A novel de novo point mutation of the OCT-binding site in the IGF2/H19-imprinting control region in a Beckwith-Wiedemann syndrome patient.

The IGF2/H19-imprinting control region (ICR1) functions as an insulator to methylation-sensitive binding of CTCF protein, and regulates imprinted expression of IGF2 and H19 in a parental origin-specific manner. ICR1 methylation defects cause abnormal expression of imprinted genes, leading to Beckwith-Wiedemann syndrome (BWS) or Silver-Russell syndrome (SRS). Not only ICR1 microdeletions involving the CTCF-binding site, but also point mutations and a small deletion of the OCT-binding site have been shown to trigger methylation defects in BWS. Here, mutational analysis of ICR1 in 11 BWS and 12 SRS patients with ICR1 methylation defects revealed a novel de novo point mutation of the OCT-binding site on the maternal allele in one BWS patient. In BWS, all reported mutations and the small deletion of the OCT-binding site, including our case, have occurred within repeat A2. These findings indicate that the OCT-binding site is important for maintaining an unmethylated status of maternal ICR1 in early embryogenesis.

Mesp2 initiates somite segmentation through the Notch signalling pathway.

The Notch-signalling pathway is important in establishing metameric pattern during somitogenesis. In mice, the lack of either of two molecules involved in the Notch-signalling pathway, Mesp2 or presenilin-1 (Ps1), results in contrasting phenotypes: caudalized versus rostralized vertebra. Here we adopt a genetic approach to analyse the molecular mechanism underlying the establishment of rostro-caudal polarity in somites. By focusing on the fact that expression of a Notch ligand, Dll1, is important for prefiguring somite identity, we found that Mesp2 initiates establishment of rostro-caudal polarity by controlling two Notch-signalling pathways. Initially, Mesp2 activates a Ps1-independent Notch-signalling cascade to suppress Dll1 expression and specify the rostral half of the somite. Ps1-mediated Notch-signalling is required to induce Dll1 expression in the caudal half of the somite. Therefore, Mesp2- and Ps1-dependent activation of Notch-signalling pathways might differentially regulate Dll1 expression, resulting in the establishment of the rostro-caudal polarity of somites.

Cryopreservation of osteoblasts by use of a programmed freezer with a magnetic field.

In order to determine a suitable condition for osteoblasts cryopreservation, murine osteoblasts were freezed by programmed freezer with a magnetic field (CAS freezer). After 7 days cryopreservation at -150°, the number of survival cells immediately after thawing and the growth rate of cultured cells for 48 hours were examined. Gene and protein expression of alkaline phosphatase (ALP), osteopontin (OPN) and bone sialoprotein (BSP) were compared between cryopreserved and non-cryopreserved groups. As a result, a plunging temperature of -30°, a hold-time at -5° for 15 minutes and a 0.1 mT of magnetic field led to the largest survival and growth rate. Moreover, there was no significant difference in ALP, OPN and BSP mRNA and protein expression between cryopreserved and control groups. From these results, it was suggested that the CAS freezer is available for osteoblast cryopreservation and bone tissue banking can be established in the future.

Effects of cryopreservation with a newly-developed magnetic field programmed freezer on periodontal ligament cells and pulp tissues.

The purpose of this study was to evaluate the effects of long-term cryopreservation on the isolated human periodontal ligament cells (PDL) and pulp tissues. In the first part of study, 10 freshly extracted teeth were selected and divided into two groups. In the cryopreserved group, the teeth were frozen for 5 years using a programmed freezer combined with a magnetic field, known as Cells Alive System "CAS". As for the control group, freshly extracted teeth were used. In each group, extracted PDL tissues were cultured and gene expression and protein concentration of collagen type I, alkaline-phosphatase (ALP) and vascular endothelial growth factor (VEGF) was compared between the two groups. In the second part, pulp tissues were obtained from 10 mature and immature third molars which were freshly extracted or cryopreserved for three months. Expression of VEGF and nerve growth factor (NGF) mRNAs and the protein concentration in the supernatant were investigated. Results indicated that long-term cryopreservation with the use of CAS freezer cannot affect the growth rate and characteristics of PDL cells. There was no significant difference in VEGF expression and VEGF and NGF protein concentration of pulp cells derived from cryopreserved teeth with immature apex and control group with mature root formation. Finally, proper PDL regeneration and appropriate apexogenesis after transplanting magnetically cryopreserved immature tooth was clinically confirmed. These findings demonstrate that teeth banking with the use of magnetic field programmed freezer can be available for future autotransplantation as a treatment modality for replacing missing teeth.

