Tmem161a regulates bone formation and bone strength through the P38 MAPK pathway.

Bone remodeling is an extraordinarily complex process involving a variety of factors, such as genetic, metabolic, and environmental components. Although genetic factors play a particularly important role, many have not been identified. In this study, we investigated the role of transmembrane 161a (Tmem161a) in bone structure and function using wild-type (WT) and Tmem161a-depleted (Tmem161aGT/GT) mice. Mice femurs were examined by histological, morphological, and bone strength analyses. Osteoblast differentiation and mineral deposition were examined in Tmem161a-overexpressed, -knockdown and -knockout MC3T3-e1 cells. In WT mice, Tmem161a was expressed in osteoblasts of femurs; however, it was depleted in Tmem161aGT/GT mice. Cortical bone mineral density, thickness, and bone strength were significantly increased in Tmem161aGT/GT mice femurs. In MC3T3-e1 cells, decreased expression of alkaline phosphatase (ALP) and Osterix were found in Tmem161a overexpression, and these findings were reversed in Tmem161a-knockdown or -knockout cells. Microarray and western blot analyses revealed upregulation of the P38 MAPK pathway in Tmem161a-knockout cells, which referred as stress-activated protein kinases. ALP and flow cytometry analyses revealed that Tmem161a-knockout cells were resistant to oxidative stress. In summary, Tmem161a is an important regulator of P38 MAPK signaling, and depletion of Tmem161a induces thicker and stronger bones in mice.

Efficacy of Diagnosing Carpal Tunnel Syndrome Using the Median Nerve Stenosis Rate Measured on Ultrasonographic Sagittal Imagery: Clinical Case-Control Study.

BACKGROUND: The purpose of the present study was to evaluate the usefulness of the median nerve stenosis rate (MNSR) measured on sagittal sonographic images of the median nerve in the diagnosis of carpal tunnel syndrome (CTS). METHODS: The study population consisted of 45 hands from 37 patients with idiopathic CTS (CTS group), and 60 hands from 35 asymptomatic healthy subjects (control group). Carpal tunnel syndrome was diagnosed by clinical findings and positive electrophysiological study results. All patients and control subjects underwent ultrasonographic examination. At the carpal tunnel level, the transducer was placed longitudinally to the median nerve, and an image of the longitudinal median nerve was obtained. The minimum median nerve diameter (MND) was measured at the middle part of the capitate level, while the maximum MND was measured at the distal radioulnar joint level. The MNSR was calculated as (1 - minimum MND/maximum MND) × 100 (%). The cross-sectional area of the median nerve was also measured at the level of the pisiform. RESULTS: On longitudinal sonographic images, the MNSR was significantly larger in the CTS group than the control group. When the cut-off value of the MNSR was 26.73%, the sensitivity and specificity were 91.1% and 80%, respectively. The area under the receiver operating characteristic curve was larger for the MNSR than for the cross-sectional area. CONCLUSION: The results suggest that the MNSR proposed in the present study may be useful as an auxiliary method for CTS diagnosis on ultrasonographic examination.

EPLINß Is Involved in the Assembly of Cadherin-catenin Complexes in Osteoblasts and Affects Bone Formation.

Epithelial protein lost in neoplasm (EPLIN) is an actin-associated cytoskeletal protein that plays an important role in epithelial cell adhesion. EPLIN has two isoforms: EPLINα and EPLINß. In this study, we investigated the role of EPLINß in osteoblasts using EPLINß-deficient (EPLINßGT/GT ) mice. The skeletal phenotype of EPLINßGT/GT mice is indistinguishable from the wildtype (WT), but bone properties and strength were significantly decreased compared with WT littermates. Histomorphological analysis revealed altered organization of bone spicules and osteoblast cell arrangement, and decreased alkaline phosphatase activity in EPLINßGT/GT mouse bones. Transmission electron microscopy revealed wider intercellular spaces between osteoblasts in EPLINßGT/GT mice, suggesting aberrant cell adhesion. In EPLINßGT/GT osteoblasts, α- and ß-catenins and F-actin were observed at the cell membrane, but OB-cadherin was localized at the perinuclear region, indicating that cadherin-catenin complexes were not formed. EPLINß knockdown in MC3T3-e1 osteoblast cells showed similar results as in calvaria cell cultures. Bone formation markers, such as RUNX2, Osterix, ALP, and Col1a1 mRNA were reduced in EPLINß knockdown cells, suggesting an important role for EPLINß in osteoblast formation. In conclusion, we propose that EPLINß is involved in the assembly of cadherin-catenin complexes in osteoblasts and affects bone formation.

