Diffusion tensor imaging in children with periventricular leukomalacia: variability of injuries to white matter tracts.

BACKGROUND AND PURPOSE: Conventional MR imaging shows evidence of brain injury and/or maldevelopment in 70%-90% of children with cerebral palsy (CP), though its capability to identify specific white matter tract injury is limited. The great variability of white matter lesions in CP already demonstrated by postmortem studies is thought to be one of the reasons why response to treatment is so variable. Our hypothesis is that diffusion tensor imaging (DTI) is a suitable technique to provide in vivo characterization of specific white matter tract lesions in children with CP associated with periventricular leukomalacia (PVL). MATERIALS AND METHODS: In this study, 24 children with CP associated with PVL and 35 healthy controls were evaluated with DTI. Criteria for identification of 26 white matter tracts on the basis of 2D DTI color-coded maps were established, and a qualitative scoring system, based on visual inspection of the tracts in comparison with age-matched controls, was used to grade the severity of abnormalities. An ordinal grading system (0=normal, 1=abnormal, 2=severely abnormal or absent) was used to score each white matter tract. RESULTS: There was marked variability in white matter injury pattern in patients with PVL, with the most frequent injury to the retrolenticular part of the internal capsule, posterior thalamic radiation, superior corona radiata, and commissural fibers. CONCLUSION: DTI is a suitable technique for in vivo assessment of specific white matter lesions in patients with PVL and, thus, a potentially valuable diagnostic tool. The tract-specific evaluation revealed a family of tracts that are highly susceptible in PVL, important information that can potentially be used to tailor treatment options in the future.

Genetic loci controlling susceptibility to gamma-ray-induced thymic lymphoma.

BALB/c is a susceptible strain for the development of gamma-ray induced mouse thymic lymphoma whereas MSM shows resistance. Association analysis of 220 backcross mice between the two strains using 67 markers was carried out to identify loci involved in the control of susceptibility. The genotype of mice with lymphoma showed excess heterozygosity relative to MSM homozygosity at D2Mit15 and D4Mit12 and was skewed toward MSM-derived alleles at D5Mit5. The P values in Mantel-Cox test were 0.0048 (D2Mit15), 0.0034 (D4Mit12) and 0.0048 (D5Mit5), suggesting association at the three loci in the susceptibility. Cooperative effect on lymphomagenesis was also observed among the three loci. To obtain independent evidence for linkage at D4Mit12, we made partially congenic mice in which a D4Mit12 region in BALB/c was replaced by MSM-derived homolog. Examination for the lymphoma susceptibility in 78 progeny of the congenic mice confirmed the effect of the locus near D4Mit12 (P=0.0037). The result, together with the linkage analysis, shows that the locus near D4Mit12 is regarded as a confirmed linkage but the other two loci as marginally suggestive.

Reduced sensitivity to and ras mutation spectrum of N-ethyl-N-nitrosourea-induced thymic lymphomas in adult C.B-17 scid mice.

Scid mice are defective in the ability to repair DNA double strand breaks and, as a consequence, their cells are radiosensitive. Further, they have been shown to be prone to develop thymic lymphomas (TLs) after small doses of ionizing radiation. Little is known, however, on the role of scid mutation in chemical carcinogenesis. To determine if scid mutation increased predisposition to chemical carcinogenesis, we examined both the susceptibility of scid mice to N-ethyl-N-nitrosourea (ENU)-induced lymphomagenesis and the involvement of ras gene activation. Adult female mice at 8 weeks of age were given ENU in their drinking water at 400 ppm for 2-10 weeks. Contrary to expectations, we observed a two to three-fold reduction in TL development in the scid mice. The highest incidence was achieved by ENU treatment for 8 weeks for scid and wild-type C.B-17 mice, of 42 and 85%, respectively (P<0.05). We investigated whether this was attributable to the usage of the ras mutation pathway. There was, however, no significant difference in the frequency and spectrum of K-ras mutation between the scid and wild-type C.B-17 mice. Most of the K-ras mutations were either GGT to GAT transition in codon 12 (11/23: 48%) or CAA to CCA transversion in codon 61 (8/23: 35%) that was independent of scid background. The incidence of N-ras mutation was very low. These results indicate that scid mice are less susceptible to ENU-induced lymphomagenesis and ras gene mutation frequently occurs in both scid and wild-type C.B-17 mice.

