Deletion of thyrotropin receptor residue Asp403 in a hyperfunctioning thyroid nodule provides insight into the role of the ectodomain in ligand-induced receptor activation.

Somatic mutations of the TSH receptor (TSHR) gene are the main cause of autonomously functioning thyroid nodules. Except for mutations in ectodomain residue S281, all of the numerous reported activating mutations are in the TSHR membrane-spanning region. Here, we describe a patient with a toxic adenoma with a novel heterozygous somatic mutation caused by deletion of ectodomain residue Asp403 (Del-D403). Subsequent in vitro functional studies of the Del-D403 TSHR mutation demonstrated greatly increased ligand-independent constitutive activity, 8-fold above that of the wild-type TSHR. TSH stimulation had little further effect, indicating that the mutation produced near maximal activation of the receptor. In summary, we report only the second TSHR ectodomain activating mutation (and the first ectodomain deletion mutation) responsible for development of a thyroid toxic adenoma. Because Del-D403 causes near maximal activation, our finding provides novel insight into TSHR structure and function; residue D403 is more likely to be involved in the ligand-mediated activating pathway than in the ectodomain inverse agonist property.

The effects of seasonal and interannual variability of oceanic structure in the western Pacific North Equatorial Current on larval transport of the Japanese eel Anguilla japonica.

As the North Equatorial Current (NEC)-bifurcation is known to be related to El Niño-Southern Oscillation (ENSO) events, the influence of the position of the NEC bifurcation on transport success of the larval Japanese eel Anguilla japonica was investigated. Using a Lagrangian modelling approach, larval transport was simulated and the relative influence of El Niño and La Niña events and the NEC-bifurcation position on the success of particle transport analysed. The number of particles transported from the NEC to the Kuroshio tended to be lowest during El Niño years, and differences between La Niña and regular years were small. The transport success observed in simulations showed some relationships to annual A. japonica glass eel recruitment to Tanegashima Island over 1993 to 2001, but not in 2002. The study shows that particle tracking simulations can be used to improve knowledge of the oceanic migration of A. japonica but further studies are required, including comparisons with the effects on larval survival of fluctuations in temperature and food availability.

SoxD: an essential mediator of induction of anterior neural tissues in Xenopus embryos.

Vertebrate neurogenesis is initiated by the organizer factors that inhibit antineuralizing activities of bone morphogenetic proteins (BMPs) in the ectoderm. Here, we report a candidate mediator of neuralization, SoxD. Expression of SoxD starts at late blastula stages widely in the prospective ectoderm and becomes restricted to the dorsal ectoderm by mid-gastrula stages. SoxD expression is enhanced by the neural inducer Chordin and is suppressed by BMP4 and its downstream genes. Microinjection of SoxD mRNA causes ectopic formation of neural tissues in vivo and induces neural and neuronal markers in the isolated animal cap. Injection of a dominant-negative form of SoxD mRNA can block neuralization of ectoderm caused by attenuation of BMP signals and can strongly suppress formation of anterior neural tissues in vivo. These data show that SoxD functions as an essential mediator of downstream signaling of neural induction.

Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity.

We have identified a stromal cell-derived inducing activity (SDIA) that promotes neural differentiation of mouse ES cells. SDIA accumulates on the surface of PA6 stromal cells and induces efficient neuronal differentiation of cocultured ES cells in serum-free conditions without use of either retinoic acid or embryoid bodies. BMP4, which acts as an antineuralizing morphogen in Xenopus, suppresses SDIA-induced neuralization and promotes epidermal differentiation. A high proportion of tyrosine hydroxylase-positive neurons producing dopamine are obtained from SDIA-treated ES cells. When transplanted, SDIA-induced dopaminergic neurons integrate into the mouse striatum and remain positive for tyrosine hydroxylase expression. Neural induction by SDIA provides a new powerful tool for both basic neuroscience research and therapeutic applications.

Strain-triggered mechanical feedback in self-organizing optic-cup morphogenesis.

Organogenesis is a self-organizing process of multiple cells in three-dimensional (3D) space, where macroscopic tissue deformations are robustly regulated by multicellular autonomy. It is clear that this robust regulation requires cells to sense and modulate 3D tissue formation across different scales, but its underlying mechanisms are still unclear. To address this question, we developed a versatile computational model of 3D multicellular dynamics at single-cell resolution and combined it with the 3D culture system of pluripotent stem cell-derived optic-cup organoid. The complementary approach enabled quantitative prediction of morphogenesis and its corresponding verification and elucidated that the macroscopic 3D tissue deformation is fed back to individual cellular force generations via mechanosensing. We hereby conclude that mechanical force plays a key role as a feedback regulator to establish the robustness of organogenesis.

Regulation of neural determination by evolutionarily conserved signals: anti-BMP factors and what next?

