bluuebunny/crossref_metadata_2025 huggingface dataset:
Difference between Maturation Division and Cleavage in Starfish Oocytes: Dependency of Induced Cytokinesis on the Size of the Aster as Revealed by Transplantation of the Centrosome
Authors: Saiki Tomoaki , Hamaguchi Yukihisa
Published: January 2005

Using the starfish oocyte and zygote, we investigated the abilities of the centrosome at maturation and cleavage divisions to form the aster and induce cytokinesis, in order to determine differences between these divisions. The transplanted centrosome originated from both maturation and cleavage, induced an additional furrow in cleavage in the recipient cells, but did not induce abnormal polar body formation at maturation. Although it organized an additional aster in the recipient cell in both divisions, a difference in size among asters formed was recognized. Therefore, mitotic asters were stabilized with hexylene glycol in order to measure their radius and clarify this difference. The mean radius (14.4 μm) of the first meiotic aster was significantly smaller than that (20.4 μm) of the aster at the first cleavage. The transplanted cleavage centrosome formed as small an aster as the recipient's own at maturation divisions. When zygotes were briefly treated with colcemid so that the zygotes could not perform cytokinesis but did perform karyokinesis, the size of aster became the same as that in meiosis. These results prove that although any centrosome functions as a microtubule organizing center independent of its origin, the size of the resultant aster decides whether or not cytokinesis would be induced.
The Role of c‐kit Proto‐oncogene during Melanocyte Development in Mouse. In vivo Approach by the In utero Microinjection of Anti‐c‐kit Antibody
Authors: Yoshida Hisahiro , Nishikawa Shin‐Ichi , Okamura Hitoshi , Sakakura Teruyo , Kusakabe Moriaki
Published: January 2005

In order to investigate the role of the c‐kit oncogene in the melanoblast development, a rat monoclonal antibody (ACK2) against the mouse c‐kit protein was used to localize cells expressing c‐kit during fetal development. ACK2 was also injected directly into the amniotic cavity of mouse fetuses at successive developmental stages. After birth, the offspring were examined to determine the resulting coat color patterns. c‐kit positive melanoblasts first appeared in dermis of fetuses at 11.5 days postcoitum (dpc). Subsequently, these cells increased in number and migrated dorsolaterally to the ventral region, and by 12.5 dpc some of them began to invade the epidermis. Treatment of fetuses by ACK2 microinjection appeared to affect the pigmentation in the coat, inducing a variety of spotting patterns in offspring, and the location of the spots was closely correlated with gestational stage. ACK2 injection of early fetuses produced major changes in coat color even though few c‐kit positive cells were detectable in the dermal mesenchyme at the time of injection. Large spots were also induced when mid‐stage fetuses with a only few c‐kit positive cells in the dorsal region were injected. By contrast, except for spot formation in the center of ventral region, ACK2 injection did not appear to affect melanogenesis in late stage fetuses that had many c‐kit positive cells.
Temporal and Spatial Expression of a Gene for the Nuclear Protein Hgv2 in Embryos and Adults of the Ascidian Halocynthia roretzi
Authors: Fujiwara Shigeki
Published: January 2005

The pHgv20 cDNA clone encodes an ascidian embryonic nuclear protein, Hgv2, that is closely related to the amphibian histone‐binding protein N1. Genomic Southern blot analysis revealed the presence of two or more genes that hybridize with the Hgv2 probe under high‐stringency conditions, although it remains to be determined whether or not each of them is actively expressed. On Northern blots prepared from embryos, a single, 2.3‐kb Hgv2 mRNA was detectable during early stages of embryogenesis. The amount of Hgv2 mRNA gradually decreased after the 64‐cell stages. Northern, Western and immunohistochemical analyses showed that the Hgv2 protein was not expressed exclusively in the oocyte: small amounts of the 2.3‐kb mRNA and of the 83‐kDa Hgv2 protein were detectable in the branchial sac of adult organisms. Weak but specific immunohistochemical staining was observed in the spermatocytes and/or spermatogonia. An Hgv2‐specific antiserum reacted specifically with the 83‐kDa protein on the Western blot of the testis. These results suggest that Hgv2 functions not only in embryonic cells but also in sperm precursor cells and some somatic cells.
Nuclear Differentiation in Paramecium caudatum: Analysis by the Monoclonal Antibody against a Micronuclear Antigen
Authors: Yanagi Akira
Published: January 2005

I obtained the monoclonal antibody 93A against a micronuclear antigen of the ciliate Paramecium caudatum. Immunocytochemical observations showed that the antigen 93A appeared in some portion of the micronucleus in every stage of life cycle. In dividing micronuclei, the antigen appeared mainly in their both poles and in fibrous structures between the poles. These results suggest that the micronuclear antigen 93A may be a component of microtubule organizing center and spindles. During nuclear differentiation in P. caudatum, four among eight postzygotic micronuclei differentiate new macronuclear anlagen and one becomes a new micronucleus and the remaining three degenerate. The micronuclear antigen 93A appeared in all of the eight nuclei in the early stage of macronuclear differentiation but then disappeared in the four macronuclear anlagen and eventually persisted only in the new micronucleus, showing that the newly developing macronuclear anlagen lose the micronuclear antigen 93A during their differentiation.
In Vivo and in Vitro Experimental Analysis of Lens Regeneration in Larval Xenopus laevis
Authors: Bosco Luigi , Valle Cristiana , Willems Daniela
Published: January 2005

