Preoperative ultrasonographic evaluation of the contralateral patent processus vaginalis at the level of the internal inguinal ring is useful for predicting contralateral inguinal hernias in children: a prospective analysis.

PURPOSE: The current study aimed to verify the usefulness of preoperative ultrasonographic evaluation of contralateral patent processus vaginalis (PPV) at the level of the internal inguinal ring. METHODS: This was a prospective study of patients undergoing unilateral inguinal hernia repair at two institutions during 2010-2011. The sex, age at initial operation, birth weight, initial operation side, and the preoperative diameter of the contralateral PPV as determined using ultrasonography (US) were recorded. We analyzed the incidence of contralateral inguinal hernia, risk factors, and the usefulness of the preoperative major diameter of the contralateral PPV. The follow-up period was 36 months. RESULTS: All 105 patients who underwent unilateral hernia repair completed 36 months of follow-up, during which 11 patients (10.5 %) developed a contralateral hernia. The following covariates were not associated with contralateral hernia development: sex (p = 0.350), age (p = 0.185), birth weight (p = 0.939), and initial operation side (p = 0.350). The preoperative major diameter of the contralateral PPV determined using US was significantly wider among patients with a contralateral hernia than those without a contralateral hernia (p = 0.001). When the 105 patients were divided into two groups according to cut-off values of the preoperative major diameter of the contralateral PPV (wide group, >2.0 mm; narrow group, ≤2.0 mm), a significant association was observed between the preoperative major diameter of the contralateral PPV and patient outcomes (p = 0.001). CONCLUSIONS: We used US and confirmed the usefulness of a preoperative evaluation of the major diameter of the contralateral PPV at the level of the internal inguinal ring in pediatric patients with unilateral inguinal hernias.

Prediction of contralateral inguinal hernias in children: a prospective study of 357 unilateral inguinal hernias.

PURPOSE: Previously, we established a pre-operative risk scoring system to predict contralateral inguinal hernia in children with unilateral inguinal hernias. The current study aimed to verify the usefulness of our pre-operative scoring system. METHODS: This was a prospective study of patients undergoing unilateral inguinal hernia repair from 2006 to 2009 at a single institution. Gender, age at initial operation, birth weight, initial operation side, and the pre-operative risk score were recorded. We analyzed the incidence of contralateral inguinal hernia, risk factors, and the usefulness of our pre-operative risk scoring system. The follow-up period was 36 months. We used forward multiple logistic regression analysis to predict contralateral hernia. RESULTS: Of the 372 patients who underwent unilateral hernia repair, 357 (96.0 %) were completely followed-up for 36 months, and 23 patients (6.4 %) developed a contralateral hernia. Left-sided hernia (OR = 5.5, 95 %, CI = 1.3-24.3, p = 0.023) was associated with an increased risk of contralateral hernia. The following covariates were not associated with contralateral hernia development: gender (p = 0.702), age (p = 0.215), and birth weight (p = 0.301). The pre-operative risk score (cut-off point = 4.5) of the patients with a contralateral hernia was significantly higher, compared with the patients without a contralateral hernia using the area under the receiver operating characteristic curve (p = 0.024). CONCLUSIONS: Using multivariate analysis, we confirmed usefulness of our pre-operative scoring system and initial side of the inguinal hernia, together, for the prediction of contralateral inguinal hernia in children.

Role of stem cells in cardiovascular biology.

This review article addresses the controversy as to whether the adult heart possesses an intrinsic growth reserve. If myocyte renewal takes place in healthy and diseased organs, the reconstitution of the damaged tissue lost upon pathological insults might be achieved by enhancing a natural occurring process. Evidence in support of the old and new view of cardiac biology is critically discussed in an attempt to understand whether the heart is a static or dynamic organ. According to the traditional concept, the heart exerts its function until death of the organism with the same or lesser number of cells that are present at birth. This paradigm was challenged by documentation of the cell cycle activation and nuclear and cellular division in a subset of myocytes. These observations raised the important question of the origin of replicating myocytes. Several theories have been proposed and are presented in this review article. Newly formed myocytes may derive from a pre-existing pool of cells that has maintained the ability to divide. Alternatively, myocytes may be generated by activation and commitment of resident cardiac stem cells or by migration of progenitor cells from distant organs. In all cases, parenchymal cell turnover throughout lifespan results in a heterogeneous population consisting of young, adult, and senescent myocytes. With time, accumulation of old myocytes has detrimental effects on cardiac performance and may cause the development of an aging myopathy.