Overexpression of human H-ras transgene is responsible for tumors induced by chemical carcinogens in mice.

Level of human prototype H-ras transgene expression in tumors induced by chemical carcinogens (N-ethyl-N-nitrosourea and N-methyl-N-nitrosourea) was analyzed in human H-ras transgenic mice (CB6F1-TgrasH2 Jic mice). All forestomach tumors examined revealed about 2-fold overexpression of the human H-ras transgene with or without point mutation at codon 12 or codon 61. However, endogenous mouse H- and K-ras genes exhibited neither point mutation nor overexpression. These results suggested that increased levels of ras gene products in the cell played an important role in facilitating chemical carcinogenesis in transgenic mice.

Genomic organization and mapping of mouse CDV (carnitine deficiency-associated gene expressed in ventricle)-1 and its related CDV-1R gene.

We have previously reported that CDV (carnitine deficiency-associated gene expressed in ventricle)-1 was a downregulated gene in the hypertrophied ventricle of carnitine-deficient juvenile visceral steatosis mice and that the related gene (CDV-1R) showed no tissue specificity and no sensitivity to carnitine deficiency. In the present paper, the CDV-1/1R gene was isolated from a mouse genomic BAC library, and the genomic structure was characterized. We found that the CDV-1/1R gene consisted of at least 19 exons and encompassed approximately 48 kb. The splice sites conformed to the GT-AG rule, and the CDV-1R mRNA containing 19 exons was processed. CDV-1 mRNA containing 5 exons was constructed from the 3' half of CDV-1R. The first exon of CDV-1 consisted of the 3' side (116 bp) of intron 14 and exon 15 (87 bp) of CDV-1R. The presumed promoter sequence for CDV-1 located in the intron 14 of CDV-1R contained the common TATA box and consensus binding sites for various transcription factors (Nkx-2.5, Spl, C/EBP, SRF, YY1, and CREB), which seem to play roles in the heart-specific expression and carnitine deficiency-associated suppression of CDV-1. In the upstream region of the CDV-1 promoter, we found two VNTRs, 13 repeats of GATA1, and 16 copies of STRE involved in yeast stress response. The CDV-1/1R gene was located close to DSMIT68 on mouse Chromosome (Chr) 5, corresponding to human Chr 12q24. All these data revealed that two mRNA species, CDV-1 and CDV-1R, are expressed tissue-specifically by using promoters peculiar to each transcript in a single gene.

Radiation-associated loss of heterozygosity at the Znfn1a1 (Ikaros) locus on chromosome 11 in murine thymic lymphomas.

Although information on the molecular pathways in radiation carcinogenesis is accumulating, the data are still relatively scanty. To find the tumor suppressor locus associated with radiation carcinogenesis, we determined the frequency and distribution of loss of heterozygosity (LOH) of X-ray-induced thymic lymphomas of B6C3F(1) mice using 58 microsatellite markers and compared the results with those for spontaneous lymphomas and N-ethylnitrosourea (ENU)-induced lymphomas. Based on the results, we describe a unique locus with frequent LOH in the centromeric region of chromosome 11 of X-ray-induced lymphomas. This locus has never been observed to be altered similarly in either ENU-induced or spontaneous lymphomas, suggesting radiation-specific molecular alteration. The LOH patterns of individual thymic lymphomas indicated that the common region of LOH was located within 1.6 cM between D11Mit62 and D11Mit204, a region syntenic to human chromosome 7p13. Linkage analysis revealed that the markers of the common LOH region were genetically linked to Ikaros (now known as Znfn1a1), a master gene of lymphopoiesis. Although the presence of radiation-associated LOH in other loci cannot be ruled out, these results suggest a novel molecular pathway in induction of thymic lymphomas by ionizing radiation.

Suppression of insulitis and diabetes in B cell-deficient mice treated with streptozocin: B cells are essential for the TCR clonotype spreading of islet-infiltrating T cells.