After lentectomy of larval Xenopus laevis, the outer cornea undergoes tissue transformation resulting in formation of a new lens. This lens regeneration is triggered and sustained by neural retina. In the present study, lens‐forming transformation of the outer cornea was completed in vitro when the outer cornea was cultured within the lentectomized eye‐cup. Well‐differentiated lens fiber cells, which showed positive immunofluorescence for total crystallins, were also formed when the outer cornea was cultivated with the retina. No lens tissue was formed when the cornea was cultured alone. Lens‐forming transformation, originating from the cornea three and five days after lentectomy, completely regressed when the tissue was isolated in vitro. Fom the present and previous findings, we concluded that, the interaction of corneal cells with the retina plays a decisive role in lens regeneration in situ.
Vimentin Expression Pattern is Different between the Flank Region and Limb Regions of Somatopleural Mesoderm in the Chicken Embryo
Authors: Hayashi Kensuke , Hagiwara Yasuko , Ozawa Eijiro
Published: January 2005

It is known that the chicken flank somatopleure also has a limb‐forming potential at early stages of development, but loses this potential later. Molecular changes during this process is, however, not well known. We obtained a monoclonal antibody which reacts to the flank somatopleure, but not to the wing bud, the leg bud and the neck somatopleure in the stage 22 chicken embryo. Further study revealed that this antibody is specific to vimentin. Time course of vimentin expression in the somatopleural mesoderm during the development was studied. It was revealed to be biphasic. Somatopleural mesoderm expressed vimentin at stage 10, but not at stage 16. Flank somatopleural mesoderm began to express vimentin again at stage 18, whereas limb bud mesenchymal cells did not until stage 27. The earlier re‐expression of vimentin at the flank somatopleure suggests that certain physiological changes take place in cells at this region.
Organization of Actin Filaments in the Axial Rod of Abalone Sperm Revealed by Quick Freeze Technique
Authors: Shiroya Yoko , Sakai Yoshi T.
Published: January 2005

An axial rod in abalone (Haliotis discus) sperm is a structure composed of a bundle of actin filaments, which elongates anteriorly to form the acrosomal process during the acrosome reaction. The ultrastructure of the actin filament bundle constituting the axial rod was examined using quick freeze technique followed by either freeze‐substitution or deep‐etch electron microscopy. Thin sections of quick freeze and freeze‐substituted sperm revealed that the actin filaments in the axial rod are hexagonally packed in a paracrystalline array through its almost entire length with an average center‐to‐center spacing of 12 nm. Periodic transverse bands were also observed across the actin filament bundle, which may reflect the cross‐bridges interconnecting the adjacent filaments. Quick‐freeze deep‐etch analysis provided the three‐dimensional view of the axial rod. Actin filaments exhibiting 5.5–6 nm spaced striations were observed to run in parallel with each other inside the axial rod. The existence of cross‐bridging structures was also displayed between adjacent filaments. These results suggest that the actin filaments in the axial rod are probably held together by regularly spaced cross‐bridges to form a well ordered hexagonally packed bundle, and also cross‐linked by fibrous structure to the lateral inner acrosomal membrane which closely surrounds the anterior half of the actin filament bundle.
Cloning and Tissue Specific Expression of the Axolotl Cellular Retinoic Acid Binding Protein
Authors: Ludolph David C. , Cameron JoAnn , Neff Anton W. , Stocum David L.
Published: January 2005

The CRABP gene encodes a cellular retinoic acid binding protein which is believed to mediate the teratogenic and pattern altering effects of retinoic acid (RA) on developing and regenerating systems. As a first step in examining the role of CRABP in transducing the effects of RA in the regenerating urodele limb, we have isolated with PCR three partial cDNAs which encode the axolotl (Ambystoma mexicanum) cellular retinoic acid binding protein (aCRABP) and analyzed its expression in various tissues by Northern analysis. Sequence analysis of the clones revealed a high degree of nucleotide and deduced amino acid homology with mammalian and avian CRABP I, showing the highest homology with mouse CRABP I (79% & 85%, respectively). The results of Northern analysis show that aCRABP I codes for a single μ 1.3 kb transcript. A strong signal is observed in the RNA of stage 40 axolotl embryos and larval brain, whereas a weaker signal is detected in the larval heart, and muscle‐skin of limbs. No message is detected in larval spleen, liver, or several stages of limb regeneration blastemas. The absence of aCRABP I message in the limb regeneration blastema suggests that CRABP II or another isoform is responsible for the observed RA binding activity of these tissues.
Changes with Development in the Expression of Cathepsin E in the Fetal Rat Stomach
Authors: Yonezawa Satoshi , Maejima Yukari , Hagiwara Naoki , Aratani Tomoko , Shoji Ryujiro , Kageyama Takashi , Tsukada Shinko , Miki Kazumasa , Ichinose Masao
Published: January 2005