Extrathymic development of V alpha 14-positive T cells.

It is known that rearrangement of the T cell antigen receptor (TCR) gene occurs in the thymus during T cell development and consequently results both in the deletion of DNA between the variable (V) and diversity/joining segments and in the formation of a circular DNA with recombination signal sequences. Here, we provide evidence that V alpha 14+ TCR gene rearrangements take place in extrathymic sites, such as bone marrow, liver, and intestine, but not in spleen, because we were able to detect frequent productive and nonproductive V alpha 14+ coding and signal sequences as a result of TCR rearrangements in extrathymic sites. Similar findings were also detected in athymic mice. Quantitative analysis shows that the relative amounts of V alpha 14 gene-mediated signal sequences in extrathymic tissues are higher than those in thymus. On the contrary, TCR rearrangements of V alpha 1.1 T cells, which are known to develop in the thymus, were mainly detected in the thymus, Peyer's patch, and spleen, but not in other extrathymic tissues, showing patterns distinct from V alpha 14 TCR rearrangements. These findings are evidence of extrathymic development of V alpha 14+ T cells. Differential characteristic TCR rearrangement patterns also indicate that distinct TCR repertoires are generated in different lymphoid tissues.

Disruption of the Bcl6 gene results in an impaired germinal center formation.

The Bcl6 gene has been identified from the chromosomal translocation breakpoint in B cell lymphomas, and its products are expressed highly in germinal center (GC) B cells. To investigate the function of Bcl6 in lymphocytes, we have generated RAG1-deficient mice reconstituted with bone marrow cells from Bcl6-deficient mice (Bcl6(-/-)RM). Lymphogenesis in primary lymphoid tissues of Bcl6(-/-)RM is normal, and Bcl6(-/-)RM produced control levels of primary IgG1 antibodies specific to T cell-dependent antigens. However, GCs were not found in these mice. This defect was mainly due to the abnormalities of B cells. Therefore, Bcl6 is essential for the differentiation of GC B cells.

Inhibition of T helper cell type 2 cell differentiation and immunoglobulin E response by ligand-activated Valpha14 natural killer T cells.

Murine Valpha14 natural killer T (NKT) cells are thought to play a crucial role in various immune responses, including infectious, allergic, and autoimmune diseases. Because Valpha14 NKT cells produce large amounts of both interleukin (IL)-4 and interferon (IFN)-gamma upon in vivo stimulation with a specific ligand, alpha-galactosylceramide (alpha-GalCer), or after treatment with anti-CD3 antibody, a regulatory role on helper T (Th) cell differentiation has been proposed for these cells. However, the identity of the cytokine produced by Valpha14 NKT cells that play a dominant role on the Th cell differentiation still remains controversial. Here, we demonstrate by using Valpha14 NKT-deficient mice that Valpha14 NKT cells are dispensable for the induction of antigen-specific immunoglobulin (Ig)E responses induced by ovalbumin immunization or Nippostrongylus brasiliensis infection. However, upon in vivo activation with alpha-GalCer, Valpha14 NKT cells are found to suppress antigen-specific IgE production. The suppression appeared to be IgE specific, and was not detected in either Valpha14 NKT- or IFN-gamma-deficient mice. Consistent with these results, we also found that ligand-activated Valpha14 NKT cells inhibited Th2 cell differentiation in an in vitro induction culture system. Thus, it is likely that activated Valpha14 NKT cells exert a potent inhibitory effect on Th2 cell differentiation and subsequent IgE production by producing a large amount of IFN-gamma. In marked contrast, our studies have revealed that IL-4 produced by Valpha14 NKT cells has only a minor effect on Th2 cell differentiation.

Identification of loss-of-function mutations of SLC35D1 in patients with Schneckenbecken dysplasia, but not with other severe spondylodysplastic dysplasias group diseases.