Role of Mel1/Prdm16 in bone differentiation and morphology.

MEL1 (MDS1/EVI1-like gene 1/PRDM16), a zinc finger protein, is located near the chromosomal breakpoint at 1p36 in human acute myeloid leukemia (AML) cells with the t (1; 3) (p36; q21) translocation. Mel1/Prdm16 is not only a causative gene of leukemia, but also has multiple regulatory functions, such as the regulation of fat metabolism. To investigate the function of Mel1/Prdm16, we generated Mel1/Prdm16-deficient mice, but homozygous deficiency (Mel1/Prdm16-/-) was embryonic lethal at E 11.5. Heterozygous mice showed abnormal cartilage and bone formation in the postnatal skull and long bones, suggesting that Mel1/Prdm16 expression plays an important role in bone development. In osteoblast and chondrocyte cell lines, Mel1/Prdm16 promotes the differentiation of chondrocytes and regulates the differentiation of osteoblasts. Transient repression of the master regulator Runx2 is required for chondrocyte differentiation at an early stage of differentiation. However, in Mel1/Prdm16-suppressed ATDC5 cells, the initial suppression of Runx2 was lacking and its expression was upregulated at the beginning of differentiation, suggesting that chondrogenic differentiation is suppressed in Mel1/Prdm16+/- mesenchymal progenitor cells because Runx2 expression is upregulated during the early stage of differentiation. Thus, the Mel1/Prdm16 gene may be involved in the early repression of Runx2 expression during osteochondral differentiation and promote chondrogenic differentiation.

Shadow pitching deviates ball release position: kinematic analysis in high school baseball pitchers.

BACKGROUND: Although shadow pitching, commonly called "towel drill," is recommended to improve the throwing motion for the rehabilitation of pitching disorders before the initiation of a throwing program aimed at returning to throwing using a ball, the motion differs from that of normal throwing. Learning improper motion during ball release (BR) may increase shoulder joint forces. Abnormal throwing biomechanics leads to injures. However, there has been no study of shadow pitching focusing on the BR position. The purpose of the present study was to evaluate the BR position and kinematic differences between shadow pitching and normal throwing. In addition, the effect of setting a target guide for BR position on throwing motion was examined in shadow pitching. METHODS: The participants included in this study were 20 healthy male students who were overhand right-handed pitchers with no pain induced by a throwing motion. Participants performed normal throwing (task 1), shadow pitching using a hand towel (task 2), and shadow pitching by setting a target of the BR position (task 3). A motion capture system was used to evaluate kinematic differences in throwing motions, respectively. Examination items comprised joint angles and the differences in BR position. RESULTS: BR position of task 2 shifted significantly toward the anterior, leftward, and downward directions compared with task 1. The distance of BR position between tasks 1 and 2 was 24 ± 10%. However, task 3 had decreased BR deviation compared with task 2 (the distance between 3 and 1 was 14 ± 7%). Kinematic differences were observed among groups at BR. For shoulder joint, task 2 showed the highest value in abduction and horizontal adduction among groups. In spine flexion, left rotation and thorax flexion, task 2 was significantly higher than task 1. Task 3 showed small differences compared with task 1. CONCLUSIONS: The BR position of shadow pitching deviated significantly in the anterior, leftward, and downward directions compared with normal throwing. Furthermore, we demonstrated that the setting of BR target reduces this deviation. Thus, the target of BR position should be set accurately during shadow pitching exercises in the process of rehabilitation.

Hemoglobin stimulates the expression of ADAMTS-5 and ADAMTS-9 by synovial cells: a possible cause of articular cartilage damage after intra-articular hemorrhage.