The changes with development in the expression of cathepsin E in the fetal rat stomach were examined immunochemically and immunohistochemically. The activity of acid proteinase in fetal gastric extracts increased dramatically during late gestational stages, rising from 0.017 units per mg of protein on day 15 of gestation to 0.591 units per mg of protein on day 21 of gestation. Electrophoretic analysis, combined with immunological tests, showed that the increase was due exclusively to increases in the activity of the monomeric and dimeric forms of cathepsin E, while SDS‐PAGE‐immunoblot analysis revealed that both forms are present as a 43‐kDa proenzyme. Immunohistochemically, cathepsin E was localized in the cytoplasm of all proliferating epithelial cells of pars glandularis on day 16 of gestation or later. As revealed by conventional histological methods, surface mucous cells and parietal cells appeared for the first time in specimens on day 19 of gestation, and all of these cells were immunopositive for cathepsin E. The present study further indicated that cathepsin E is the predominant aspartic proteinase in the stomach of young rats, until pepsinogen C appears. Based on these results, possible roles of gastric cathepsin E are discussed.
Specific Developmental Defects in Molluscs after Treatment with Retinoic Acid during Gastrulation
Authors: Créton Robbert , Zwaan Gideon , Dohmen René
Published: January 2005

All‐trans retinoic acid is known as a teratogen in vertebrate development. To study whether molluscan morphogenesis is sensitive to retinoic acid, the development of Lymnaea stagnalis, Physa fontinalis and Bithynia tentaculata was examined after treatment with retinoic acid. Low concentrations retinoic acid (10–7M) specifically affected eye formation in each of these species. In Lymnaea, it was shown that 10–6M retinoic acid resulted in a wider spectrum of deficiencies, including eye defects, arrested development and shell deformations. Pulse treatments revealed that embryos were most sensitive during gastrulation. Soon after gastrulation the embryos had lost their sensitivity to retinoic acid, which indicates that the observed defects do not result from a general non‐specific toxic effect on cells. Even though molluscan development differs in numerous respects from vertebrate development, the present results suggest that molluscs share common features with vertebrates in morphogenetic processes that operate in early development.
Gene Knockouts and Murine Development
Authors: Gatherer Derek
Published: January 2005

A considerable quantity of data has been generated using the technique of in vivo gene knockout in mice, much of which is of relevance to the developmental biologist. Null mutations in Hox genes at the 3′‐end of the clusters create complex irregularities at the rostral end of the embryo, including defects in the middle ear and the large blood vessels, suggesting that Hox genes may be involved in pattern specification of these structures in addition to the anteroposterior axis. Null mutations in oncogenes either cause wide pleiotropic effects, or act in a restricted manner on the haematopoietic system. Null mutations in growth factors and related molecules cause failure of proliferation in restricted areas of the embryo in some cases, but have little phenotype in others. There is as yet no null mutation which supports the idea that growth factors are involved in mesoderm induction in mammals. A surprising variety of genes have no null phenotype, or one less severe than might have been previously predicted on the basis of their known function in vitro and pattern of expression. This leads to the possibility that genetic redundancy exists in development.
Nonuniformity within Embryonic Somites: Differental Response to Retinoic Acid in Vitro
Authors: Vasan Nagaswami S.
Published: January 2005

Recent studies have shown that in the developing chick embryo, at physiological level retinoic acid (RA) causes mirror‐image duplication of limb skeletal elements. This has led to the suggestion that RA could be the endogenous morphogen or isgnal substance. In this study, in order to explore the effect of RA on somite chondrogenesis, we have standardized a serum‐free chemically defined medium that supports the growth of somite explants in vitro. The results indicate that in somites RA at 10 ng/ml level induces cell proliferation, DNA and sulfated proteoglycan synthesis, and at higher concentrations is toxic. The results further show that RA induced stimulation of somite chondrogenesis is sclerotomal specific and the dermamyotemal portion of the somites does not exihibit a similar response. Retinoic acid also increases heparan sulfate synthesis and aggregation of isolated sclerotomal cells in culture. These results thus suggest that in amplifying chondrogenesis, RA acts at all phases such as cell proliferation (may increase cell viability) and aggregation, increased DNA synthesis and increased synthesis of matrix components. In otherwords, RA seems to initiate a chain of inter‐related events.
Cell Sorting and Chondrogenic Aggregate Formation in Limb Bud Recombinants and in Culture
Authors: Wada Naoyuki , Uchiyama Kohji , Ide Hiroyuki
Published: January 2005

The cartilage pattern of the developing chick limb changes along the proximal‐distal (PD) axis. It is assumed that these spatial changes are brought about by differences in the cellular properties of distal mesoderm, the progress zone (PZ). To examine whether these differences are actually maintained in the individual cells composing the PZ, we dissociated early (stage 20) and late (stage 25) PZ tissues into single cells, then mixed and recombined them with ectodermal jackets. The recombinants were grafted to limb bud stumps and allowed to develop into limb‐like structures. Early PZ cells were distributed within whole cartilage elements along the PD axis of the limb‐like structures, while cells from late PZ participated only in the formation of distal cartilage elements.