BACKGROUND: Schneckenbecken dysplasia (SBD) is an autosomal recessive lethal skeletal dysplasia that is classified into the severe spondylodysplastic dysplasias (SSDD) group in the international nosology for skeletal dysplasias. The radiological hallmark of SBD is the snail-like configuration of the hypoplastic iliac bone. SLC35D1 (solute carrier-35D1) is a nucleotide-sugar transporter involved in proteoglycan synthesis. Recently, based on human and mouse genetic studies, we showed that loss-of-function mutations of the SLC35D1 gene (SLC35D1) cause SBD. OBJECT: To explore further the range of SLC35D1 mutations in SBD and elucidate whether SLC35D1 mutations cause other skeletal dysplasias that belong to the SSDD group. METHODS AND RESULTS: We searched for SLC35D1 mutations in five families with SBD and 15 patients with other SSDD group diseases, including achodrogenesis type 1A, spondylometaphyseal dysplasia Sedaghatian type and fibrochondrogenesis. We identified four novel mutations, c.319C>T (p.R107X), IVS4+3A>G, a 4959-bp deletion causing the removal of exon 7 (p.R178fsX15), and c.193A>C (p. T65P), in three SBD families. Exon trapping assay showed IVS4+3A>G caused skipping of exon 4 and a frameshift (p.L109fsX18). Yeast complementation assay showed the T65P mutant protein lost the transporter activity of nucleotide sugars. Therefore, all these mutations result in loss of function. No SLC35D1 mutations were identified in all patients with other SSDD group diseases. CONCLUSION: Our findings suggest that SLC35D1 loss-of-function mutations result consistently in SBD and are exclusive to SBD.

Cryopreservation of periodontal ligament cells with magnetic field for tooth banking.

The purpose of this study was to establish a long-term tooth cryopreservation method that can be used for tooth autotransplantation. Human periodontal ligament (PDL) cells were frozen in 10% dimethyl sulfoxide (Me(2)SO) using a programmed freezer with a magnetic field. Cells were cryopreserved for 7 days at -150 degrees C. Immediately after thawing, the number of surviving cells was counted and the cells were cultured; cultured cells were examined after 48 h. Results indicated that a 0.01 mT of a magnetic field, a 15-min hold-time, and a plunging temperature of -30 degrees C led to the greatest survival rate of PDL cells. Based on these findings, whole teeth were cryopreserved under the same conditions for 1 year. The organ culture revealed that the PDL cells of cryopreserved tooth with a magnetic field could proliferate as much as a fresh tooth, although the cells did not appear in the cryopreserved tooth without a magnetic field. Histological examination and the transmission electron microscopic image of cryopreserved tooth with a magnetic field did not show any destruction of cryopreserved cells. In contrast, severe cell damage was seen in cells frozen without a magnetic field. These results indicated that a magnetic field programmed freezer is available for tooth cryopreservation.

Airborne particle dispersion around the feet of surgical staff while walking in and out of a bio-clean operating theatre.

BACKGROUND: Bacterial contamination by airborne particles is one of the most important factors in the pathogenesis of surgical-site infections. AIM: This study aimed to identify the generation and behaviour of airborne particles around the feet of surgical staff while walking in and out of an operating theatre. METHODS: Two physicians and two nurses walked in and out of a bio-clean theatre under laminar airflow, either individually or as a group. The generation and behaviour of airborne particles was filmed using a fine-particle visualization system, and the number of airborne particles per 2.83 m3 of air was counted using a laser particle counter. Each action was repeated five times, and particle counts were evaluated statistically. FINDINGS: Airborne particles were generated from the floor and by the shoes and gown hems of the participants, whether walking individually or as a group. Numerous airborne particles were generated by the group, and significantly more particles, especially those measuring 0.3-0.5 µm, were carried up to the level of the operating table by the group than by individuals (P<0.01). CONCLUSIONS: The results of this study provide a clearer picture of the dispersion and distribution of airborne particles around the feet of staff walking in and out of an operating theatre. The findings suggest that to reduce the incidence of bacterial contamination and risk of surgical site infections, surgical staff should walk calmly and independently, if possible, near sterile areas.

Requirement for Valpha14 NKT cells in IL-12-mediated rejection of tumors.

A lymphocyte subpopulation, the Valpha14 natural killer T (NKT) cells, expresses both NK1.1 and a single invariant T cell receptor encoded by the Valpha14 and Jalpha281 gene segments. Mice with a deletion of the Jalpha281 gene segment were found to exclusively lack this subpopulation. The Valpha14 NKT cell-deficient mice could no longer mediate the interleukin-12 (IL-12)-induced rejection of tumors. Although the antitumor effect of IL-12 was thought to be mediated through natural killer cells and T cells, Valpha14 NKT cells were found to be an essential target of IL-12, and they mediated their cytotoxicity by an NK-like effector mechanism after activation with IL-12.

CD1d-restricted and TCR-mediated activation of valpha14 NKT cells by glycosylceramides.