In order to clarify the role of B cells in the development of insulitis and diabetes, B cell-deficient (B(-)) mice treated with streptozocin (STZ) were studied. The extent of insulitis and the cumulative incidence of diabetes were significantly suppressed in B(-) mice (P < 0.0001), indicating that B cells are crucial for the progression of insulitis and diabetes. Accumulation of both CD4(+) T cells and B cells was observed in islets of B(+) mice, while CD4(+) T cells but not B cells were found in B(-) mice. A few CD8(+) T cells and macrophages were detectable in both types of mice. The immunohistochemical study did not reveal any change in the subpopulations of infiltrating lymphocytes except for the absence of B cells in the B(-) mice. TCR V(beta) gene repertoire usage of islet-infiltrating T cells was restricted to some extent in the B(+) or B(-) mice, but there was no significant difference between the B(+) and B(-) mice, suggesting that the initial islet-reactive T cell response can occur in the absence of B cells. In contrast, TCR clonotype spreading of islet-infiltrating T cells was significantly suppressed in B(-) mice compared with B(+) mice (P < 0.0001). These data suggest that initial priming of T cells is not impaired and TCR V(beta) repertoire usage is not limited by the lack of B cells, while B cells are important essentially for the spreading of islet-infiltrating clonal T cells in autoimmune diabetic mice induced with STZ.

YAC/BAC-based physical and transcript mapping around the gracile axonal dystrophy (gad) locus identifies Uchl1, Pmx2b, Atp3a2, and Hip2 genes.

We generated a yeast artificial chromosome (YAC)/bacterial artificial chromosome (BAC)-based physical and transcript map of a region containing the gracile axonal dystrophy (gad) locus on mouse chromosome 5. The YAC/BAC contig consists of 13 YAC and 49 BAC clones onto which 4 genes, 40 expressed sequence tags, and 7 new DNA polymorphisms were ordered. Using this physical map, we mapped Uchl1 encoding ubiquitin carboxyl-terminal hydrolase I, whose deletion has been determined to cause the gad mutation. We also mapped three other recently identified genes: Hip2, encoding Huntingtin interacting protein 2; Atp3a2, encoding a P-type ATPase; and Pmx2b, encoding PHOX2b.

Molecular phylogeny of East Asian moles inferred from the sequence variation of the mitochondrial cytochrome b gene.

Taxonomic analysis has previously revealed that the species of moles that inhabit Japan are characterized by exceptional species richness and a high level of endemism. Here, we focused on the evolutionary history of the four Japanese mole species of the genera Euroscapter and Mogera, examining mitochondrial cytochrome b (cyt b) gene sequences and comparing them with those of continental Mogera wogura (Korean and Russian populations), M. insularis from Taiwan, and Talpa europaea and T. altaica from the western and central Eurasian continent, respectively. Our data support the idea that in a radiation center somewhere on the Eurasian continent, a parental stock evolved to modern mole-like morph and radiated several times intermittently during the course of the evolution, spreading its branches to other peripheral geographic domains at each stage of the radiation. Under this hypothesis, the four lineages of Japanese mole species, E. mizura, M. tokudae, M. imaizumii, and M. wogura, could be explained to have immigrated to Japan in this order. Mogera wogura and M. imaizumii showed substantial amounts of geographic variation and somewhat complicated distributions of the cyt b gene types. These intraspecific variations are likely to be associated with the expansion processes of moles in the Japanese Islands during the Pleistocene glacial ages.

Rapid induction of uterine tumors with p53 point mutations in heterozygous p53-deficient CBA mice given a single intraperitoneal administration of N-ethyl-N-nitrosourea.