A difference in distribution pattern between the cells of early and late PZ in mixed culture was also observed. Cells of early PZ aggregated rapidly in patches and formed cartilage nodules, while the cells of late PZ distributed in regions surrounding these cell aggregates and gradually differentiated to cartilage cells. These results suggest that the cellular properties in the PZ concerning the rate of chondrogenic aggregate formation change during limb bud development, and that this change may relate to the cartilage pattern formation along the PD axis.
The Primary Egg Envelope of the Anuran Lepidobatrachus laevis: Physicochemical and Macromolecular Alterations During Development
Authors: Peavy Thomas R. , Carroll Edward J.
Published: January 2005

The coelomic egg envelope (CE) of the frog Lepidobatrachus laevis has a network of fibrillar bundles which disperse after transit through the oviduct. Following oviposition, the egg vitelline envelope (VE) has an additional amorphous zone on the exterior surface. The fertilization envelope (FE) formed after fertilization, appears to be very similar to the VE. The CEs, VEs, FEs and hatched envelopes (FEh) were manually isolated. The CE, VE and FE were solubilized at 100° using denaturing conditions, but were only partially solubilized in phosphate buffer, pH 7.0. All envelopes and several purified polypeptides from the VE and FE were analyzed using gel electrophoresis and one‐dimensional peptide mapping. Each of the envelopes contained 9 major polypeptides ranging from 118.5 to 22 kD and 8–12 minor polypeptides. Several envelope components were added/removed in the conversions based on the results of experiments in which preparations were incubated with activated egg exudate and crude hatching enzyme; some of these transformations were mimicked by tryptic and chymotryptic digestions. Therefore, serine proteases may be involved in envelope processing in vivo. Lepidobatrachus CE polypeptides and several major components from the VE, FE and FEh were crossreactive with antibodies against Xenopus VE*.
Identification of Early Oogenetic Cells in the Solitary Ascidians, Ciona savignyi and Ciona intestinalis: An Immunoelectron Microscopic Study
Authors: Okada Toshiaki , Yamamoto Masamichi
Published: January 2005

We raised monoclonal antibodies against homogenates of ovaries of Ciona intestinalis. We obtained an antibody named GC‐1 which specifically recognized early germ cells in C. intestinalis and C. savignyi. Using GC‐1 as a marker in immunoelectron microscopy, we determined the morphological sequence of early oogenesis in the ovaries of Ciona. In the stratified epithelium composing the wall of the ovarian tubes, the oocytes were identifiable at the early stages of meiotic prophase according to nuclear features such as condensed chromatin with synaptonemal complexes. GC‐1 recognized these early oocytes. We found round cells with large and homogeneous nuclei clustered at the marginal end of the stratified epithelium. We identified these cells as oogonia on the basis of: (1) features of the nucleus, (2) reactivity to GC‐1, and (3) early emergence in the developing ovaries. The oogonia were classified into three types: type A was large (7–9 μm in diameter) and clear, type B was intermediate in size (5–6 μm) and electron‐density, and type C was small (4–5 μm) and dark. In the developing ovaries of juvenile C. intestinalis, type A oogonia appeared first (before 11 days after settlement) and types B and C followed (15 days after settlement). Thus we see that the type A is the oogenetic stem cell, type B is the proliferating oogonium, and type C is the final oogonium just before meiosis. The oocytes appeared 18 days after metamorphosis.
Developmental Plasticity of the Prospective Dermatome and the Prospective Sclerotome Region of an Avian Somite
Authors: Aoyama Hirohiko
Published: January 2005

The ventro‐medial wall of a somite gives rise to the sclerotome and then to cartilaginous axial skeleton, while the dorso‐lateral wall differentiates into the dermomyotome to form dermal mesenchyme and muscle. Although previous studies suggested pluri‐potency of somite cell differentiation, apparent pluri‐potency may be the result of migration of predetermined cells. To investigate whether the developmental fate of any region is determined, I isolated fragments of a region of a quail somite and transplanted them into chick embryos. When a fragment of the ventral wall of a quail somite, the prospective sclerotome, was transplanted into a chick embryo between the ectoderm and a newly formed somite, the transplanted quail cells were shown to form myotome and mesenchyme in 4‐day chimera embryos and to form muscle and dermal tissue in 9‐day chimeras. On the other hand, when a fragment of the dorsal wall of a quail somite, the prospective dermomyotome, was transplanted into a chick embryo between the neural tube and a newly formed somite, the graft gave rise to mesenchyme around the neural tube and notochord and then to vertebral cartilage. Thus the developmental fate of a region of a somite was shown not to be determined at the time of somite segmentation, confirming previous observations.
The Structure and Development of the Apical Ganglion in the Sea Urchin Pluteus Larvae of Strongylocentrotus droebachiensis and Mespilia globulus
Authors: Nakajima Yoko , Burke Robert D. , Noda Yoshio
Published: January 2005

The structure of the apical ganglion is described using transmission electron microscopy and ultrastructural immunohistochemical localization with anti‐serotonin, and the development of these nerve cells in animalized and vegetalized embryos, and embryos treated with Ca2+‐deficient sea water are demonstrated with immunofluorescence microscopy. The axons within the neuropile contain clear and dense‐core vesicles, but all the vesicles appear anti‐serotonin immunoreactive. The principal type of neuron within the apical ganglions is anti‐serotonin immunoreactive. In the animalized embryos, serotonergic neuroblasts and neurons appear in the second quarter of animal‐vegetal axis. Serotonergic cells were not detected in vegetalized exogestrulae. Mespilia globulus embryos treated with Ca2+‐deficient sea water ruptured to form a cellular sheet on the substratum. In ruptured embryos serotonergic neurons formed a ring around the area corresponding to the apical plate of normal embryos. These findings indicated that the serotonergic preoral nerve cells of echinopluteus derive from cells on the periphery of the apical plate.
Protein Phosphorylation during the Process of Prestalk‐to‐Stalk Conversion in Dictyostelium discoideum
Authors: Maeda Mineko , Kubohara Yuzuru
Published: January 2005