Natural killer T (NKT) lymphocytes express an invariant T cell antigen receptor (TCR) encoded by the Valpha14 and Jalpha281 gene segments. A glycosylceramide-containing alpha-anomeric sugar with a longer fatty acyl chain (C26) and sphingosine base (C18) was identified as a ligand for this TCR. Glycosylceramide-mediated proliferative responses of Valpha14 NKT cells were abrogated by treatment with chloroquine-concanamycin A or by monoclonal antibodies against CD1d/Vbeta8, CD40/CD40L, or B7/CTLA-4/CD28, but not by interference with the function of a transporter-associated protein. Thus, this lymphocyte shares distinct recognition systems with either T or NK cells.

Aberrant quality control in the endoplasmic reticulum impairs the biosynthesis of pulmonary surfactant in mice expressing mutant BiP.

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR), which alleviates protein overload in the secretory pathway. Although the UPR is activated under diverse pathological conditions, its physiological role during development and in adulthood has not been fully elucidated. Binding immunoglobulin protein (BiP) is an ER chaperone, which is central to ER function. We produced knock-in mice expressing a mutant BiP lacking the retrieval sequence to cause a defect in ER function without completely eliminating BiP. In embryonic fibroblasts, the UPR compensated for mutation of BiP. However, neonates expressing mutant BiP suffered respiratory failure due to impaired secretion of pulmonary surfactant by alveolar type II epithelial cells. Expression of surfactant protein (SP)-C was reduced and the lamellar body was malformed, indicating that BiP plays a critical role in the biosynthesis of pulmonary surfactant. Because pulmonary surfactant requires extensive post-translational processing in the secretory pathway, these findings suggest that in secretory cells, such as alveolar type II cells, the UPR is essential for managing the normal physiological ER protein overload that occurs during development. Moreover, failure of this adaptive mechanism may increase pulmonary susceptibility to environmental insults, such as hypoxia and ischemia, ultimately leading to neonatal respiratory failure.

A novel pathogenesis of megacolon in Ncx/Hox11L.1 deficient mice.

The Ncx/Hox11L.1 gene, a member of the Hox11 homeobox gene family, is mainly expressed in neural crest-derived tissues. To elucidate the role of Ncx/Hox11L.1, the gene has been inactivated in embryonic stem cells by homologous recombination. The homozygous mutant mice were viable. These mice developed megacolon with enteric ganglia by age 3-5 wk. Histochemical analysis of the ganglia revealed that the enteric neurons hyperinnervated in the narrow segment of megacolon. Some of these neuronal cells degenerated and neuronal cell death occurred in later stages. We propose that Ncx/Hox11L.1 is required for maintenance of proper functions of the enteric nervous system. These mutant mice can be used to elucidate a novel pathogenesis for human neuronal intestinal dysplasia.

Crosstalk between NRF2 and HIPK2 shapes cytoprotective responses.

Homeodomain interacting protein kinase-2 (HIPK2) is a member of the HIPK family of stress-responsive kinases that modulates cell growth, apoptosis, proliferation and development. HIPK2 has several well-characterised tumour suppressor roles, but recent studies suggest it can also contribute to tumour progression, although the underlying mechanisms are unknown. Herein, we have identified novel crosstalk between HIPK2 and the cytoprotective transcription factor NRF2. We show that HIPK2 is a direct transcriptional target of NRF2, identifying a functional NRF2 binding site in the HIPK2 gene locus and demonstrating for the first time a transcriptional mode of regulation for this kinase. In addition, HIPK2 is required for robust NRF2 responsiveness in cells and in vivo. By using both gain-of-function and loss-of-function approaches, we demonstrate that HIPK2 can elicit a cytoprotective response in cancer cells via NRF2. Our results have uncovered a new downstream effector of HIPK2, NRF2, which is frequently activated in human tumours correlating with chemoresistance and poor prognosis. Furthermore, our results suggest that modulation of either HIPK2 levels or activity could be exploited to impair NRF2-mediated signalling in cancer cells, and thus sensitise them to chemotherapeutic drugs.

Insulin resistance and increased pancreatic beta-cell proliferation in mice expressing a mutant insulin receptor (P1195L).

