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Y Wakamatsu

Publications and source records attributed to Y Wakamatsu.

At least 19 recordsLinked to original sources
PubMed1999

Mannosylerythritol lipid is a potent inducer of apoptosis and differentiation of mouse melanoma cells in culture.

Malignant melanomas are tumors that are well known to respond poorly to treatment with chemotherapeutic reagents. We report here that mannosylerythritol lipid (MEL), an extracellular glycolipid from yeast, markedly inhibited the growth of mouse melanoma B16 cells in a dose-dependent manner. Exposure of B16 cells to MEL at 10 microM and higher concentrations caused the condensation of chromatin, DNA fragmentation, and sub-G1 arrest, all of which are hallmarks of cells that are undergoing apoptosis. Analysis of the cell cycle also suggested that both the MEL-mediated inhibition of growth and apoptosis were closely associated with growth arrest in the G1 phase. Moreover, MEL exposure stimulated the expression of differentiation markers of melanoma cells, such as tyrosinase activity and the enhanced production of melanin, which is an indication that MEL triggered both apoptotic and cell differentiation programs. Forced expression of Bcl-2 protein in stably transformed B16 cells had a dual effect: it interfered with MEL-induced apoptosis but increased both tyrosinase activity and the production of melanin as compared with these phenomena in vector-transfected MEL-treated control B16 cells. These results provide the first evidence that growth arrest, apoptosis, and the differentiation of mouse malignant melanoma cells can be induced by a microbial extracellular glycolipid.

Animals↗
PubMed1999

Transplantation of blastula nuclei to non-enucleated eggs in the medaka, Oryzias latipes.

Studies of nuclear transplantation were conducted to establish methods for the production of clones of fish, using a small laboratory fish, medaka, Oryzias latipes. As the first step of the study, single-blastula nuclei of an inbred strain with the wild-type body color were transplanted into non-enucleated unfertilized eggs of an outbred orange red strain. Of 845 operated eggs, 45 hatched into fry exhibiting the wild-type body color, one of the donor markers. Twenty-seven of these nuclear transplants grew to the adult stage and clearly exhibited external secondary sexual characteristics. Fourteen were females and 13 were males. The allozyme analysis of phosphoglucomutase, measurements of relative DNA content by microfluorometry and chromosome counts consistently indicated that the nuclear transplants were triploids that originated from both the diploid donor nuclei and the haploid recipient pronuclei. In the crossing experiments between the nuclear transplants and the orange-red strain, most of the male nuclear transplants were sterile, whereas one male produced a viable offspring with wild-type body color. All of the female nuclear transplants were sterile. Macroscopic observations of their gonads showed that the testes appeared normal and the ovaries appeared degenerated. These features of the reproductive potential and the morphology of gonads also indicated that the nuclear transplants were triploids. These results demonstrated that a basic technique for nuclear transplantation in medaka was established.

Animals↗
PubMed1998

Avian neural crest-derived neurogenic precursors undergo apoptosis on the lateral migration pathway.

Neural crest cells of vertebrate embryos disperse on distinct pathways and produce different derivatives in specific embryonic locations. In the trunk of avian embryos, crest-derived cells that initially migrate on the lateral pathway, between epidermal ectoderm and somite, produce melanocytes but no neuronal derivatives. Although we found that melanocyte precursors are specified before they disperse on the lateral pathway, we also observed that a few crest-derived neuronal cells are briefly present on the same pathway. Here, we show that neuronal cells are removed by an episode of apoptosis. These observations suggest that localized environmental factor(s) affect the distribution of fate-restricted crest derivatives and function as a 'proof-reading mechanism' to remove 'ectopic' crest-derived cells.

Animals↗
PubMed1998

Usefulness of the medaka beta-actin promoter investigated using a mutant GFP reporter gene in transgenic medaka (Oryzias latipes).