We have previously shown that 8‐bromo cAMP (Br‐cAMP) efficiently induces prestalk‐to‐stalk conversion in in vitro cultures of the cellular slime mold Dictyostelium discoideum, and that protein kinase A (PKA) plays an important role(s) in the conversion process (Kubohara et al. Exp. Cell Res. vol. 207, pp. 107–114, 1993). In the present study, we analyzed protein phosphorylation during the Br‐cAMP‐induced process, and identified two phosphoproteins, p60 and p40, that were specifically induced (phosphorylated) by Br‐cAMP. The protein kinase inhibitors, staurosporine and K252a, which blocked the Br‐cAMP‐induced stalk cell formation, also inhibited the phosphorylations of these proteins. These results suggest that p60 and p40 may be the target proteins of PKA and thus be involved in the prestalk‐to‐stalk conversion.
Temporal Characteristics of the Differentiation of Embryonic Erythroid Cells in Fetal Peripheral Blood of the Syrian Hamster
Authors: Morioka Kiyokazu , Minamikawa‐Tachino Reiko
Published: January 2005

The morphological changes in erythroid cells and their nuclei in the circulation of fetuses of the Syrian hamster were investigated by use of an image‐processing system. The analysis included monitoring of nuclear condensation, nuclear periphralization (access of the nucleus to the cell membrane), enucleation, density of cells, and changes in cell size from day 9 of gestation to day 5 after birth. The yolk‐sac‐derived erythroid cells made rapid progress in nuclear condensation on day 11, while this process proceeded at a much lower rate after day 12 of gestation. The peripheralization of nuclei started on day 10 and reached a maximum on day 11. The frequency of enucleated cells was below 2% on day 11, while it increased to 30% on day 12. Extruded nuclei, most of which were accompanied by a small quantity of cytoplasm, appeared in the circulation on day 12. The most frequently observed diameter of enucleated erythrocytes, which was 10–10.5 μm on day 12, fell gradually to 8–9 μm on day 14. By contrast, the shift from fetal liver erythrocytes to adult erythrocytes occurred in a discontinuous manner. Adult‐type erythrocytes were detected after birth with diameters of 5.5–6 μm. Our data allows us to present the schedule of morphological changes during embryonic erythropoiesis and show that the developmental behavior of “primitive” yolk‐sac‐derived erythroid cells is more closely correlated with that of the “definitive” fetal liver cells than has been considered to be the case to date.
Nonradioactive In Situ Hybridization Methods for Drosophila Embryos Detecting Signals by Immunogold‐Silver or Immunoperoxidase Method for Electron Microscopy
Authors: Amikura Reiko , Kobayashi Satoru , Endo Keita , Okada Masukichi
Published: January 2005

We present details of in situ hybridization methods for electron microscopy applicable for Drosophila embryos. Improvements upon the foregoing methods were made at 1) hybridization and visualization of signals were carried out with whole embryos that were then processed for electron microscopy, and 2) digoxigenin‐labeled probes were detected by the immunogold silver enhancement method or by the immunoperoxidase method. Using these methods. we demonstrated the localization of fushi tarazu transcripts in the apical region of blastodermal cells. We also showed that mitochondrial large ribosomal RNA is associated with polar granules in pole plasm of cleavage embryos. These methods will make a useful tool to determine the precise subcellular distribution of specific transcripts in Drosophila embryos.
Molecular Characterisation of Three Murine HOX11‐Related Homeobox Genes, Tlx‐1, −2, and −3, and Restricted Expression of Tlx‐1 during Embryogenesis
Authors: Cheng Shuk Han , Mak Tak Wah
Published: January 2005

Elevated expression of the human homeobox gene HOX11 has been implicated in T cell acute lymphoblastic leukemia. We report here the isolation and characterisation of three murine genes, Tlx‐1, −2, and −3, which are related to HOX11 and constitute a new class of homeobox genes. Sequence analysis indicates that Tlx‐1 is a murine homolog of HOX11. The expression during development of Tlx‐1 was studied by in situ hybridization. Tlx‐1 expression was found in the first branchial arch, the pancreas, the hindbrain and the epidermis of the ear of the developing embryos. Localized expression of Tlx‐1 in these developing structures suggests that it might play a specific role during post‐implantation development and its aberrant expression might be involved in oncogenesis. The homeodomains of Tlx‐2 and −3 are found to be 75% and 80% identical to that of Tlx‐1, respectively. In addition to the homeodomain, the three Tlx proteins also share four conserved motifs (TH1–TH4). Interestingly, these motifs are also found in other developmental control genes and transcription factors, suggesting a functional role in transcriptional regulation.
A Novel Substance Localizing on the Apical Surface of the Ectodermal and the Esophageal Epithelia of Sea Urchin Embryos
Authors: Yazaki Ikuko
Published: January 2005

A new substance (ES‐1) which localizes on the ectodermal and espophageal epithelia of sea urchin embryos was identified by a monoclonal antibody, McA ES‐1. McA ES‐1 recognized a 175 KDa protein of fertilized and 200 KDa in proteins of unfertilized egg‐cortices. By indirect fluorescent antibody staining, ES‐1 was found on the plasma membrane of fertilized eggs and in the cortical region of unfertilized eggs. ES‐1 was not contained in the cortical granules and remained fixed in the cortex after centrifugation of unfertilized eggs for 30 min at 20,000 g. The polarized localization of ES‐1 on the apical surface of ectodermal epithelial cells continued to the metamorphosis. It disappeared from mesenchyme cells and other migrating cells of the gastrula, while ES‐1 was reexpressed in the presumptive esophagus to be connected with ectodermal epithelium. This may suggest a functional significance of ES‐1 in establishment of cell polarity in the epithelium of larvae. In metamorphosing larvae and adults, the apical localization of ES‐1 could no longer be found, and it was found in coelomocytes. From these findings, it is concluded that ES‐1 was a novel surface substance of embryos and is probably phagocytosed at metamorphosis.
cis‐Elements and Protein Factors Related to Regulation of Transcription of Arylsulfatase Gene during Sea Urchin Development
Authors: Yamada Kazumi , Yamamoto Takashi , Akasaka Koji , Shimada Hiraku
Published: January 2005