Several mutations of the tyrosine kinase domain of insulin receptor (IR) have been clinically reported to lead insulin resistance and insulin hypersecretion in humans. However, it has not been completely clarified how insulin resistance and pancreatic beta-cell function affect each other under the expression of mutant IR. We investigated the response of pancreatic beta-cells in mice carrying a mutation (P1195L) in the tyrosine kinase domain of IR beta-subunit. Homozygous (Ir(P1195L/P1195L)) mice showed severe ketoacidosis and died within 2 days after birth, and heterozygous (Ir(P1195L/wt)) mice showed normal levels of plasma glucose, but high levels of plasma insulin in the fasted state and after glucose loading, and a reduced response of plasma glucose lowering effect to exogenously administered insulin compared with wild type (Ir(wt/wt)) mice. There were no differences in the insulin receptor substrate (IRS)-2 expression and its phosphorylation levels in the liver between Ir(P1195L/wt) and Ir(wt/wt) mice, both before and after insulin injection. This result may indicate that IRS-2 signaling is not changed in Ir(P1195L/wt) mice. The beta-cell mass increased due to the increased numbers of beta-cells in Ir(P1195L/wt) mice. More proliferative beta-cells were observed in Ir(P1195L/wt) mice, but the number of apoptotic beta-cells was almost the same as that in Ir(wt/wt) mice, even after streptozotocin treatment. These data suggest that, in Ir(P1195L/wt) mice, normal levels of plasma glucose were maintained due to high levels of plasma insulin resulting from increased numbers of beta-cells, which in turn was due to increased beta-cell proliferation rather than decreased beta-cell apoptosis.

Expression of the Dan gene during chicken embryonic development.

The Dan gene was first identified as the putative rat tumor suppressor gene and encodes a protein structurally related to Cerberus and Gremlin in vertebrates. Xenopus DAN, as with Cerberus and Gremlin, was demonstrated to block bone morphogenetic protein (BMP) signaling by binding BMPs, and to be capable of inducing additional anterior structures by ectopic overexpression in Xenopus embryos. DAN, thus, is suggested to play pivotal roles in early patterning and subsequent organ development, as in the case of other BMP antagonists. In this report, we isolated the chicken counterpart of Dan. Chicken Dan is mainly expressed in the cephalic and somitic mesoderm and several placodes during organ development.

Zic1 regulates the patterning of vertebral arches in cooperation with Gli3.

Skeletal abnormalities are described that appeared in Zic1-deficient mice. These mice show multiple abnormalities in the axial skeleton. The deformities are severe in the dorsal parts of the vertebrae, vertebral arches, but less so in the vertebral bodies (spina bifida occulta). The proximal ribs are deformed having ectopic processes. The abnormalities found in the vertebral arches can be traced back to disturbed segmental patterns of dorsal sclerotome. The Zic1/Gli3 double mutants showed severe abnormalities of vertebral arches not found in single mutants. The abnormalities in the vertebral arches were less severe in Zic1/Pax1 mutants than Zic1/Gli3 mutants, but significantly more pronounced than in Zic1 single mutants. The three genes may act synergistically in the development of the vertebral arches.

The role of Bcl6 in mature cardiac myocytes.

OBJECTIVE: The Bcl6 gene encodes a sequence-specific transcriptional repressor and is ubiquitously expressed in adult murine tissues including heart muscle. The objective of this study was to examine the role of Bcl6 in cardiac myocytes. METHOD: We developed Bcl6-deficient (Bcl6-/-) mice and histologically examined hearts from these mice. RESULTS: Massive myocarditis with eosinophilic infiltration occurred in Bcl6-/- mice after 4-6 weeks of age. Since expression of the Bcl6 gene was induced in normal cardiac myocytes after 2 weeks of age and thereafter detected through adulthood, loss of Bcl6 in mature cardiac myocytes may be related to the induction of eosinophilic myocarditis. To examine the effects of eosinophils from Bcl6-/- mice on normal hearts, bone marrow cells from Bcl6-/- mice were adoptively transferred into sublethally irradiated RAG1-deficient mice. Although massive eosinophilic infiltration was detected in conjunctivas and spleens from the chimeric mice, myocarditis was never observed. Electron microscopic analysis of cardiac myocytes from Bcl6-/- mice revealed a spectrum of degenerative changes prior to eosinophilic infiltration. CONCLUSION: Bcl6 maynot be essential for the maturation of cardiac myocytes but may play a role in protecting mature cardiac myocytes from eosinophilic inflammation.

Genomic DNA with transformation-related activity and melanoma antigen expression.

We have analyzed the mouse melanoma antigen with monoclonal antibodies established by syngeneic immunization. To further understand the structure of this antigen at a molecular level, we have cloned the genomic DNA controlling the expression of melanoma antigen by cosmid library transfection and a monoclonal antibody. In the process of analyzing this DNA fragment we found that it contained a gene related with transformation, which was proved by tumor formation in nude mice inoculated with NIH/3T3 transfectants of this DNA fragment. We discuss the structure of the gene product based on the deduced amino acid sequence of cDNA, which maps the genome bearing transformation-related activity.