Induction of immunological tolerance by oral, but not intravenous and intraportal, administration of ovalbumin and the difference between young and old mice.

BACKGROUND: Induction of immunological tolerance is dependent on the route of antigenic administration, the dose of an antigen and the age of animals. OBJECTIVES: We investigated the effect of age on the tolerance induction in mice by administration of antigen through different routes and at different doses. DESIGN: Young and old BDF1 mice were orally, intraportally or intravenously administrated with a low or a high dose of ovalbumin (OVA). Then, delayed-type hypersensitivity (DTH) responses and serum anti-OVA antibody levels were assessed after systemic immunization of OVA with alum after appropriate intervals. RESULTS: In the young mice, oral administration of OVA suppressed DTH response and anti-OVA IgG1, IgG2b, IgM and IgE level in a dose-dependent manner. In the old mice, however, the suppression of IgG1 and IgE levels was induced by oral administration of a low dose of OVA, but no suppression by a high dose. On the other hand, intraportal or intravenous injection of OVA did not suppress DTH response and enhanced anti-OVA antibody levels in a dose-dependent manner in both young and old mice. Production of anti-OVA IgG2a antibody after systemic injection of OVA was detected in the mice, which had been treated with intraportal or intravenous injection of OVA, but not detected in the mice, which had been treated with oral administration of OVA. On the contrary, suppression of anti-OVA IgE antibody was observed only in the mice, which had been treated with oral administration of OVA. CONCLUSION: The oral administration of OVA, neither intravenous nor intraportal, induced immunological tolerance to OVA. An adequate dose of OVA for the tolerance induction and the suppression of antibody production are different between young and old mice. The suppression of IgE antibody was observed only by oral administration of OVA, much obviously in young mice than in the old. The results also indicated that the antigen processing in the liver did not play a major role in the induction of oral tolerance to OVA.

Independent nonframeshift deletions in the MC1R gene are not associated with melanistic coat coloration in three mustelid lineages.

Sequence variation within the 5' flanking (about 240 bp) and exon regions (426 bp) of the melanocortin-1 receptor (MC1R) gene was examined to determine the potential role of this protein in the melanistic coat coloration of 17 mustelid species in four genera: Gulo (wolverines), Martes (martens), Mustela (weasels), and Meles (badgers). Members of the genera Mustela and Meles, together with Martes flavigula and Martes pennanti, were shown to have intact gene sequences. However, several "in frame" deletions of the MC1R gene region implicated in melanism of other species were detected within members of the genera Martes and Gulo. For instance, Gulo gulo possessed a 15 bp deletion in the second transmembrane domain coding region, while Martes americana, Martes melampus, Martes zibellina, and Martes martes shared a 45 bp deletion overlapping this area. In addition, Martes foina was found to possess a 10 bp insertion followed closely by a 28 bp deletion immediately downstream of the deletion found in other martens. Notably, none of these indels was associated with a melanistic phenotype. Phylogenetic analysis revealed that each of these nonrandomly distributed deletions arose independently during the evolution of this family. Specific indel-neighboring motifs appear to largely account for the biased and repeated occurrence of deletion events in the Martes/Gulo clade.

Analysis of the effect of leptin on immune function in vivo using diet-induced obese mice.

Leptin can regulate several immune functions. However, the role of leptin on lymphocyte function has not been recognized in vivo. Accordingly, we have investigated the effect of leptin on starvation-induced immune dysfunction using diet-induced obese mice. To induce obesity, C57BL/6J mice were fed a high-fat diet for 14 weeks and control mice were fed a standard diet for the same period. The obese and control groups of mice were then starved for 48 h, and received intraperitoneal injections of recombinant leptin or phosphate-buffered saline four times during starvation. Other control mice in both diet groups were free fed without being starved. Although starvation of the control mice dramatically reduced the weights of the immune organs, cytokine production and increased proliferation of cultured splenocytes, these levels returned to those of the free-feeding groups with exogenous leptin administration. However, these effects of leptin were not observed in obese mice. These findings provide some evidence that leptin can regulate the immune function in vivo. It is also suggested that the action of leptin might not appear in obesity.