BACKGROUND: ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) proteins play an important pathological role in matrix degeneration. Aggrecan degradation is a significant and critical event in early-stage osteoarthritis. To determine the effect of hemoglobin (Hb) on the ability of synovial tissues to produce ADAMTS family members, we examined the influence of Hb by synovial cells in an in vitro experimental system. METHODS: Synovial tissues were obtained from five young patients with meniscal injury under arthroscopic surgery. Primary cultures of human knee synovial cells were treated with different doses of human Hb (0, 25, 50, 100 µg/ml). The culture media were collected 24 h after Hb-treatment. In the time-course studies, cells were treated with and without 100 µg/ml Hb, and culture media were taken at 6, 12, and 24 h. To identify the proteins responsible for aggrecanase activity, Western blot analysis using antibodies against human ADAMTS-5, -8, -9, and -10; enzyme-linked immunosorbent assay (ELISA); and gene expression for ADAMTS-5 and -9 were examined. Statistical comparisons between each group were performed using paired t-tests. RESULTS: Western blot analysis revealed that Hb-treatment resulted in the expression of ADAMTS-5 and -9. Neither control group nor Hb-treated medium showed immunoreactivity against ADAMTS-8 or -10. In a dose-dependency study, the Hb-treated group showed significantly higher levels of ADAMTS-5 and -9 compared with the control (p < 0.05). There was no significant difference between 25, 50, and 100 µg/ml Hb-treated groups. In a time-course study, the ADAMTS-5 and -9 levels in the conditioned medium had significantly increased expression at 6, 12, and 24 h in the Hb-treated group (p < 0.05). Hb evoked significant expression of ADAMTS-9 mRNA at 12 and 24 h (p < 0.05). CONCLUSIONS: These findings indicate that Hb induces the expression of ADAMTS-5 and -9 by synovial cells at low doses, even at an acute phase, and suggests a possible role for Hb in cartilage damage after intra-articular hemorrhage. The results also suggest a new potential therapeutic target by inhibiting the activities of ADAMTS-5 and -9 to prevent cartilage damage after intra-articular hemorrhage.

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.

Angiotensin II Stimulation of Cardiac Hypertrophy and Functional Decompensation in Osteoprotegerin-Deficient Mice.

Circulating and myocardial expressions of receptor activator of nuclear factor-κb ligand and osteoprotegerin are activated in heart failure; however, it remains to be determined their pathophysiological roles on left ventricular structure and function in interaction with renin-angiotensin system. We conducted experiments using 8-week-old osteoprotegerin(-/-) mice and receptor activator of nuclear factor-κb ligand-transgenic mice to assess whether they affect the angiotensin II-induced left ventricular remodeling. Subcutaneous infusion of angiotensin II to osteoprotegerin(-/-) mice progressed the eccentric hypertrophy, resulting in left ventricular systolic dysfunction for 28 days, and this was comparable with wild-type mice, showing concentric hypertrophy, irrespective of equivalent elevation of systolic blood pressure. The structural alteration was associated with reduced interstitial fibrosis, decreased procollagen α1 and syndecan-1 expressions, and the increased number of apoptotic cells in the left ventricle, compared with wild-type mice. In contrast, angiotensin II infusion to the receptor activator of nuclear factor-κb ligand-transgenic mice revealed the concentric hypertrophy with preserved systolic contractile function. Intraperitoneal administration of human recombinant osteoprotegerin, but not subcutaneous injection of anti-receptor activator of nuclear factor-κb ligand antibody, to the angiotensin II-infused osteoprotegerin(-/-) mice for 28 days ameliorated the progression of heart failure without affecting systolic blood pressure. These results underscore the biological activity of osteoprotegerin in preserving myocardial structure and function during the angiotensin II-induced cardiac hypertrophy, independent of receptor activator of nuclear factor-κb ligand activity. In addition, the antiapoptotic and profibrotic actions of osteoprotegerin that emerged from our data might be involved in the mechanisms.

Cardiac hypertrophy is exacerbated in aged mice lacking the osteoprotegerin gene.