To investigate the sensitivity of heterozygous p53-deficient CBA mice to carcinogens, 20 female mice [p53(+/-)] and 20 wild-type littermates [p53(+/+)] were given an intraperitoneal injection of 120 mg/kg body wt of N-ethyl-N-nitrosourea (ENU) and were maintained without any other treatment for a further 26 weeks. Histopathology showed that uterine tumors (endometrial polyps and stromal sarcomas) and lung adenomas were induced in both p53(+/-) and p53(+/+) mice. The incidence of uterine tumors and lung adenomas (94% and 81%, respectively) in p53(+/-) mice was significantly greater than that in p53 (+/+) mice (37% and 42%, respectively). Malignant lymphomas were only induced in p53(+/-) mice, at an incidence of 31%. Concerning uterine tumors and preneoplastic lesions, there were endometrial stromal sarcomas and atypical hyperplasias of the endometrial gland in 90% and 63%, respectively, of p53(+/-) mice, with significantly greater incidences than in p53(+/+) mice. Gene analysis revealed GCG-->GTG point mutations in codon 135 of exon 5 of the p53 allele in all of the uterine endometrial stromal sarcomas examined. Our results suggest that female p53(+/-) CBA mice are very susceptible to uterine carcinogenesis, providing a useful model for ENU-induced uterine tumors.

Identification of an imprinted gene, Meg3/Gtl2 and its human homologue MEG3, first mapped on mouse distal chromosome 12 and human chromosome 14q.

BACKGROUND: The paternal duplication of mouse distal chromosome 12 leads to late embryonal/neonatal lethality and growth promotion, whereas maternal duplication leads to late embryonal lethality and growth retardation. Human paternal or maternal uniparental disomies of chromosome 14q that are syntenic to mouse distal chromosome 12 have also been reported to show some imprinting effects on growth, mental activity and musculoskeletal morphology. For the isolation of imprinted genes in this region, a systematic screen of maternally expressed genes (Megs) was carried out by our subtraction-hybridization method using androgenetic and normally fertilized embryos. RESULTS: We have isolated seven candidate clones of the mouse Meg gene. Among them, we identified a novel maternally expressed imprinted gene, Meg3, on mouse distal chromosome 12 and showed that it was identical to the Gtl2 gene. We also found that the human homologue MEG3 on chromosome 14q was also monoallelically expressed. CONCLUSIONS: This is the first identification of the imprinting gene, both on mouse distal chromosome 12 and on human chromosome 14q, respectively. Because there are no obvious open reading frames in either the mouse Meg3/Gtl2 or human MEG3, the function of these genes remains unclear. However, this result will provide a good basis for the further investigation of several important imprinted genes in this chromosomal region.

Molecular cloning of a novel NF2/ERM/4.1 superfamily gene, ehm2, that is expressed in high-metastatic K1735 murine melanoma cells.

We have cloned a novel gene, Ehm2, that is expressed in high-metastatic but not in low-metastatic K-1735 murine melanoma cells. The Ehm2 gene encodes a protein of 527 amino acid residues, showing up to 41% amino acid identity with the FERM domain of NF2/ERM/4.1 superfamily proteins, which have the function of connecting cell surface transmembrane proteins to cytoskeletal molecules. The Ehm2 gene was mapped to chromosome 4 and was expressed in the liver, lung, kidney, and testis and in 7- to 17-day embryos. The highest level of homology was observed with NBL4, which is a new subfamily protein of the NF2/ERM/4.1 superfamily. A human homologue of the mouse Ehm2 gene, showing significant homology (83% identity), was identified in the genomic DNA and EST databases. Furthermore, seven rat EST clones and one pig EST clone in the GenBank EST database were identified as having 83-92% sequence homology with the cDNA sequence of the mouse Ehm2 gene. Thus, Ehm2 is a highly conserved gene that encodes a novel member of the NF2/ERM/4.1 superfamily proteins.

Gene mapping of SEZ group genes and determination of pentylenetetrazol susceptible quantitative trait loci in the mouse chromosome.

Gene mapping of the newly discovered SEZ genes (seizure-related genes) in the mouse was performed by linkage analysis. SEZ6 was on chromosome 11, SEZ12 on chromosome 16, SEZ15 on chromosome 3 and SEZ17 (PTZ17) on chromosome 18. The mouse chromosomal locus related to high susceptibility to pentylenetetrazol (PTZ) was also determined by linkage analysis using the recombinant inbred mouse, BXD (C57BLxDBA). A significant level of PTZ susceptibility was found on chromosome 2. Chromosomal loci of the newly discovered SEZ genes were not coincident with the significant chromosomal loci to PTZ susceptibility. Since epilepsy is assumed to be a disease syndrome which is probably manifested by abnormal expression of multifocal genes, determination of the role of each chromosomal locus in the provocation of seizure activity is important.

