Impaired DNA damage repair as a common feature of neurodegenerative diseases and psychiatric disorders.

Impaired DNA damage repair is a common pathological endophenotype of some types of neurodegenerative diseases, intellectual disabilities, and psychiatric diseases. Dysfunctional DNA repair and DNA damage, including DNA double-stranded breaks, are linked to transcriptional dysfunction and abnormal DNA methylation. Impaired DNA repair in neural stem cells leads to microcephaly or cerebellar ataxia. Furthermore, DNA repair defects and DNA damage in mature neurons lead to progressive cognitive impairment, which might be a common feature of Alzheimer's disease, Huntington's disease, and other polyglutamine diseases. Oxidative DNA damage and altered DNA repair gene expression are observed in GABAergic neurons in schizophrenia. These findings indicate that impaired DNA repair is a common pathological endophenotype of neurological diseases, and that DNA damage might lead to diverse disease symptoms dependent on timing and the affected cell type.

In utero gene therapy rescues microcephaly caused by Pqbp1-hypofunction in neural stem progenitor cells.

Human mutations in PQBP1, a molecule involved in transcription and splicing, result in a reduced but architecturally normal brain. Examination of a conditional Pqbp1-knockout (cKO) mouse with microcephaly failed to reveal either abnormal centrosomes or mitotic spindles, increased neurogenesis from the neural stem progenitor cell (NSPC) pool or increased cell death in vivo. Instead, we observed an increase in the length of the cell cycle, particularly for the M phase in NSPCs. Corresponding to the developmental expression of Pqbp1, the stem cell pool in vivo was decreased at E10 and remained at a low level during neurogenesis (E15) in Pqbp1-cKO mice. The expression profiles of NSPCs derived from the cKO mouse revealed significant changes in gene groups that control the M phase, including anaphase-promoting complex genes, via aberrant transcription and RNA splicing. Exogenous Apc4, a hub protein in the network of affected genes, recovered the cell cycle, proliferation, and cell phenotypes of NSPCs caused by Pqbp1-cKO. These data reveal a mechanism of brain size control based on the simple reduction of the NSPC pool by cell cycle time elongation. Finally, we demonstrated that in utero gene therapy for Pqbp1-cKO mice by intraperitoneal injection of the PQBP1-AAV vector at E10 successfully rescued microcephaly with preserved cortical structures and improved behavioral abnormalities in Pqbp1-cKO mice, opening a new strategy for treating this intractable developmental disorder.

Is the prognostic impact of tumor location in patients with surgically resected esophageal squamous cell carcinoma affected by surgical approach?

AIM: The aim of the present study was to clarify differences in node metastasis mode and clinical outcomes based on tumor location in patients with esophageal squamous cell carcinoma (ESCC). PATIENTS AND METHODS: Participants comprised 228 patients with ESCC who underwent radical esophagectomy without preoperative supplement therapies. Lymph nodes were harvested from three fields: the neck, thorax, and abdomen. Patients were divided into three groups depending on tumor location [upper esophagus (UE), middle esophagus, or lower esophagus (LE)] and analyzed clinicopathologically. RESULTS: The LE group showed significantly more progressive ESCC in terms of tumor invasion (p = 0.025), node metastasis (p = 0.0071), and TNM stage (p = 0.0043). The LE group revealed a tendency to metastasize to extrathoracic (especially abdominal) nodes (p = 0.0008). Recurrent laryngeal node metastasis was increased in the UE group (p = 0.016). However, no prognostic differences were detected between groups according to tumor location. Likewise, subgroup analyses by surgical approach (open thoracotomy vs. thoracoscopy) and cancer stage (stage I/II, III, and IV) did not reveal any significant prognostic impact of tumor location. CONCLUSION: Lymphatic spread varied by tumor location, but no prognostic impact of tumor location could be detected in patients with ESCC in spite of surgical approach or cancer stage.

AURKA is one of the downstream targets of MAPK1/ERK2 in pancreatic cancer.

DUSP6/MKP-3, a specific inhibitor of MAPK1/ERK2, frequently loses its expression in primary pancreatic cancer tissues. This evidence suggests that constitutive activation of MAPK1 synergistically induced by frequent mutation of KRAS2 and the loss of function of DUSP6 plays key roles in pancreatic carcinogenesis and progression. By profiling of gene expressions associated with downregulation of MAPK1 induced by exogenous overexpression of DUSP6 in pancreatic cancer cells, we found that AURKA/STK15, the gene encoding Aurora-A kinase, which plays key roles in cellular mitosis, was among the downregulated genes along with its related genes, which included AURKB, TPX2 and CENPA. An association of expression and promoter activity of AURKA with MAPK activity was verified. Knockdown of ETS2 resulted in a reduction of AURKA expression. These results indicate that AURKA is a direct target of the MAPK pathway and that its overexpression in pancreatic cancer is induced by hyperactivation of the pathway, at least via ETS2.