Haemoglobin biosynthesis site in rabbit embryo erythroid cells

Properly metabolized globin synthesis and iron uptake are indispensable for erythroid cell differentiation and maturation. Mitochondrial participation is crucial in the process of haeme synthesis for cytochromes and haemoglobin. We studied the final biosynthesis site of haemoglobin using an ultrastructural approach, with erythroid cells obtained from rabbit embryos, in order to compare these results with those of animals treated with saponine or phenylhydrazine. Our results are similar to those obtained in assays with adult mammals, birds, amphibians, reptiles and fish, after induction of haemolytic anaemia. Therefore, the treatment did not interfere with the process studied, confirming our previous findings. Immunoelectron microscopy showed no labelling of mitochondria or other cellular organelles supposedly involved in the final biosynthesis of haemoglobin molecules, suggesting instead that it occurs free in the cytoplasm immediately after the liberation of haeme from the mitochondria, by electrostatic attraction between haeme and globin chains.

Effect of obesity and insulin on immunity in non-insulin-dependent diabetes mellitus.

OBJECTIVE: To understand the effect of obesity and insulin on immune functions in non-insulin-dependent diabetes mellitus (NIDDM). SUBJECT: Fourteen obese NIDDM (body mass index (BMI)=30.6+/-1.1), seven non-obese NIDDM (BMI=24.2+/-0.5) and five obese non-NIDDM (BMI=28.3+/-0.67). INTERVENTIONS: We first examined the influence of insulin on the proliferation of several human cell lines. Second, we compared several immune functions between obese and non-obese NIDDM, and obese non-NIDDM patients using peripheral blood mononuclear cells. RESULT: Insulin decreased proliferation of T-cell lines but not that of other types of cell lines. Furthermore, obesity augmented the production of IL-1beta which could have cytotoxity against islet beta cells in NIDDM. CONCLUSION: Our data suggested that the pathophysiology of NIDDM could be affected by the change of immunity due to obesity, and the treatment of obesity in NIDDM may be important from an immunological aspect.

Estrogen protects neuronal cells from amyloid beta-induced apoptotic cell death.

Accumulating studies have shown that estrogen replacement therapy reduces the risk of Alzheimer's disease. In this study, we clarified that 17beta-estradiol (E2) significantly rescues PC12 neuronal cells from amyloid beta protein (Abeta)-induced cell death. We found that the amino acid residues of 25 to 35 (Abeta25-35) were more cytotoxic than the full length protein (Abeta1-40) and these residues induced DNA fragmentation typical for apopto- sis. In addition, E2 was confirmed to inhibit calcium influx and cytochrome c release induced by Abeta25-35. Since these sequential events cause apoptosis, the protective effect of E2 may be exerted not by the direct interaction with Abeta, but by the blockade of the mitochondrial apoptotic pathway induced by Abeta.

Alzheimer's disease and estrogen.

The preventive effect of estrogen on Alzheimer's disease (AD) has become clear with epidemiological data. Therapeutic effects of estrogen have not yet been established. In this presentation, we report our new basic and clinical data. The estrogen receptor, (ER)alpha, and ERbeta mRNA were investigated in rat brain. Estradiol-17beta (E(2)) treatment following OVX reduced the levels of ERalpha mRNA in the hypothalamus. In the substantia innominata (SI), the number of choline acetyltransferase immunoreacive cells increased significantly in the estrogen treatment rat. The neurons in SI projecting to the forebrain cortex contained ERalpha. Increasing amounts of intracellular calcium, peroxidation, and apoptosis with amyloid beta were suppressed in neuronal cells from rat pheochromocytoma (PC12) cells with E(2). ERalpha cDNA transfected PC 12 cells elaborated more neurite-like processes with E(2). In clinics, we are currently preparing vaginal progesterone tablets, which essentially may concentrate in the endometrium to prevent endometrial cancer, with few general circulation of progesterone inviting less depression. The therapeutic effects of cyclic estrogen, such as its preventive effect, are suggested in these studies, at least on mild AD.

A novel myocyte-specific gene Midori promotes the differentiation of P19CL6 cells into cardiomyocytes.