Intragenic deletion in the gene encoding ubiquitin carboxy-terminal hydrolase in gad mice.

The gracile axonal dystrophy (gad) mouse is an autosomal recessive mutant that shows sensory ataxia at an early stage, followed by motor ataxia at a later stage. Pathologically, the mutant is characterized by 'dying-back' type axonal degeneration and formation of spheroid bodies in nerve terminals. Recent pathological observations have associated brain ageing and neurodegenerative diseases with progressive accumulation of ubiquitinated protein conjugates. In gad mice, accumulation of amyloid beta-protein and ubiquitin-positive deposits occur retrogradely along the sensory and motor nervous systems. We previously reported that the gad mutation was transmitted by a gene on chromosome 5 (refs 10,11). Here we find that the gad mutation is caused by an in-frame deletion including exons 7 and 8 of Uchl1, encoding the ubiquitin carboxy-terminal hydrolase (UCH) isozyme (Uch-l1) selectively expressed in the nervous system and testis. The gad allele encodes a truncated Uch-l1 lacking a segment of 42 amino acids containing a catalytic residue. As Uch-l1 is thought to stimulate protein degradation by generating free monomeric ubiquitin, the gad mutation appears to affect protein turnover. Our data suggest that altered function of the ubiquitin system directly causes neurodegeneration. The gad mouse provides a useful model for investigating human neurodegenerative disorders.

Low frequency of Ras gene mutation in spontaneous and gamma-ray-induced thymic lymphomas of scid mice.

Scid mice, which have a defect in the capacity to repair DNA double-strand breaks, were highly prone to the induction of thymic lymphomas after exposure to ionizing radiation; approximately 70% of mice developed lymphomas within 1 year after exposure to 1-3 Gy, whereas approximately 20% of unirradiated control mice developed lymphomas. To gain information on the possible role of Ras activation in development of thymic lymphomas in scid mice, we have examined both the frequency and the spectrum of Kras and Nras mutations in spontaneous and radiation-induced lymphomas. Neither activated Kras nor Nras genes were detected in spontaneous lymphomas, while Kras mutations increased in a dose-dependent manner in radiation-induced lymphomas. However, Kras mutations were infrequent (6% in lymphomas in mice exposed to 1 Gy, 12.5% in those exposed to 2 Gy, 16.7% in those exposed to 3 Gy), and no mutations were detected in Nras genes, suggesting that Ras mutation was not significantly involved in the development of thymic lymphomas in scid mice. Analysis of the spectrum of Kras mutations demonstrated unique mutations in both codons 13 (GGC to GAC) and 61 (CAA to CTA) in addition to the commonly identified substitution of GAT for GGT in codon 12 of Kras.

Molecular cloning of rat and mouse membrane cofactor protein (MCP, CD46): preferential expression in testis and close linkage between the mouse Mcp and Cr2 genes on distal chromosome 1.

Human membrane cofactor protein (MCP, CD46) is widely distributed and is one of the plasma membrane complement inhibitors. We isolated cDNA clones encoding genetic homologues of human MCP from a rat testis cDNA library. Northern blot analysis indicated that rat MCP is preferentially expressed in testis, similar to what is found with guinea pig MCP. We identified several different cDNAs, which were presumably generated by alternative splicing from a single-copy gene. The most prevalent isoform corresponded to the Ser/Thr/Pro-rich C type of human MCP. Mouse MCP cDNA was cloned by polymerase chain reaction based on the nucleotide sequence of rat MCP. The deduced amino acid sequence showed 77.8% identity to rat MCP. Mouse MCP was also preferentially expressed in testis. Unique expression in testis in rat and mouse as well as guinea pig suggests that MCPs in these species not only act as complement regulatory proteins but may also have more specialized functions in fertilization or reproduction. Genetic mapping by linkage analysis indicated that the mouse Mcp gene is located on distal chromosome 1, closely linked to the complement receptor 2 (Cr2) gene.