Application of synchrotron X-ray imaging to phase objects in orthopedics.

Novel imaging of the fine structures of the ribs of a pig and a specimen of human osteosarcoma utilizing the spatial coherence of X-rays was successfully performed with an incident X-ray energy of 30 keV at SPring-8, Japan. The image contrast appearing at the periphery of trabecular bone, small calcifications and small fractures is caused by the phase shift of the X-rays at the boundary of these objects that have different X-ray refractive indices. The image is recorded on mammography film without an intensifying screen. Comparison of the image contrast using different sample-to-film distances, Z, such as Z = 5 m and Z approximately 0 m, showed that the former images were always more informative, i.e. better in resolution and/or image contrast when imaging trabecular bone, bone marrow and small fractures in compact bone, and for imaging cartilage. Radiography using synchrotron X-rays for phase objects should be a powerful tool for diagnosis in orthopedics, especially for bone disease.

X-ray absorption study of molten yttrium trihalides.

The local structure and structural changes in molten YCl3-LiCl-KCl and molten YBr3-LiBr systems have been investigated by using a high-temperature extended X-ray absorption fine structure (EXAFS) technique. The behaviour of octahedral coordination of the halide ion (Cl(-) and Br(-)) around the Y(3+) ion has been studied by EXAFS of the Y K-absorption edge. The nearest Y(3+)-Cl(-) and Y(3+)-Br(-) distances and coordination numbers of halide ions around the Y(3+) ion do not change by mixing with the alkali halides. The stabilization of the (YCl6)(3-) and (YBr6)(3-) octahedral coordination by adding alkali halides was suggested by decreasing the Debye-Waller factor and the anharmonicity in the nearest Y(3+)-Cl(-) and Y(3+)-Br(-) interactions. The bridging structure of the (YBr6)(3-) octahedra sharing a Br(-) ion in the molten YBr3-LiBr system was studied by EXAFS of the Br K-absorption edge. The coordination number of Y(3+) around the Br(-) ion decreases from 2 in the pure melt to almost 1 in the 30mol% and 15mol% YBr3 melts. This suggests that the bridging is almost broken and the stable octahedron exists freely in the LiBr-rich melts.

Deletion mapping of 14q32 in human neuroblastoma defines an 1,100-kb region of common allelic loss.

BACKGROUND: We analyzed loss of heterozygosity (LOH) in 54 primary neuroblastomas (NBs) using 12 microsatellite markers on 14q32, and found that 31% (17/54) NBs showed LOH. PROCEDURE: The smallest region of overlap (SRO) was identified between D14S62 and D14S987. RESULTS: There was no statistical correlation between LOH and any clinicopathologic features, including age, stage, amplification of MYCN, and ploidy. A sequence-ready bacterial artificial chromosome (BAC) contig was constructed, and the minimum tiling path of six BACs covered the SRO; the physical length of this region was no larger than 1,000 kb. CONCLUSIONS: Our findings support the existence of a putative tumor-related gene on 14q32 for the tumorigenesis of NB.

Roles of endogenous retroviruses and platelets in the development of vascular injury in spontaneous mouse models of autoimmune diseases.

MRL/MpJ-lpr/lpr (MRL/lpr) mice spontaneously develop immune complex-mediated glomerulonephritis, granulomatous arteritis, and thrombocytopenia. Recent genetic analyses in a few different strains of lupus-prone mice have pointed out a close correlation between autoantibodies reactive with the endogenous retroviral env gene product, gp70, and the development and severity of glomerulonephritis. We have also shown that autoantibodies reactive with endogenous retroviral gp70 are closely correlated with the development of necrotizing polyarteritis in another lupus-prone strain of mice, SL/Ni. However, suggested pathogenicity of anti-gp70 autoantibodies has not yet been directly tested. To examine if anti-gp70 autoantibodies induce glomerular and vascular pathology, we established from unmanipulated MRL/lpr mice hybridoma clones that secrete monoclonal antibodies reactive with endogenous xenotropic viral env gene products. As reported separately, a high proportion of these anti-gp70 antibody-producing hybridoma clones induced in syngeneic non-autoimmune and severe combined immunodeficiency mice proliferative or wire loop-like glomerular lesions with granular deposits of gp70, IgG, and C3 in affected glomeruli. Some mice transplanted with these anti-gp70 autoantibody-producing hybridoma cells also showed massive subendothelial deposition of electron-dense materials in small arterioles in the kidneys. Furthermore, we identified an IgG2a-producing anti-gp70 hybridoma clone that induced microvascular intraluminal platelet aggregation, thrombocytopenia, and amenia upon transplantation into syngeneic non-autoimmune mice. This anti-gp70 autoantibody bound onto the surfaces of mouse platelets, and specifically precipitated a platelet protein with an approximate relative molecular mass of 40000. Attachment of activated platelets to the intimal surfaces of small arteries was also observed by electron microscopy in mice transplanted with the pathogenic anti-gp70 IgG2a-producing hybridoma cells, suggesting an interaction between antibody-bound platelets and endothelial cells.