Although several cardiac-specific transcription factors have been shown to play vital roles in various steps during the heart formation, the precise mechanism of the early stage of cardiogenesis has yet to be elucidated. By differential display technique, we tried to identify molecules that are expressed earlier than cardiac transcription factors such as CSX/NKX2-5 and GATA-4 and are involved in cardiomyocyte differentiation using the P19CL6 cell line, which efficiently differentiates into cardiomyocytes when treated with dimethyl sulfoxide. We isolated a novel gene designated Midori. Its deduced amino acid sequence contained an ATP/GTP-binding site, Ig-like domain, and Kringle-like domain. Northern blot analysis revealed that expression of Midori was restricted to the fetal and adult heart and adult skeletal muscle in mice. In whole mount in situ hybridization, Midori was expressed in cardiac crescent and developing heart but not in somites. The MIDORI protein was localized in the nucleus and overexpression of Midori induced expression of endogenous Midori itself, suggesting that MIDORI may act as a transcriptional regulator. Permanent P19CL6 cell lines overexpressing Midori more efficiently differentiated into cardiomyocytes than did parental cells, whereas those overexpressing the antisense Midori less efficiently differentiated. These results suggest that Midori may promote the differentiation of P19CL6 into cardiomyocytes.

Smads, TAK1, and their common target ATF-2 play a critical role in cardiomyocyte differentiation.

We previously demonstrated that bone morphogenetic proteins (BMPs) induce cardiomyocyte differentiation through the mitogen-activated protein kinase kinase kinase TAK1. Transcription factors Smads mediate transforming growth factor-beta signaling and the ATF/CREB family transcription factor ATF-2 has recently been shown to act as a common target of the Smad and the TAK1 pathways. We here examined the role of Smads and ATF-2 in cardiomyocyte differentiation of P19CL6, a clonal derivative of murine P19 cells. Although P19CL6 efficiently differentiates into cardiomyocytes when treated with dimethyl sulfoxide, P19CL6noggin, a P19CL6 cell line constitutively overexpressing the BMP antagonist noggin, did not differentiate into cardiomyocytes. Cooverexpression of Smad1, a ligand-specific Smad, and Smad4, a common Smad, restored the ability of P19CL6noggin to differentiate into cardiomyocytes, whereas stable overexpression of Smad6, an inhibitory Smad, completely blocked differentiation of P19CL6, suggesting that the Smad pathway is necessary for cardiomyocyte differentiation. ATF-2 stimulated the betaMHC promoter activity by the synergistic manner with Smad1/4 and TAK1 and promoted terminal cardiomyocyte differentiation of P19CL6noggin, whereas overexpression of the dominant negative form of ATF-2 reduced the promoter activities of several cardiac-specific genes and inhibited differentiation of P19CL6. These results suggest that Smads, TAK1, and their common target ATF-2 cooperatively play a critical role in cardiomyocyte differentiation.

[Study on the effects of walking on functional fitness in aged patients with the hand-arm vibration syndrome].

PURPOSE: This article reports the effectiveness of mild walking exercise for maintaining functional fitness in aged patients with the hand-arm vibration syndrome (HAVS). SUBJECTS AND METHODS: Fifty-two patients suffering from vibration syndrome, for which they received annual compulsory examination from December 1998 to March 1999 at the San-in Rosai Hospital, were examined. They all were male, with a mean age (standard deviation) of 69.1 (7.3) years, and were randomly allocated to an intervention group (N = 26) and a control group (N = 26). The goal of the intervention was to achieve and maintain at least 30 minutes of walking a day. Functional fitness was assessed by a sitting and standing test, a zigzag walking test, a hand working test with a pegboard for dexterity, and a self-care working test proposed by the Physical Fitness Research Institute, Meiji Life Foundation of Health and Welfare. RESULTS: There were no significant differences between the two groups regarding baseline characteristics. The proportions of subjects with regular exercise habits after the intervention were 84.6% (22/26) and 53.8% (14/26) in the intervention and control groups, respectively, the difference being significant. The total scores for functional fitness were improved in the intervention, while decline was noted in the control group. Sitting and standing and self-care working ability were also improved in the intervention group as against the deterioration with age in the control group. CONCLUSIONS: Our findings show to some extent that intervention using mild exercise, walking for 30 min, is effective for aged patients with HAVS to maintain and improve functional fitness.

Change of cytokine balance in diet-induced obese mice.

