Tomohisa Sekimoto

Targeted disruption of the Tab1 gene causes embryonic lethality and defects in cardiovascular and lung morphogenesis

The transforming growth factor-beta (TGF-beta) superfamily consists of a group of secreted signaling molecules that perform important roles in the regulation of cell growth and differentiation. TGF-beta activated kinase-1 binding protein-1 (TAB1) was identified as a molecule that activates TGF-beta activated kinase-1 (TAK1). Recent studies have revealed that the TAB1-TAK1 interaction plays an important role in signal transduction in vitro, but little is known about the role of these molecules in vivo. To investigate the role of TAB1 during development, we cloned the murine Tab1 gene and disrupted it by homologous recombination. Homozygous Tab1 mutant mice died, exhibiting a bloated appearance with extensive edema and hemorrhage at the late stages of gestation. By histological examinations, it was revealed that mutant embryos exhibited cardiovascular and lung dysmorphogenesis. Tab1 mutant embryonic fibroblast cells displayed drastically reduced TAK1 kinase activities and decreased sensitivity to TGF-beta stimulation. These results indicate a possibility that TAB1 plays an important role in mammalian embryogenesis and is required for TAK1 activation in TGF-beta signaling.

Region‐specific gastrointestinal Hox code during murine embryonal gut development

Hox genes encode transcription factors, and they are involved in the specification of each body part along the anteroposterior (AP) body axis during embryogenesis. To clarify AP pattern formation of the digestive tract, the expression patterns of Hox genes belonging to paralogous groups 4 and 5, and parts of groups 6 and 7, were systematically examined by whole‐mount and section in situ hybridization. The Hox gene expression pattern of paralogous groups 4–9 in the developing gut at 12.5 days post‐coitum was fully examined. All HoxA and HoxB genes in paralogous groups 4–8 were expressed in the stomach, in contrast to the HoxC and HoxD genes. In the midgut region, all Hox cluster genes showed colinear expression within each cluster, yielding the Hox code; the more 3′ located genes were expressed more rostrally and the 5′ group genes more caudally. The colinear expression of HoxA and HoxB cluster genes started from the duodenum, that of HoxC cluster genes started from the jejunum, and HoxD cluster genes were expressed in the caudal part of the midgut, ileum and cecum. In the hindgut region, HoxD cluster genes and Abd‐B family genes were expressed. Thus, a different Hox code seems to exist in each subdomain of developing gut (foregut, midgut and hindgut). The visceral mesoderm restricted expression also suggested that the Hox code primarily functions in mesenchymal specification, and then leads to the regional differentiation of gut subdomains as the result of epithelial– mesenchymal interactions.

Characterization of an exchangeable gene trap using pU-17 carrying a stop codon-βgeo cassette

We have developed a new exchangeable gene trap vector, pU‐17, carrying the intron‐lox71‐splicing acceptor (SA)‐βgeo‐loxP‐pA‐lox2272‐pSP73‐lox511. The SA contains three stop codons in‐frame with the ATG of βgalactosidase/neomycin‐resistance fusion gene (βgeo) that can function in promoter trapping. We found that the trap vector was highly selective for integrations in the introns adjacent to the exon containing the start codon. Furthermore, by using the Cre‐mutant lox system, we successfully replaced the βgeo gene with the enhanced green fluorescent protein (EGFP) gene, established mouse lines with the replaced clones, removed the selection marker gene by mating with Flp‐deleter mice, and confirmed that the replaced EGFP gene was expressed in the same pattern as the βgeo gene. Thus, using this pU‐17 trap vector, we can initially carry out random mutagenesis, and then convert it to a gain‐of‐function mutation by replacing the βgeo gene with any gene of interest to be expressed under the control of the trapped promoter through Cre‐mediated recombination.

Region-specific expression of murine Hox genes implies the Hox code-mediated patterning of the digestive tract

Hox genes encode transcription factors which are involved in the establishment of regional identities along the anteroposterior (AP) body axis. To elucidate the AP patterning of the digestive tract, we have systematically examined the expression patterns of Hox genes belonging to paralogue groups 6, 7, 8 and 9 by whole‐mount in situ hybridization and by section in situ hybridization analyses.

Evaluation of circulatory compromise in the leg in lumbar spinal canal stenosis.

To evaluate whether hemoglobin oxygen saturation and hemoglobin concentration of the leg are useful indicators for circulatory compromise in patients with lumbar spinal canal stenosis, we investigated the changes in the indices during level gait using reflectance spectrophotometry. Thirty-three patients with lumbar spinal stenosis were studied. Preoperatively, the hemoglobin oxygen saturation was greater in the 33 patients than in the control subjects. The indices increased in the control subjects more than those in the patients. Postoperatively, the increases in hemoglobin oxygen saturation were greater in the patients with lumbar spinal canal stenosis than before decompression and the hemoglobin concentration tended to approximate that in the control subjects. The results suggest these indices might be useful for monitoring disease severity in patients with lumber spinal canal stenosis. In addition to stenotic ischemia in the spinal canal, it is thought that the neurogenic intermittent claudication is secondarily caused by circulatory failure in the lower extremities attributable to the autonomic nervous dysfunction. Level of Evidence: Prognostic study, Level I-1 (prospective study)

Evaluation of human beta-enolase as a serum marker for exercise-induced muscle damage.