Eight restriction fragments (I–VIII) were prepared to cover a whole span of the enhancer region in the upstream of the Ars gene of the sea urchin, Hemicentrotus pulcherrimus, and their abilities to influence on the Ars gene expression were estimated by CAT assay. Only three fragments (III, IV and V) encompassing a 0.6 kb region between −2.8 kb and −2.2 kb stimulated CAT expression. By mobility shift assays, it was found that the Ars enhancer region is composed of multiple cis‐acting elements that interact with nuclear proteins in a sequence‐specific manner. Among them, two sequences, a G‐string and a GATCTCCCC, were determined by DNA footprinting as sites of protein‐DNA interaction. The DNA‐binding factor prevalence changed ontogenically in three different patterns. Possible activation of DNA‐binding proteins through their modification is discussed.
Expression of Regeneration‐Associated Antigens in Normal and Retinoid‐Treated Regenerating Limbs of Ambystoma mexicanum
Authors: Koshiba Kazuko , Tamura Koji , Ide Hiroyuki
Published: January 2005

The expression of two regeneration‐associated antigens in the blastemas of normal and retinoid‐treated regenerating limbs of axolotl (Ambystoma mexicanum) was examined.

One antigen, 55C12, which was similar to tenascin in expression pattern and molecular weight profile, was weakly expressed in the perichondrium and tendon of normal limbs. In the regenerating limbs, the amount of 55C12 antigen increased near the amputation site within 7 days and almost all cells of the blastema mesenchyme came to be positive to the antigen at 20 days, although those of epidermis and most stump tissues were negative. When the regenerating limbs were treated with Am80, a synthetic retinoid, which induced proximo‐distal duplication, the expression of 55C12 antigen in the blastema became weak temporarily and was reactivated in the anterior region of the blastema. This expression pattern suggests that the duplicated limb is formed by the preferential growth of this 55C12‐positive anterior blastema region.

The other antigen, 117C1, was faintly expressed in the epidermis, dermis, muscle, perichondrium and cartilage of normal limbs, and intensely expressed in the blastema mesenchyme and wound epidermis. The Am80 treatment, however, induced no changes in the expression pattern of 117C1.

These results suggest that these antigens may distinguish two different regions of the blastema in normal regeneration and retinoid‐induced duplication.
Acceleration of Retinol‐Induced Epidermal Mucous Metaplasia by Stimulating the Dermal Adenylate Cyclase‐cAMP System in Chick Embryonic Skin: Appearance of cAMP‐Dependent Phosphorylated Proteins in Dermis of Retinol‐Pretreated Skin after 2 h‐Treatment with cAMP
Authors: Obinata Akiko
Published: January 2005

Epidermal mucous metaplasia of cultured 13‐day‐old chick embryonic tarsometatarsal skin can be induced by culture in medium containing excess retinol (20 μM) for only 8–24 h and then in a chemically defined medium with Bt2cAMP (0.2–2 mM) and without retinoids or serum for 2 days. In this work, stimulation of the adenylate cyclase‐cAMP system in retinol‐pretreated skin by forskolin, pertussis toxin, cholera toxin or AIF4– was found to accelerate the synthesis of epidermal sulfated glycoprotein (mucin). In skin induced toward mucous metaplasia by retinol, treatment with forskolin for 1 day increased the cAMP content 10‐fold in the dermis but only 2‐fold in the epidermis over the control levels. The cAMP level of Bt2cAMP (0.2 mM)‐treated skin was 18 times higher in the dermis but rather lower in the epidermis than untreated skin. These results suggest the importance of an adenylate cyclase‐cAMP system in the dermis of skin in stimulating mucous metaplasia induced by retinoids. In fact, cAMP‐dependent protein phosphorylation was seen only in the dermis of retinol‐pretreated skin after 2 h‐treatment with cAMP. As no transfer of cAMP from the dermis to the epidermis of forskolin‐treated skin was detected, there may be no gap junctional communication between the epidermis and the dermis, while the basement membrane becomes discontinuous during mucous metaplasia.
The Developmental and Morphological Studies on the Neural and Skeletal Abnormalities in the T/btm Tailless Mice
Authors: Fujimoto Atsushi , Wakasugi Noboru , Tomita Takeshi
Published: January 2005

The crosses, T/+ or T/tw2×btm/btm, give rise to 50% incidence of the tailless mice, development of which was investigated. No difference was seen in external appearance of the embryos at 9 days of gestation. However, some embryos showed fusion of the notochord and the neural tube at the posterior part of the body on the histological examination. The prospective tailless individuals were distinguishable from the normal littermates by the constriction of the root of the tail at 10 days of gestation. Thereafter, they showed several abnormalities such as the poor growth of the posterior part of the body, thinning of the tail and a blood blister at the tail tip or in the lumbosacral region. The abnormal embryos of 11–12 days showed severer abnormalities in the medio‐dorsal area, i.e., the notochord was branched or degenerated at several places and the neural tube was distorted, duplicated or fused with the mesenchyme. All the tailless newborn young had blood blisters or red scars on the dorsal skin at the middle of the lumbosacral region.