AIMS: Osteoprotegerin (OPG) may play a role in the progression of cardiac hypertrophy and heart failure. However, its pathophysiological role in changes in cardiac structure and function with ageing remains to be elucidated. METHODS AND RESULTS: We conducted experiments using 2.5- and 12-month-old OPG(-/-) mice and age-matched wild-type (WT) mice and compared the morphology and function of the left ventricle (LV). Both 2.5- and 12-month-old OPG(-/-) mice showed a higher systolic blood pressure and a greater heart weight/body weight ratio than age-matched WT mice. Twelve-month-old OPG(-/-) mice had a significantly larger LV chamber and reduced wall thickness compared with age-matched WT mice, and contractile function was decreased. The morphological differences were accompanied by an increase in the number of apoptotic cells and activation of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) in the LV of 12-month-old OPG(-/-) mice. Correspondingly, OPG small interfering RNA induced the expressions of TRAIL and cleaved caspase-3 in cultured cardiac myocytes. In addition, these mice revealed a decrease in interstitial fibrosis, activation of matrix metalloproteinase (MMP)-2 and tissue inhibitors of MMP-1 and -2, and inactivation of procollagen α1 synthesis. Moreover, intraperitoneal administration of recombinant OPG to either 2.5- or 12-month-old OPG(-/-) mice for 28 days led to partial improvement of LV structure and function without affecting systolic blood pressure. CONCLUSION: These results suggest that OPG plays a role in preserving myocardial structure and function with ageing through a reduction in apoptosis and preservation of the matrix structure. In addition, this appears to be independent of effects on the vasculature.

Influence of leg-length discrepancy on anterior acetabular coverage using false profile image.

BACKGROUND: Leg-length discrepancy (LLD) occurs commonly in patients with osteoarthritis (OA) of the hip. Although some investigators argue that LLD in a weight-bearing position may influence lateral acetabular coverage, there have been no reports on the influence of LLD on anterior acetabular coverage and the relationship between LLD and vertical center anterior margin (VCA) angle before and after LLD correction. Anterior acetabular coverage is an important index for diagnosis, treatment, and surgery for OA of the hip. Therefore, we investigated the influence of LLD in a weight-bearing position on VCA angle. METHODS: There were 154 patients with LLD in OA of the hip and 146 healthy individuals without LLD. The sole of the short-leg side in patients was adjusted with an acrylic plate, and the LLD revision value was calculated in the anteroposterior (AP) view in a weight-bearing position. Calculated revision value was applied to individuals and VCA angles in false profile images before and after correction was measured. For healthy individuals, we corrected the sole of the nonexamined side with an acrylic plate to artificially increase LLD and then measured VCA angles in false profile images before and after correction. RESULTS: Significant difference was found in VCA angles between before and after LLD correction in patients and healthy individuals (p < 0.05). Difference in VCA angles before and after LLD correction in both patients and health individuals highly correlated with LLD level in both short- and long-leg sides. CONCLUSIONS: This study clarified that LLD in a weight-bearing position influenced VCA angle. Results suggested that comparison of images before and after correction increases diagnostic accuracy. Assessing anterior acetabular coverage before and after LLD correction is valuable in evaluating the need for surgery, suitable correction of osteotomized acetabular fragments in periacetabular osteotomy, and determining acetabular cup angle in artificial joint replacement.

Segmental copy number loss in the region of Semaphorin 4D gene in patients with acetabular dysplasia.

Acetabular dysplasia (AD) appears to be a multi-factorial disease, which may involve both genetic and environmental factors and whose pathogenesis remains obscure. The present study aims to identify a genetic variation that might confer risk of AD. We performed whole-genome screening of a copy number variation (CNV) using a deCODE-Illumina CNV beadchip with 20 female AD patients and 131 control subjects. Subsequently, Agilent's region-targeted high-density oligonucleotide tiling microarray was used to analyze 64 female AD patients and 32 female control subjects. By sequential analyses, we found a copy number loss in 18 of 64 AD patients, but none in the 32 controls. The loss occurred within a 472 kb region on 9q22.2, which harbors the gene for Semaphorin 4D (Sema4D; 18/64 vs. 0/32, p = 4.81 × 10(-4) , OR = 25.86). We suggest that a copy number loss of the Sema4D gene region may play a role in the etiology of AD.