The activity of the medaka beta-actin promoter as a ubiquitous expression vector in transgenic medaka was examined using complementary DNA of the green fluorescent protein (GFP). Plasmid pOBA-GFP contained both the medaka beta-actin promoter and cDNA of the wild-type GFP, while pOBA-hGFP contained the medaka beta-actin promoter and cDNA of the mutant GFP in which serine was substituted for threonine at position 65 and codon usage was humanized to promote translation in vertebrate cells. The ApaI-SmaI fragment of both plasmids was microinjected into the nuclei of oocytes or the cytoplasm of embryos at the one-cell stage. The gene expression was detected, using a fluorescent stereomicroscope, from early stages of development to 1 week after hatching. The expression of the wild-type GFP was detected in early embryos, in the yolk sac and in small portions of the muscle and epidermis. This expression pattern was similar to that of the Escherichia coli beta-galactosidase reporter gene (lacZ), driven by the medaka beta-actin promoter, which was examined in our previous studies. The mutant GFP was expressed in early embryos and in many tissues such as the epidermis, blood vessels, muscle, notochord, fin ray, gut, eyes, and yolk sac, and the fluorescence was much stronger than that of the wild-type GFP. Thus, the usefulness of the medaka beta-actin promoter as a ubiquitous expression vector was confirmed using the mutant GFP as a reporter gene.

Actins↗
PubMed1997

An embryological study of ventralization of dorsal structures in the tail of medaka (Oryzias latipes) Da mutants.

In adult Da (double anal fin) mutants of medaka (Oryzias latipes), structures such as the dorsal fin and the dorsal half of the caudal fin are ventralized in adult fish. However, there have been few embryological studies of the development of mutant phenotypes except those of the caudal fin. In this study, development of mutant phenotypes of the tail where they typically develop was examined morphologically at various stages of embryogenesis. The arrangement of melanocytes along the dorsal midline, the shape of the dorsal fin fold, and the shape of the dorsal myotome exhibited a ventral pattern in the tail at various embryonic stages in Da mutants.

Animals↗
PubMed1997

Regulation of the neural crest cell fate by N-myc: promotion of ventral migration and neuronal differentiation.

During neural crest development in avian embryos, transcription factor N-myc is initially expressed in the entire cell population. The expression is then turned off in the period following colonization in ganglion and nerve cord areas except for the cells undergoing neuronal differentiation. This was also recapitulated in the culture of Japanese quail neural crest, and the cells expressing N-myc eventually coincided with those expressing neurofilaments. These findings suggested that N-myc is involved in regulation of neuronal differentiation in the neural crest cell population. In fact, transient overexpression of N-myc in the neural crest culture by transfection resulted in a remarkable promotion of neuronal differentiation. An experimental procedure was developed to examine the effect of exogenous N-myc expression in the neural crest cells in embryos. Neural crest cell clusters still attached to the neural tube were excised from Japanese quail embryos, transfected and grafted into chicken host embryos. Using this chimera technique, we were able to analyze the consequence of transient high N-myc during the early phase of neural crest migration. Two effects were demonstrated in the embryos: first, high N-myc expression provoked massive ventral migration of the neural crest population and, second, those cells that migrated to the ganglion-forming areas underwent neuronal differentiation with the cell type determined by the nature of the ganglion. Thus, N-myc is involved in regulation of the neural crest fate in two different aspects: ventral migration and neuronal differentiation.

Animals↗
PubMed1997

Sequential expression and role of Hu RNA-binding proteins during neurogenesis.

We have identified three avian (chicken) Hu/elav family RNA-binding protein genes. cHuD and cHuC are expressed specifically in neurons of both the central and peripheral nervous systems. Although cHuA is expressed in a wide variety of tissues, including neurogenic precursor cells, it is transiently down-regulated, and is then re-expressed in maturing neurons. Misexpression of cHuD in cultured neural crest cells results in a dramatic increase in the proportion of cells exhibiting neuronal morphology, molecular markers for neurons, and neurotrophin dependence. These data confirm that cHuD protein is involved in regulating neuronal differentiation.

3T3 Cells↗
PubMed1996

Transgenic expression of L-gulono-gamma-lactone oxidase in medaka (Oryzias latipes), a teleost fish that lacks this enzyme necessary for L-ascorbic acid biosynthesis.

Transfer of the gene for L-gulono-gamma-lactone oxidase, the missing enzyme in L-ascorbic acid biosynthesis in scurvy-prone animals, into medaka (Oryzias latipes) was successfully done. The expression plasmid pSVL-GLO, carrying rat liver L-gulono-gamma-lactone oxidase cDNA, was microinjected into the cytoplasm of fertilized eggs during the one-cell stage. Four male F0 fish having the transgene in their germ cells came to maturity, and F1 progeny derived from one of the F0 fish possessed L-gulono-gamma-lactone oxidase activity, indicating that the transgene was functionally expressed in the fish. Genomic Southern blot analysis demonstrated that the transgene existed in both chromosome-integrated and extrachromosomal forms.