OBJECTIVE To evaluate whether the serum beta-enolase level is a useful indicator of exercise-induced muscle damage in athletes. DESIGN Blood samples were taken from 49 adult amateur marathon runners before and immediately after a marathon race, and the serum levels of beta-enolase and creatine phosphokinase were measured. SETTING The Aoshima Taiheiyo Marathon 2000, Miyazaki, Japan, on a cloudy day in December with an ambient temperature of 18 degrees C. SUBJECTS Forty-nine adult amateur marathon runners (42 men and 7 women) who regularly participated in runs. INTERVENTION The intervention was a marathon run. MAIN OUTCOME MEASURES Serum beta-enolase was measured using a sensitive sandwich enzyme-linked immunosorbent assay. Serum creatine phosphokinase was measured using a standard procedure. RESULTS The mean beta-enolase concentration was 9.45 +/- 3.11 ng/mL before the race. It increased to 22.11 +/- 8.80 ng/mL after the race, representing a proportional increase of 1.57 +/- 1.46. The serum concentration of beta-enolase after the race was significantly higher than that before the race (P < 0.0001). Moreover, the serum beta-enolase level increased as much as the creatine phosphokinase level after the race, and strongly correlated with creatine phosphokinase (r = 0.828, P < 0.0001). The proportional increase of beta-enolase also correlated with that of creatine phosphokinase (r = 0.441, P < 0.005). CONCLUSIONS Our data suggest that the absolute values of the serum beta-enolase are more appropriate to relate to muscle damage.

Naso-maxillary deformity due to frontonasal expression of human transthyretin gene in transgenic mice.

Background Retinoic acid, a metabolic product of retinol, is essential for craniofacial morphogenesis. Transthyretin (TTR) is a plasma protein delivering retinol to tissues. We produced several transgenic mouse lines using the human mutant TTR (hTTRMet30) gene to establish a mouse model of familial amyloidotic polyneuropathy. One of the lines showed an autosomal dominant inheritance of naso‐maxillary deformity termed Nax.

Development of an efficient screening system to identify novel bone metabolism-related genes using the exchangeable gene trap mutagenesis mouse models.

Despite numerous genetic studies on bone metabolism, understanding of the specific mechanisms is lacking. We developed an efficient screening system to identify novel genes involved in bone metabolism using mutant mouse strains registered with the Exchangeable Gene Trap Clones (EGTC) database. From 1278 trap clones in the EGTC database, 52 candidate lines were selected in the first screening, determined based on “EST profile”, “X-gal”, “Related article”, and “Novel gene”. For the second screening, bone morphometric analysis, biomechanical strength analysis, bone X-gal staining, etc. were performed on candidate lines. Forty-two male trap lines (80.8%) showed abnormalities with either bone morphometric analysis or biomechanical strength analysis. In the screening process, X-gal staining was significantly efficient (P = 0.0057). As examples, Lbr and Nedd4 trap lines selected using the screening system showed significant bone decrease and fragility, suggesting a relationship with osteoblast differentiation. This screening system using EGTC mouse lines is extremely efficient for identifying novel genes involved in bone metabolism. The gene trap lines identified as abnormal using this screening approach are highly likely to trap important genes for bone metabolism. These selected trap mice will be valuable for use as novel bio-resources in bone research.

Results of Overhead Traction in Congenital Dislocation of the Hip

当科では先天性股関節脱臼に対しRBによる整復を試みるが，整復困難例や初診時に生後1年以上経過している例に対してはオーバーヘッドトラクション（OHT）による整復を行う．これらの保存的治療に抵抗する例に対しては手術療法を考慮する．今回当科にてOHTによる整復を行って1年以上経過した例について検討した．【対象】平成3年から平成19年までに当科外来を受診した6例7関節（全例女児）を対象とした．初診時平均月齢は1歳（5ヶ月～1歳10ヶ月），牽引開始平均年齢は1歳2ヶ月（8ヶ月～2歳）平均観察期間は8年（1年～15年）である．【結果と考察】牽引期間は平均で65日（51日～74日）であり，7関節全例に整復が得られた．しかしOHT開始が1歳以降の4例中2例に補正手術が必要であった．また，残りの2例も1例は補正手術を予定し，1例は外転装具装着中である．早期の脱臼整復が予後に関係するものと考えられた．