Although decreased T-cell function has been observed in obese human subjects and genetically obese animals, the precise role of immune functions in obesity is still unclear. To investigate immune functions in obesity, we examined the proliferative responses of splenic lymphocytes and their capacity to produce cytokines in the presence or absence of leptin, the protein produced by the obese gene, in diet-induced obese and control mice. For induction of obesity, C57BL/6J mice were fed a high-fat diet for 13 weeks. In mice fed the high-fat diet, body weight, fat pad weight, and tumor necrosis factor (TNF) alpha production by adipocytes were significantly increased relative to mice fed the normal diet. Lipopolysaccharide (LPS) stimulated proliferation of cultured splenocytes from diet-induced obese mice was also increased. However, production of interleukin (IL)-2 by splenic lymphocytes from obese mice was suppressed, whereas interferon (IFN)-gamma and IL-4 production was increased. Exogenous lepitn regulated the cytokine production by cultured splenocytes from control and obese mice, respectively (upregulation of IFN-gamma and downregulation of IL-2 in control mice, and downregulation of IL-4 in obese mice). These results suggest that changes in cytokine production by splenic lymphocytes in obesity are indicative of altered immune functions that might contribute to related complications, although the effect of difference in nutrient intake (macro and micro) may also have contributed to the changes.

Cloning of murine glycosyl phosphatidylinositol anchor attachment protein, GPAA1.

Glycosyl phosphatidylinositols (GPIs) are used to anchor many proteins to the cell surface membrane and are utilized in all eukaryotic cells. GPI anchoring units are attached to proteins via a transamidase reaction mediated by a GPI transamidase complex. We isolated one of the components of this complex, mGPAA1 (murine GPI anchor attachment), by the signal sequence trap method. mGPAA1 cDNA is about 2 kb in length and encodes a putative 621 amino acid protein. The mGPAA1 gene has 12 small exons and 11 small introns. mGPAA1 mRNA is ubiquitously expressed in mammalian cells, and in situ hybridization analysis revealed that it is abundant in the choroid plexus, skeletal muscle, osteoblasts of rib, and occipital bone in mouse embryos. Its expression levels and transamidation efficiency decreased with differentiation of embryonic stem cells. The 3T3 cell lines expressing antisense mGPAA1 failed to express GPI-anchored proteins on the cell surface membrane.

Determination of the complete nucleotide sequence and haplotypes in the D-loop region of the mitochondrial genome in the oriental white stork, Ciconia boyciana.

The complete nucleotide sequence of the mitochondrial genome of the Oriental white stork, Ciconia boyciana, has been determined from captive storks by a novel method incorporating Long PCR and shotgun sequencing. 13 protein-coding genes, two ribosomal RNA genes and 22 transfer RNA genes were identified as in other vertebrate mitochondrial genomes. The position and direction of the NADH6 and tRNA-Glu genes were the same as previously reported for avian mitochondrial genomes. A 71 bp direct repeat and long CAAA repeat sequences were found at the 3' end of the D-loop region, together with SCB-1, SCB-2, SCB-3, and three TAS sequences. Direct sequencing of the PCR fragments in the D-loop region in 26 captive Oriental white storks originating from Japan, China, and Russia revealed nucleotide differences at 18 sites along 1,248 bp, and a total of nine haplotypes have been identified. It was found that one pair of individuals in the Japanese captive breeding program were of the same haplotype, suggesting that they were caught from the same nest. The pair has since been dissolved in consideration of the possibility of inbreeding depression.

Atheroprotective effect of estriol and estrone sulfate on human vascular smooth muscle cells.

In patients with atherosclerosis, fibrosclerotic focuses are induced by multiplication of vascular smooth muscle cells (VSMC), and they are regulated by cytokines and regulators. There have been few reports about the atheroprotective effect of estriol (E(3)). Estrone sulfate (E(1)-S) is the predominant estrogen of conjugated equiline estrogens, which is commonly used in hormone replacement therapy, but it should be hydrolyzed by steroid sulfatase (STS) to enter the cells of target tissues. The purpose of this study was to detect STS in VSMC and to investigate whether E(3) and E(1)-S have atheroprotective effects like E(2). First, we detected the presence of STS mRNA in VSMC by in situ hybridization. We then examined the changes in the expression of mRNAs of cytokines, namely, PDGF-A chain, IL-1, IL-6 and TGF-beta, in VSMC, in the presence and absence of E(3) and estrogens. As a result, the expression of PDGF-A chain, IL-1 and IL-6 mRNAs was suppressed by E(3) (P<0.05 vs control) significantly like E(1)-S and E(2), but that of TGF-beta mRNA was not significantly affected by any estrogen. These results indicate that E(1)-S can be hydrolyzed by STS in VSMC, and that E(3) may regulate the cytokines by suppressing the production of mRNAs. It is suggested that there is a possibility of E(1)-S and E(3) having a direct effect on vessels in atherogenesis.

Evolutionary trends of the mitochondrial lineage differentiation in species of genera Martes and Mustela.