Histologically, the spinal cord posterior to the lumbosacral level was revealed to be severely distorted or duplicated and completely devoid of the bony vertebrae, and the dorsal blood blister was found to be the meningomyelocele derived from the abnormal development of the spinal cord. Skeletal abnormalities of the tailless young were as follows. The sacral and caudal vertebrae were absent. The cervical vertebrae were mostly normal, but the thoracic and lumbar vertebrae showed several abnormalities such as fusion of the ribs, lack of the vertebral body and vertebral arch.
Localization of Type IV Collagen in the Myotome Cells during the Somite Differentiation in the Chick Embryo
Authors: Itoh Masaaki , Arai Katsuhiko , Uehara Kohkichi
Published: January 2005

Type IV collagen is a major structural component of basement membranes which play various roles upon their adjacent cells. In order to better understand roles of type IV collagen during the somite differentiation, we produced anti‐rat type IV collagen polyclonal antibodies and demonstrated spacial and temporal distribution of type IV collagen in somites of the chick embryo by immunohistochemical procedure. Type IV collagen was detected in the basal surface and the cytoplasm of epithelial dermatome cells at early stage of the somite differentiation, and then detected in myotome cells overlying apical surface of dermatomes, but not in migratory mesenchymal dermatome cells. With the appearance of type IV collagen‐expressing myotome cells, epithelial dermatome cells showed the decrease in immunoreaction with anti‐type IV collagen antibodies, the disappearance of their basal‐apical polarity and their epithelial shape. From these results, it was suggested that type IV collagen is an early marker for myotome cells, and that type IV collagen and/or other factors co‐expressed by myotome cells might provide an accelerative signal for epithelial/mesenchymal conversion of dermatome cells.
Branching Morphogenesis of Mouse Embryonic Submandibular Epithelia Cultured under Three Different Conditions
Authors: Mori Yasuko , Yoshida Keiichi , Morita Toshiteru , Nakanishi Yasuo
Published: January 2005

To investigate how the mesenchyme interacts with the epithelium, we employed three different culture systems: System A, in which intact submandibular gland rudiments at the mid 13‐day stage were cultured on Millipore filters; System B, in which the 13‐day epithelium and mesenchyme were separated once with dispase, recombined again, and cultured on the filter; System C, in which the separated 13‐day epithelium was clotted with Matrigel and cultured with the mesenchyme across the filter or in the presence of EGF instead of the mesenchyme. In Systems A and B, 13‐day epithelia expanded and produced similar lobules with narrow clefts and stalk. When the 13‐day epithelium was cultured in System C under the influence of the mesenchyme, it formed rather oval lobules with stalk that were superficially similar to those in System A, but narrow clefts, as seen in the intact early 13‐day gland, were rarely found in System C. Furthermore, no long stalk formation was observed when EGF was introduced in place of the mesenchyme. A bacterial collagenase from Clostridium histolyticum gave a considerable inhibition of branching of the 13‐day epithelium in Systems A and B, but no significant inhibition was observed in System C when the mesenchyme or EGF was employed as the source of diffusible factor(s). In contrast, although the 13‐day epithelium was significantly resistant to the action of heparitinase I from Flavobacterium heparinum in Systems A and B, the enzyme almost completely inhibited the expansion and branching of the epithelium in System C. Judging from these observations, we conclude that the mechanisms of lobular formation in Systems A and B are not the same as those in System C, where the epithelium is clotted with basement membrane matrix components during tissue culture.
Nesiritide during extracorporeal membrane oxygenation
Authors: Smith Todd , Rosen David A. , Russo Pierantonio , Berkenbosch John W. , Gustafson Robert A. , Siu Benjamin L. , Rosen Kathleen R. , Tobias Joseph D.
Published: January 2005

Nesiritide is a recombinant formulation of B‐type natriuretic peptide (BNP). Preliminary experience in the adult population has shown nesiritide to be an effective agent in the treatment of decompensated congestive heart failure (CHF) in adults. Given its physiological effects, it may be an effective agent in other clinical scenarios. We report the use of nesiritide in two infants during extracorporeal membrane oxygenation (ECMO). In one patient, nesiritide in doses up to 0.09 μg·kg−1·min−1 were used to control mean arterial pressure while in the other patient, doses of 0.01–0.03 μg·kg−1·min−1 were used to augment urine output. The potential applications of nesiritide and dosing regimens for this agent in the ECMO population are discussed.
The optimal insertion length of central venous catheter via the femoral route for open‐heart surgery in infants and children1
Authors: SHINOHARA YUKA , ARAI TOSHIMI , YAMASITA MASAO
Published: January 2005

Background:  The recommended insertion length of central venous (CV) catheter via the internal jugular or subclavian vein has been determined in infants and children. However, the insertion length via the femoral vein has not been well‐studied. This study determined the optimal insertion length of CV catheter via the femoral vein.

Methods:  Infants and children, who had undergone cardiac catheterization via the right femoral vein, were the subjects of the study. After routine cardiac catheterization, the distance from the femoral puncture site to the third lumbar vertebral body (L3) level, was measured and recorded. The femoral‐L3 length was termed as the optimal insertion length.