The evolutionary conservation of Chordin/bone morphogenetic protein (BMP) signaling supports the hypothesis of dorsal-ventral axis inversion of vertebrates and invertebrates, and implies that the invention of a central nervous system occurred only once during animal evolution. This hypothesis is further strengthened by recent findings of the conservation of downstream genes and modifier genes of neural induction. On the other hand, in contrast with such gross conservation, recent data suggest that the requirement for some signals in neural determination may differ even within the vertebrate subphylum.

Xenopus Xenf: an early endodermal nuclear factor that is regulated in a pathway distinct from Sox17 and Mix-related gene pathways.

We report a novel zygotic gene encoding a Xenopus endodermal nuclear factor, Xenf. Expression of Xenf starts at the late blastula stages and is decreased after gastrulation. Xenf shows no structural homology to any known proteins. When GFP-tagged Xenf is overexpressed in Xenopus cells, Xenf protein is localized to the nucleus, associating closely with the chromosomes. In animal cap assays, Xenf expression is strongly activated by mRNA injection of Vg1 and VegT, maternal vegetal genes that can induce endodermal differentiation. In contrast, Xenf is not induced by endoderm-inducing zygotic transcription factors such as Sox17 and Mix-related genes. In turn, Xenf does not activate expression of Sox17, Mixer or Milk. Thus, Xenf is regulated by maternal vegetal positional information in a parallel manner to Sox17 and Mix-related gene pathways.

Tandem duplications of the FLT3 receptor gene are associated with leukemic transformation of myelodysplasia.

We recently reported an internal tandem duplication of the human flt3 receptor gene (FLT3) as a somatic mutation in 17% of acute myelogenous leukemia (AML). The present study revealed the duplication at the juxtamembrane and the first tyrosine kinase domains of FLT3 in seven of 92 (8%) patients with myelodysplastic syndrome (MDS) and AML with trilineage myelodysplasia (AML/TMDS), the diseases which may represent neoplastic changes of pluripotent stem cells. A tandem duplication of exon 11 of FLT3 was harbored by two of 58 (3%) patients with MDS and five of 34 (15%) with overt leukemia, including MDS-derived leukemia, AML/TMDS and therapy-related leukemia. Although the duplicated regions varied within exon 11 in each case, they occurred in-frame, and altered mRNA expressions were demonstrated by reverse-transcription polymerase chain reaction. Two cases of MDS with a FLT3 duplication transformed to overt leukemia within a few months. Longitudinal analyses in two other patients with leukemia revealed that the duplication was a late genetic event during the disease course; one of whom showed two independent duplications of FLT3 at the terminal therapy-resistant phase. Of seven patients with the FLT3 duplication, six had abnormal karyotypes, and four harbored a point mutation of the N-RAS and/or TP53 genes. Patients with FLT3 mutations have poor prognoses. This study uncovered the fact that the accumulation of genetic events, including FLT3 duplication, correlates with leukemic transformation from antecedent myelodysplasia and with subsequent disease progression.

Distinct genetic involvement of the TP53 gene in therapy-related leukemia and myelodysplasia with chromosomal losses of Nos 5 and/or 7 and its possible relationship to replication error phenotype.

We examined chromosomes and molecular aberrations in 21 patients with therapy-related leukemias (t-AML) or myelodysplastic syndromes (t-MDS). All patients showed abnormal karyotypes, and chromosomal losses of No. 5 and/or No. 7 (-5/5q- and/or -7/7q-) were identified in 12 patients. Among these 12, six patients (50%) harbored a TP53 mutation, and two of five examined showed microsatellite instability, suggesting replication error (RER+) phenotype. Meanwhile, among the other nine patients without -5/5q- and/or -7/7q-, none harbored a TP53 mutation, and none of five examined showed RER+ phenotype. Thus, TP53 mutations and RER+ phenotype were preferentially associated with specific chromosomal losses in t-AML/MDS. We then screened for mutational events in representative DNA mismatch repair genes; exons 5-7 and 12-15 of the hMSH2 gene and exon 9 of hMLH1. Notably, two unrelated patients showing RER+ phenotype had an identical missense alteration at codon 419 of hMSH2 in their marrow cells and fibroblasts, which were not found in 120 DNA samples from healthy volunteers or patients with other hematological disorders. Consequently, this study revealed a possible relationship of RER+ phenotype accompanying an hMSH2 alteration to the development of therapy-related AML/MDS in association with TP53 mutations and specific chromosomal losses, and suggests that some patients may be predisposed to myelodysplasia after chemotherapy for their primary tumor.

Directed differentiation of neural and sensory tissues from embryonic stem cells in vitro.

