual difference in the liver weight was most predominant in the third to fifth decades, but the sexual difference was not marked in the older age groups. The highest liver weight to body weight ratio was observed in the fifth decade of both sexes, and a total decadal pattern of the ratio was similar to a parabola. An interesting finding was that the male liver weights in the third to fifth decades considerably increased in recent years, but the female liver weights in the third decade were almost the same despite the difference in investigation period. We suggest the data of this study may be a standard for Japanese people.