We compared partial sequences (402 bp) of the mitochondrial cytochrome b gene in 68 individuals of martens (Martes), weasels (Mustela) and their relatives from the Northern Hemisphere to identify the modes of geographic differentiation in each species. We then compared complete sequences (1140 bp) of the gene in 17 species of the family Mustelidae to know the spatial and temporal modes of speciation, constructing linearized trees with transversional substitutions for deeper lineage divergences and with transversions and transitions for younger lineages. Our data suggested that these lineages of Martes and Mustela differentiated in a stepwise fashion with five radiation stages from the generic divergences (stage I) to the intraspecific divergences (stage V), during the last 10 or 20 million years as the fossil evidence suggests. In the lineage of Martes, the first offshoots are of Martes flavigula, M. pennanti, and Gulo gulo (stage II), the second is M. foina (stage III), and the third are M. americana, M. martes, M. melampus, and M. zibellina (stage IV). The divergence of the lineages of Mustela is likely to have taken place concurrently with the radiations of the Martes. These divergence processes are attributable in part to the geographic allocation along the two continents, North America and Eurasia, as well as among peripheral insular domains, such as Taiwan and the Japanese Islands. In addition, the Eurasian continent itself was shown to have been involved in the species diversification in the martens and weasels.

Differential age effect of oral administration of an antigen on antibody response: an induction of tolerance in young mice but enhancement of immune response in old mice.

Sheep red blood cells (SRBC) were orally administered to young (4 months old) and old (22 months old) mice, and its effect on the antibody production after systemic immunization was compared between young and old mice. The results showed that the dose-dependent suppression of antibody response (oral tolerance) was observed in young mice which had been previously treated with oral administration of SRBC. On the contrary, the enhancement of antibody production was observed in old mice which had been treated in the same way. The enhanced level of IgG antibody in old mice was higher than that of young mice. The critical age determining either suppression or enhancement of antibody response after the oral administration of the antigen was present between 6.5 and 10.5 months of age. When the oral administration of the antigen was performed in young (3 months old) and middle-aged mice (12 months old), the oral tolerance for young and the enhanced antibody response for middle-aged mice were observed even at 6 months after the treatment. The analysis by in vitro antibody response using T and B cells prepared from young and old mice showed that age-related alteration of T and B cells is responsible for the suppression and the enhancement of antibody response after oral administration of SRBC, respectively.

Bone morphogenetic proteins induce cardiomyocyte differentiation through the mitogen-activated protein kinase kinase kinase TAK1 and cardiac transcription factors Csx/Nkx-2.5 and GATA-4.

Bone morphogenetic proteins (BMPs) have been shown to induce ectopic expression of cardiac transcription factors and beating cardiomyocytes in nonprecardiac mesodermal cells in chicks, suggesting that BMPs are inductive signaling molecules that participate in the development of the heart. However, the precise molecular mechanisms by which BMPs regulate cardiac development are largely unknown. In the present study, we examined the molecular mechanisms by which BMPs induce cardiac differentiation by using the P19CL6 in vitro cardiomyocyte differentiation system, a clonal derivative of P19 embryonic teratocarcinoma cells. We established a permanent P19CL6 cell line, P19CL6noggin, which constitutively overexpresses the BMP antagonist noggin. Although almost all parental P19CL6 cells differentiate into beating cardiomyocytes when treated with 1% dimethyl sulfoxide, P19CL6noggin cells did not differentiate into beating cardiomyocytes nor did they express cardiac transcription factors or contractile protein genes. The failure of differentiation was rescued by overexpression of BMP-2 or addition of BMP protein to the culture media, indicating that BMPs were indispensable for cardiomyocyte differentiation in this system. Overexpression of TAK1, a member of the mitogen-activated protein kinase kinase kinase superfamily which transduces BMP signaling, restored the ability of P19CL6noggin cells to differentiate into cardiomyocytes and concomitantly express cardiac genes, whereas overexpression of the dominant negative form of TAK1 in parental P19CL6 cells inhibited cardiomyocyte differentiation. Overexpression of both cardiac transcription factors Csx/Nkx-2.5 and GATA-4 but not of Csx/Nkx-2.5 or GATA-4 alone also induced differentiation of P19CL6noggin cells into cardiomyocytes. These results suggest that TAK1, Csx/Nkx-2.5, and GATA-4 play a pivotal role in the cardiogenic BMP signaling pathway.