We have recently identified a stromal cell-derived inducing activity (SDIA), which induces differentiation of neural cells from mouse embryonic stem (ES) cells. Particularly, midbrain TH+ dopaminergic neurons are generated efficiently in this system. These dopaminergic neurons are transplantable and survive well in the 6-OHDA-treated mouse striatum. SDIA induces co-cultured ES cells to differentiate into rostral central nervous system (CNS) tissues containing both ventral and dorsal cells. While early exposure of SDIA-treated ES cells to BMP4 suppresses neural differentiation and promotes epidermogenesis, late BMP4 exposure after the 4th day of co-culture causes differentiation of neural crest cells and dorsal-most CNS cells, with autonomic system and sensory lineages induced preferentially by high and low BMP4 concentrations, respectively. In contrast, Sonic Hedgehog (Shh) suppresses differentiation of neural crest lineages and promotes that of ventral CNS tissues such as motor neurons and HNF3beta+ floor plate cells with axonal guidance activities. Thus, SDIA-treated ES cells generate naïve precursors that have the competence of differentiating into the "full" dorsal-ventral range of neuroectodermal derivatives in response to patterning signals. I also discuss the role of SDIA and the mode of rostral-caudal specification of neuralized ES cells. In addition, I would like to discuss them in the light of control of in vitro neural production for the use in regenerative medicine for parkinsonism and retinal degeneration.

Roles of Sox factors in neural determination: conserved signaling in evolution?

Neural differentiation in amphibian embryos is initiated by the neural inducers emanating from the Spemann-Mangold organizer. The fate of uncommitted ectoderm is determined by graded BMP activity along the dorsal-ventral axis. Several transcriptional regulators acting in early neural differentiation have been identified, including Sox, Zic, Pou, HLH and Fox factors. In this paper, I review recent molecular studies on neural determination, focusing mainly on Sox factors. I also discuss the possible conservation of regulatory factors in neural differentiation, comparing Xenopus and Drosophila counterparts.

Contribution of T cell-mediated immunity to the resistance to staphylococcal infection.

Abscess formation in nude mice after subcutaneous inoculation of Staphylococcus aureus (S. aureus) was more extensive and prolonged as compared with that in phenotypically normal littermates. Abscess formation in nude mice was augmented markedly by whole-body irradiation. Not only T cell-mediated immunity but also radiosensitive, nonimmune phagocytosis appear to contribute to the resistance against staphylococcal infection.

Regulation of mammalian neural development by helix-loop-helix transcription factors.

Understanding of the molecular mechanisms of mammalian neural development has been greatly advanced by identification and characterization of the molecules homologous to the factors regulating Drosophila neurogenesis, which provides a powerful model system. Studies of Drosophila show that transcription factors with a helix-loop-helix (HLH) domain play an essential role in neurogenesis. Several lines of evidence demonstrate that mammalian homologues of the Drosophila HLH factors do also play an essential role in neural development. Mash-1, a mammalian HLH factor homologous to the products of Drosophila proneural genes achaete-scute complex, is a positive regulator of neurogenesis and required for differentiation of olfactory and autonomic neurons. In addition, HES-1, another mammalian HLH factor homologous to the products of Drosophila hairy and Enhancer of split, antagonizes the activity of Mash-1 and negatively regulates neurogenesis. Thus, positive and negative HLH factors interact with each other, and the balance between them is important for the developmental processes. Recent studies show that many other HLH factors exist expressed in the developing mammalian nervous system. In this article, the authors review mammalian HLH factors expressed in the nervous system and discuss the molecular aspect of mammalian neurogenesis.

Genotype of glutathione S-transferase and other genetic configurations in myelodysplasia.

We examined polymorphisms of glutathione S-transferase (GST) genes in 159 Japanese patients with myelodysplasia and compared the incidence with that in 43 normal individuals to clarify their pathogenetic significance in myelodysplasia. In individuals with the GSTT1 null genotype, the odds ratios for disease risk were elevated to 2.65 (95%CI; 1.27-5.52) in de novo MDS, 4.62 (1.48-14.4) in therapy-related AML, and 2.94 (1.07-8.07) in AML with triliniage dysplasia. Other representative polymorphisms of GSTs had a similar incidence among patients with myelodysplasia, and those of the controls and other hematological disorders. To further investigate the genetic pathway of myelodysplasia, the association between GST genotype and karyotype or configurations of TP53 and NRAS was evaluated, but no relationship was noted. These results suggest that the GSTT1 null genotype may play a role in an increased risk of myelodysplasia unrelated to other mechanisms of myelodysplasia, such as chromosomal alterations or mutation of TP53 or NRAS.

Significance of chromosomal alterations and mutations of the N-RAS and TP53 genes in relation to leukemogenesis of acute myeloid leukemia.