Results:  This length was measured in 78 infants and children (age: 1–101 months, weight: 3.1–33.8 kg). The body weight of the patient and the length correlated well: the optimal insertion length (cm) = 0.45 × body weight (kg) + 8.13, coefficient of determination (R2) = 0.84.

Conclusions:  It has been recommended to place the tip of the catheter below the level of renal veins to avoid blocking free flow of those veins. Therefore, we chose the mid‐point, L3 level as the optimal tip position of the femoral venous catheter. The length derived from the above formula could be used as a guideline for CV catheter insertion via the femoral vein in infants and children.
Estimation of circulating blood volume in infants using the pulse dye densitometry method
Authors: NAGANO KEIKO , KUSAKA TAKASHI , OKUBO KENSUKE , YASUDA SANEYUKI , OKADA HITOSHI , NAMBA MASANORI , KAWADA KOU , IMAI TADASHI , ISOBE KENICHI , ITOH SUSUMU
Published: January 2005

Background:  Estimation of hemodynamics is important for critically ill infants. Pulse dye densitometry (PDD) using indocyanine green (ICG), which enables measurements of circulating blood volume at the bedside, has recently been developed for adults.

Methods:  We conducted a basic investigation to determine whether this method can be applied to infants and measured circulating blood volume in 25 infants whose gestational ages ranged from 24 to 40 weeks (median, 32 weeks). At first, to validate the accuracy of measurements, arterial ICG concentrations determined by blood sample measurements were compared using a spectrophotometer ([ICG blood]) and by noninvasive measurement using PDD ([ICG pdd]) in seven infants. Next, blood volumes in 25 infants were estimated by the PDD method.

Results:  There was a positive relationships between [ICG blood] and [ICG pdd] (r = 0.913, P < 0.0001). Using Bland Altman analysis, the bias between the two methods was 0.24 ± 0.30 mg·l−1 (95% confidence interval: 0.39–0.09 mg·l−1) and the limits of agreement (2 sd) were −0.36  and 0.84 mg·l−1, respectively. Mean (sd) blood volume was 94.9 ml·kg−1 (24.3). The values obtained by this study are almost the same as previously reported values obtained by using other methods.

Conclusions:  PDD using ICG can be used to monitor of hemodynamics in infants.
Ketorolac after congenital heart surgery: does it increase the risk of significant bleeding complications?
Authors: GUPTA ANUJA , DAGGETT CASEY , LUDWICK JOSEPH , WELLS WINFIELD , LEWIS ALAN
Published: January 2005

Background:  The routine use of ketorolac after congenital heart surgery in infants and children is limited by concerns for postoperative bleeding complications. The object of this study was to determine if the use of ketorolac is associated with an increased risk of significant postoperative bleeding after congenital heart surgery in infants and children.

Methods:  A retrospective chart review was performed. The exposure of interest was postoperative use of ketorolac after congenital heart surgery in infants and children. The outcome measured was postoperative bleeding requiring surgical exploration. The patients who received ketorolac were compared with an age‐ and diagnosis‐matched comparison group who did not receive ketorolac.

Results:  Records of 842 infants and children who underwent congenital heart surgery between July 2001 and October 2002 were reviewed. 94 (11.1%) patients were treated with ketorolac postoperatively. The comparison group consisted of 94 matched subjects selected from the patients that did not receive ketorolac. The mean age of patient in the ketorolac group was 8.5 (±6.1) years. No (0%) patients in the ketorolac group and four (4.2%) patients in the nonketorolac group developed postoperative bleeding requiring surgical exploration. The relative risk for postoperative bleeding that required surgical exploration in the ketorolac group compared with the nonketorolac group was 0.2 (95% CI 0.02–1.67).

Conclusions:  The use of ketorolac after congenital heart surgery in infants and children does not significantly increase the risk of bleeding complications requiring surgical exploration.
Topiramate and metabolic acidosis: a case series and review of the literature
Authors: GROEPER KELLY , McCANN MARY ELLEN
Published: January 2005

Topiramate is a new anticonvulsant drug recommended for treatment of partial and generalized seizures in children and adults. It has been found to cause a nonanion gap metabolic acidosis in some patients, which is related to carbonic anhydrase inhibition. This adverse reaction is more common in children than adults and is rarely symptomatic. Clinicians need to be aware of this potential side effect especially in children undergoing major surgery. Children who are treated with topiramate should have a careful history taken preoperatively looking for signs of a metabolic acidosis and baseline blood chemistries should be measured prior to surgery to detect an asymptomatic metabolic acidosis.
A. R. Lord and Later Idealist Political Philosophy
Authors: Sweet William
Published: January 2005

This essay introduces and discusses some central themes in the political philosophy of the British/South African idealist, Arthur Ritchie Lord (1880–1941). Lord was the author of the most substantial text of third generation ‘British idealist’ political thought, The Principles of Politics (1921) , but many of his manuscripts from the 1920s to early 1940s have only recently been prepared for publication. I argue that Lord's work provides valuable insights into the development of later idealist political philosophy, particularly on two themes that distinguish it from that of Lord's idealist predecessors, T. H. Green and Bernard Bosanquet—namely, democracy and freedom. This study also raises the question of whether the distinctively pluralistic context of South Africa had an influence on Lord's analysis of these themes.