Detailed deletion mapping of chromosome band 14q32 in human neuroblastoma defines a 1.1-Mb region of common allelic loss.

Neuroblastoma (NB) is a well-known malignant disease in infants, but its molecular mechanisms have not yet been fully elucidated. To investigate the genetic contribution of abnormalities on the long arm of chromosome 14 (14q) in NB, we analysed loss of heterozygosity (LOH) in 54 primary NB samples using 12 microsatellite markers on 14q32. Seventeen (31%) of 54 tumours showed LOH at one or more of the markers analysed, and the smallest common region of allelic loss was identified between D14S62 and D14S987. This region was estimated to be 1-cM long from the linkage map. Fluorescence in situ hybridization also confirmed the loss. There was no statistical correlation between LOH and any clinicopathologic features, including age, stage, amplification of MYCN and ploidy. We further constructed a contig spanning the lost region using bacterial artificial chromosome and estimated this region to be approximately 1.1-Mb by pulsed-field gel electrophoresis. Our results will contribute to cloning and characterizing the putative tumour-associated gene(s) in 14q32 in NB.

Experimental lupus nephritis in severe combined immunodeficient (SCID) mice: remodelling of the glomerular lesions by bystander IgM antibodies.

MRL/Mp-lpr/lpr (MRL/lpr) mice develop glomerular lesions with regular variations in their histopathological manifestations, similar to those in lupus nephritis. These lesions are mainly either cell-proliferative or wire loop-like and are associated with glomerular deposits of immunoglobulins, most frequently IgG and IgM. We previously established a nephritogenic IgG3-producing hybridoma clone, B1, from an MRL/lpr mouse, which induces only a 'wire loop-like' type of glomerular lesion when injected into SCID mice. Injection of SCID mice with an anti-trinitrophenyl IgM antibody-producing hybridoma clone, Sp6, following injection of the B1 clone, however, resulted in the development of a 'cell-proliferative' type of glomerular lesion, associated with an accumulation of both antibodies in glomeruli. This accumulation occurred even though Sp6 IgM antibodies did not react with B1 IgG3 antibodies and vice versa. A mutant clone of Sp6, T/13microE/3.1, which produces antibodies deficient in C1q binding, produced a similar effect as that of the Sp6 clone, i.e. 'cell-proliferative' lesions. Again the B1 antibodies did not react with T/13microE/3. 1-IgM antibodies and vice versa. We therefore conclude that bystander IgM antibodies contribute to the remodelling of glomerular lesions in situ, following glomerular injury by the nephritogenic antibodies.

Expression of the DMBT1 gene is frequently suppressed in human lung cancer.

DMBT1 (deleted in malignant brain tumors) is a candidate tumor suppressor gene that has been mapped to chromosome 10q25.3-q26.1, a region in which frequent loss of heterozygosity (LOH) has been observed in several human tumors. Since DMBT1 is highly expressed in the lung, we analyzed LOH at the DMBT1 locus and expression of this gene in lung cancer. Thirty-five (53%) of 66 primary lung cancers showed LOH, and diminished expression of DMBT1 was observed in 20 (91%) of 22 lung cancer cell lines: three (14%) of them showed loss of expression. We further determined the primary structure of DMBT1 and analyzed genetic alterations in this gene using 23 lung cancer cell lines. Two (9%) of them had homozygous deletion within the gene, and two cell lines had genetic aberrations: one was a rearrangement involving exons 5 and 6, and the other was a missense mutation at codon 52. These results suggest that inactivation of the DMBT1 gene plays an important role in human lung carcinogenesis.

Identification of a 100-kb region of common allelic loss on chromosome bands 10q25-q26 in human endometrial cancer.

In human endometrial cancer, we have previously identified a 790-kb region of common allelic loss in chromosome bands 10q25-q26, flanked by D10S587 and D10S1723. We constructed a contig covering the entire deleted region using YACs, PACs, and BACs. Five overlapping cosmid clones derived from YAC clones completely covered the entire deleted region: its size was estimated to be no larger than 200 kb. We further performed two-color fluorescence in situ hybridization (FISH) analysis to confirm the deletion and narrowed down the deleted region to 100 kb or less; it was covered by three overlapping cosmid clones that were included in one BAC clone. Restriction endonuclease mapping identified a region in which NotI, SalI, SmaI, and Xhol were clustered, suggesting the possible existence of a CpG island.