Effect of genu recurvatum on the anterior cruciate ligament-deficient knee during gait.

PURPOSE: This study aimed to investigate the effects of genu recurvatum, which is considered to carry a high risk for anterior cruciate ligament (ACL) injury, on healthy and post-ACL injury gait and lower extremity muscle strength. METHODS: Subjects were 36 patients with ACL-deficient knee and 40 healthy controls without pain or restricted range of motion of the lower extremity during gait. The knee joints of all subjects were examined; those with over 10° hyperextension of both knees were defined as exhibiting genu recurvatum. On this basis, the subjects were further subdivided into two groups: with or without genu recurvatum. A three-dimensional motion analysis system and force plates were used for gait analysis. Isokinetic dynamometers were used to measure knee muscle strength. RESULTS: There were no differences in joint angles, joint moments, or components of ground reaction force during gait or in knee strength for the healthy control subjects with and without genu recurvatum. ACL-deficient subjects without genu recurvatum showed a decrease in knee angles during the stance phase and a decrease in extension moments during the early stance phase compared with ACL-deficient subjects with genu recurvatum and controls. In contrast, neither knee angles nor extension moments during the stance phase differed significantly between ACL-deficient subjects with genu recurvatum and controls. CONCLUSIONS: This study provides clinically relevant information regarding the effects of genu recurvatum on gait parameters. The results suggest that in ACL injuries, the presence of genu recurvatum alters gait pattern. Consideration of the presence of genu recurvatum would be useful during rehabilitation following ACL injuries or ACL reconstruction. LEVEL OF EVIDENCE: II. Prospective comparative study.

Roles of the endoplasmic reticulum stress transducer OASIS in fracture healing.

A new type of endoplasmic reticulum (ER) stress transducer, Old Astrocyte Specifically Induced Substance (OASIS), which is induced by bone morphogenetic protein-2 (BMP2), has been reported to activate the transcription of type I collagen and contribute to the secretion of bone matrix proteins in osteoblasts. Here, we examined the role of OASIS in fracture healing using the fracture models in wild-type (WT) and OASIS(-/-) mice. We found that the expression of OASIS mRNA was induced after fracture. Micro-computed tomography indicated that the callus density of OASIS(-/-) mice was less than that of WT mice, and the newly formed bone in OASIS(-/-) mice exhibited a decrease of the bone volume by bone morphometric analysis. Biomechanically, the callus bone strength of OASIS(-/-) mice was inferior to that of WT mice. Based on RT-PCR, in situ hybridization, immunohistochemical, and electrophoretic analyses, it was clarified that the synthesis of type I collagen was impaired in OASIS(-/-) mice. Electron microscopic analysis revealed that OASIS(-/-) osteoblasts in the fracture callus contained the abnormal expansion of the ERs, similar to OASIS(-/-) osteoblasts in the normal skeletal development. Thus, OASIS may play a role in bone formation through the expression of type I collagen and the secretion of bone matrix proteins in fracture healing.

Plasma adrenomedullin and proadrenomedullin N-terminal 20 peptide in patients diagnosed as having early rheumatoid arthritis.

The aim of this study was to investigate plasma adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) level in patients diagnosed with early rheumatoid arthritis (RA). Furthermore, several inflammatory cytokines were measured in those patients to clarify the roles of AM and PAMP. Forty patients diagnosed with early RA (women 46 +/- 8.5 years old) and 10 healthy controls (women 57 +/- 5 years old) were studied. Plasma levels of AM, PAMP, matrix metalloprotease 3 (MMP-3), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and C-reactive protein (CRP) were measured using an immunoradiometric assay and enzyme-linked immunosorbent asay (ELISA) methods. The plasma levels of AM (17.5 +/- 8.4 fmol/ml) and PAMP (2.01 +/- 0.57 fmol/ml) in patients exceeded those in healthy controls (AM 8.6 +/- 1.7, PAMP 1.17 +/- 0.34 fmol/ml). Moreover, plasma AM and PAMP levels demonstrated a significantly positive correlation with plasma MMP-3 and IL-6 levels. Nevertheless, CRP and TNF-alpha levels in these patients showed no significant correlation with plasma AM and PAMP levels. These data support the possible role for AM and PAMP in the pathophysiology of early RA.