We examined chromosomes and mutations of the N-RAS and TP53 genes in 73 patients with acute myeloid leukemia (AML). Twenty-six patients showed a reciprocal chromosomal translocation or an inversion, and 34 patients showed only unbalanced aberrations. Balanced aberrations were predominantly detected in the AML patients who did not have myelodysplasia, preceding myelodysplastic syndrome, and a history of chemotherapy or radiation therapy. In contrast, unbalanced aberrations were more frequently seen in the patients with AML with trilineage myelodysplasia, AML transformed from MDS, and therapy-related AML. Twenty-two mutations of the N-RAS and TP53 genes were detected, and these mutations were frequently associated with unbalanced chromosomal aberrations. Furthermore, the spectrum of mutations was suggestive of an exposure to alkylating chemicals.

FK506 and cyclosporin A inhibit growth factor-stimulated human keratinocyte proliferation by blocking cells in the G0/G1 phases of the cell cycle.

FK506, a new immunosuppressive agent, is effective in treating patients with psoriasis. A major feature of psoriasis vulgaris is the hyperproliferation of keratinocytes together with inflammation. To determine the effect of FK506 or cyclosporin A (CsA) on the keratinocyte cell cycle, flow cytometry and the growth factor free normal human keratinocyte-arrested system were used to assess keratinocyte proliferation. FK506 and CsA inhibit keratinocyte proliferation induced by EGF, TGF-alpha or IL-6. The antiproliferative effects of FK506 and CsA directly correlated with blockade of the keratinocyte cell cycle at the G0/G1 phases. These findings might indicate that the effects of FK506 and CsA on proliferation of cultured normal human keratinocytes are probably related to direct effects on growth regulation of keratinocytes via EGF, TGF-alpha or IL-6 stimulation.

Purification and characterization of prolyl endopeptidase from rat skin.

An enzyme with the specificity of a prolyl endopeptidase was purified approximately 329-fold from rat skin. The enzyme has a molecular weight of 70,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pH optimum of 5.8 as checked with 7-(Succinyl-Gly-Pro)-4- methylcoumarinamide (Suc-Gly-Pro-MCA) as the substrate. The optimal temperature for the enzyme activity was 40 degrees C. The Km and Vmax values for Suc-Gly-Pro-MCA were 0.7 mM and 68 nmol/min per mg protein, respectively. The enzyme activity was markedly inhibited by diisopropyl fluorophosphate, p-chloromercuribenzoic acid, N-ethylmaleimide, Zn2+ and Cu2+, while it was partially inhibited by phenylmethanesulphonyl fluoride. The purified enzyme was shown to release the N-terminal tetrapeptide, Arg-Pro-Lys-Pro, from substance P producing the C-terminal heptapeptide, Gln-Gln-Phe-Phe-Gly-Met- CONH2. In the skin, this enzyme might be related to the inactivation of substance P.

Purification of human blood eosinophils by a combination method using anti-CD16 monoclonal antibody, immunobeads, and Nycodenz density gradient.

A simple method is described for the procurement of human blood eosinophil phenotypes by combining an anti-CD16 monoclonal antibody, immunobeads, and a non-toxic and non-ionic density gradient medium, Nycodenz. The purification depends on the removal of mononuclear cells using a 1.076/1.102 g/ml Nycodenz density gradient, partial removal of neutrophils based on different binding to plastic dishes, interaction of residual neutrophils with immunobeads via an anti-CD16 monoclonal antibody and, finally, extraction of eosinophil phenotypes by sifting the immunobeads-loaded neutrophils through an 1.080/1.102 g/ml Nycodenz density gradient. This method permits simultaneous preparation of highly purified normodense (> 1.080 g/ml) and hypodense eosinophils (< 1.080 g/ml) with reasonable chemiluminescence responses to opsonized zymosans and helminthotoxic activity to opsonized schistosomula corresponding to their own immunocytological properties.

The incidence of internal malignancies in pemphigus and bullous pemphigoid in Japan.

To evaluate the significance of the association of malignancy with autoimmune blistering diseases, we studied the incidence of internal malignancies in pemphigus and bullous pemphigoid based upon 496 cases of pemphigus and 1113 cases of bullous pemphigoid in Japan. Results showed that (1) an association between internal malignancies and pemphigus was observed in 25 out of 496 cases (5.0%), while that with bullous pemphigoid was seen in 64 out of 1113 cases (5.8%). Such association ratios were significantly higher than that of the controls aged over 70 years old (0.61%); (2) The average ages of pemphigus/bullous pemphigoid with malignancy were 64.7 and 69.2 years, respectively. The association ratio of malignancy with pemphigus increased by age, while that with pemphigoid was not correlated with aging; (3) Lung cancer was most common in pemphigus and gastric cancer in bullous pemphigoid; (4) There were no significant differences in the titers of circulating antibody, the presence or extent of mucous involvement or annular erythema between bullous pemphigoid patients with malignancy and without malignancy. Our results indicated that detailed examination for internal malignancy is essential for those patients with pemphigus or bullous pemphigoid.