Electronics for SPring-8 X-ray beam monitors.

A sensitive current-measuring system is required to construct a highly sensitive X-ray beam-position monitor (XBPM). A current-voltage converter (I/V) which can measure currents between 0.1 nA and 10 mA was designed, and the signal processing system of the XBPM was constucted using this I/V. This system was used in beamline commissioning. Beam-position data standard deviations of sigma approximately 3 micro m for the bending-magnet beamline, and sigma(x) approximately 3 micro m and sigma(y) approximately 1 micro m for the insertion-device beamline were obtained during the beamline commissioning.

Present status and performance of SPring-8 front ends.

SPring-8 front ends have a novel structure which makes it easy to rearrange them and exchange the components. The structure has a common support for all the components except X-ray beam-position monitors and lead collimators. The alignment of the common support as well as the components was carried out with an accuracy of 0.25 mm in the vertical and horizontal directions. Replaceable pumping systems have also been placed on the common support and have achieved a vacuum of 2 x 10(-8) Pa at the upstream part of the front ends without synchrotron radiation. During the commissioning with synchrotron radiation, the pumping systems displayed good pumping-down characteristics. Commissioning has been successfully performed for four standard in-vacuum X-ray undulators and three bending-magnet-beamline front ends up to July 1997. Measurements of temperature rise show that absorber, pre-slits and XY slits can handle the anticipated maximum heat load at a ring current of 100 mA.

Diamond beam-position monitor for undulator radiation and tests at the Tristan Super Light Facility.

A photon beam-position monitor using a diamond foil as a position-sensitive device has been developed for use on insertion-device beamlines of third-generation synchrotron radiation facilities such as SPring-8, and was tested on the undulator beamline of the Tristan Super Light Facility at KEK. The beam test results show that the diamond monitor can be operated in a photoconductive mode like a semiconductor detector. It has a linear working range of about +/-1 mm and a position sensitivity of less than 3 mum. The stability of the monitor was confirmed by continuous operation under low photon beam intensity conditions.

The insulin-like growth factor II receptor gene is mutated in genetically unstable cancers of the endometrium, stomach, and colorectum.

Disruption of the DNA mismatch repair system, characterized by microsatellite instability (MI), plays an important role in the course of human carcinogenesis. Repetitive sequences constitute targets for mutation in MI+ cells, and frequent mutations have indeed been reported in such regions within the transforming growth factor beta receptor II (RII) gene in genetically unstable colorectal and gastric cancers. However, other genes that are targets for mutations in MI+ cells during the course of carcinogenesis have proven elusive. Because the insulin-like growth factor II receptor (IGFIIR) gene contains several repetitive sequences within its coding region, we examined mutations of this gene in MI+ cancers occurring at various primary sites. We found frameshift mutations in the poly(G)8 tract of IGFIIR in eight tumors, all of which were MI+: 4 of 26 (15%) MI+ endometrial cancers, 3 of 12 (25%) MI+ gastric cancers, and 1 of 18 (6%) MI+ colorectal cancers. In contrast, no mutation was found in 51 pancreatic cancers, 7 of which (14%) were MI+. These results implicate abnormal IGFIIR-mediated growth control in carcinogenesis involving the endometrium, stomach, and colorectum but not the pancreas.

Alternative splicing of hMSH2 in normal human tissues.

hMSH2 is a homolog of bacterial mutS and yeast Msh2, a member of the group of mismatch repair genes whose products bind to mismatched regions of double-stranded DNA. We analyzed expression of hMSH2 in normal human organs by the polymerase chain reaction coupled with reverse transcription and found two novel types of alternatively spliced mRNAs that were expressed in normal human organs. One lacked exon 13, and the other lacked a portion from the second nucleotide of codon 633 to the second nucleotide of codon 719. In the latter transcript, intro 12 started with TA and ended with TT (TA-TT intron) which did not meet the GT-AG rule. Both types of transcript resulted in frameshifts which generated truncated hMSH2 proteins lacking the main part of the highly conserved region. The biological significance of the alternative splicing remains to be elucidated.

Alternative splicing of GTBP in normal human tissues.

GTBP is one of the functional homologues of the bacterial mismatch repair gene mutS, whose product forms a heterodimer with hMSH2 to bind to the mismatched region of double-stranded DNA. We determined the expression of the GTBP gene and found that two forms of the transcripts were ubiquitously expressed in normal human tissues. These transcripts, termed GTBP-N and GTBP-ALT, were generated by the alternative splicing machinery. These two transcripts share 3172 bp from the translation initiation codon that codes for 1057 amino acids. GTBP-ALT lacked a region that codes for a highly conserved region between GTBP-N, hMSH2, and hMSH3 in their C-termini.