Regulation of ER molecular chaperone prevents bone loss in a murine model for osteoporosis.

Endoplasmic reticulum (ER) stress response is important for protein maturation in the ER. Some murine models for bone diseases have provided significant insight into the possibility that pathogenesis of osteoporosis is related to ER stress response of osteoblasts. We examined a possible correlation between osteoporosis and ER stress response. Bone specimens from 8 osteoporosis patients and 8 disease-controls were used for immunohistochemical analysis. We found that ER molecular chaperones, such as BiP (immunoglobulin heavy-chain binding protein) and PDI (protein-disulfide isomerase) are down-regulated in osteoblasts from osteoporosis patients. Based on this result, we hypothesized that up-regulation of ER molecular chaperones in osteoblasts could restore decreased bone formation in osteoporosis. Therefore, we investigated whether treatment of murine model for osteoporosis with BIX (BiP inducer X), selective inducer BiP, could prevent bone loss. We found that oral administration of BIX effectively improves decline in bone formation through the activation of folding and secretion of bone matrix proteins. Considering these results together, BIX may be a potential therapeutic agent for the prevention of bone loss in osteoporosis patients.

Therapeutic efficacy of intra-articular adrenomedullin injection in antigen-induced arthritis in rabbits.

INTRODUCTION: Adrenomedullin is a potent vasodilatory and hypotensive peptide as well as an endogenous immunomodulatory factor with predominantly anti-inflammatory effects. The purpose of the present study was to evaluate the therapeutic effects of adrenomedullin in rabbits with antigen-induced arthritis, an experimental model of rheumatoid arthritis. METHODS: Following the induction of arthritis in both knee joints by ovalbumin injection into the joint spaces of pre-immunized rabbits, increasing daily doses of adrenomedullin were injected into the knee joint spaces or saline was injected into the contralateral knee joint spaces as the control. For time-course experiments, adrenomedullin and saline were injected into the knee joint spaces daily for 7 days and 20 days. The degree of joint swelling and the histological change in the knee joints injected with adrenomedullin were compared with the control knee joints. Histological evaluation of the infrapatellar fat pads and synovial tissue was performed. TNFalpha, IL-6, vascular endothelial growth factor and transforming growth factor-beta mRNA levels in the synovial tissue were measured using real-time quantitative PCR. RESULTS: Daily injections of adrenomedullin into the knee joint spaces of rabbits with antigen-induced arthritis decreased joint swelling. Histological examination revealed that adrenomedullin reduced edematous changes and the infiltration of inflammatory cells in the synovial tissues. Analysis of mRNA levels showed that adrenomedullin significantly reduced TNFalpha mRNA expression by 21% to 49% in a dose-dependent manner, and dose-dependently increased IL-6 mRNA expression by 45% to 121%. CONCLUSIONS: These results suggest that daily injections of adrenomedullin into the knee joint spaces of rabbits with antigen-induced arthritis ameliorated the inflammatory response in arthritic joints. Adrenomedullin may thus be useful as a treatment for rheumatoid arthritis; however, the effect of adrenomedullin on IL-6 production in the synovial tissue may be an undesirable adverse effect in rheumatoid arthritis therapy.

Characterization of an exchangeable gene trap using pU-17 carrying a stop codon-beta geo cassette.

We have developed a new exchangeable gene trap vector, pU-17, carrying the intron-lox71-splicing acceptor (SA)-beta geo-loxP-pA-lox2272-pSP73-lox511. The SA contains three stop codons in-frame with the ATG of beta galactosidase/neomycin-resistance fusion gene (beta geo) 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 beta geo 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 beta geo 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 beta geo gene with any gene of interest to be expressed under the control of the trapped promoter through Cre-mediated recombination.

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.

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.

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.