Animals↗
PubMed1996

Regionalized expression of the Dbx family homeobox genes in the embryonic CNS of the mouse.

Here we report the identification of a novel homeobox gene family Dbx in mouse, which consists of Dbx and Dbx2. The two genes share similar structural organization and are encoded by different chromosomes. The predicted Dbx and Dbx2 proteins share 85% identity in their homeodomain amino acid sequences, but otherwise showed no significant similarity. Characterization of the expression of these two genes in the embryos suggested their role in the development of the CNS. In the forebrain, Dbx is expressed in various regions, while Dbx2 showed a more restricted pattern of expression. In the midbrain, the expression domains of Dbx and Dbx2 overlap along the dorso-lateral wall of the ventricle. In the hindbrain and spinal cord, both genes are expressed in the boundary separating the basal and alar plates, which seems to correspond to the sulcus limitans. Expression of the Dbx/Dbx2 genes is restricted to the ventricular region of the embryonic CNS except for that of Dbx in the septum of the telencephalon. Together these observations indicate possible participation of the members of the Dbx family in regionalization of the CNS. While the expression of Dbx was restricted to the CNS, Dbx2 was also expressed in some of the mesenchymal cells, such as limb buds and tooth germs.

Amino Acid Sequence↗
PubMed1995

Initiation of the transgenic lacZ gene expression in medaka (Oryzias latipes) embryos.

In total, 4165 medaka (Oryzias latipes) oocytes were injected with three DNA constructs separately, and results showed that exogenous lacZ expression was transient and stage-dependent. The initiation of the transgene expression was at the mid-blastula stage for embryos derived from oocytes injected with pmiwZ, containing the long terminal repeat (LTR) of the Rous sarcoma virus, and with pCAGGS-lacZ, containing the enhancer and promoter of the immediate early gene of the human cytomegalovirus, respectively, whereas embryos derived from oocytes injected with pMoZtk, containing the LTR of the Moloney murine leukemia virus, started expression at the late-blastula stage. These reveal that the earliest onset of the exogenous lacZ gene should be by the mid-blastula stage. Therefore the mid-blastula transition phenomenon in embryogenesis known in other animal species exists in medaka embryos.

Animals↗
PubMed1994

Establishment of a pluripotent cell line derived from a medaka (Oryzias latipes) blastula embryo.

A pluripotent cell line, OLES1, was established from a blastula embryo of a small freshwater fish, medaka (Oryzias latipes). Cells of this cell line were small and round, and they grew actively and stably in culture as dense clusters. They exhibited a positive alkaline phosphatase activity upon histochemical staining. When the cells were treated with retinoic acid, differentiation into various types of cells, including melanocytes, dopa-positive precursors of melanocytes, and cells with a molecular marker of skeletal muscles, troponin T, was induced in vitro. The present study opens a way to establishing embryonic stem cell lines in fish.

Alkaline Phosphatase↗
PubMed1994

An efficient expression vector for transgenic medaka construction.

The transparency and external fertilization of the eggs of medaka (Oryzias latipes) make them ideally suitable for investigating molecular interactions that occur during vertebrate development. Genetically engineered medaka is a potential tool for such studies. It requires several types of suitable expression vectors. To obtain abundant and ubiquitous expression of foreign genes in medaka embryos, we have designed an expression vector that contains the proximal promoter and enhancer elements and polyadenylation signal of the medaka beta-actin gene. The utility of this "all-medaka" expression vector was examined using the Escherichia coli lacZ gene as a reporter gene. Most of the injected embryo showed high gene expression, and several embryos showed ubiquitous expression even at six days after injection. Of nine individuals derived from the injected embryos and grown until adult stage, one produced expression-positive F1 fish. The transgene was identified in these F1 using polymerase chain reaction (PCR). These data revealed that the expression vector based on the expression cassette from the medaka beta-actin gene should be useful for making transgenic medaka. The cloned gene in this cassette vector is stably transmittable and efficiently expressible.

Actins↗
PubMed1993

A mouse homologue of the Drosophila tumour-suppressor gene l(2)gl controlled by Hox-C8 in vivo.

The homeobox is a 183-base-pair DNA sequence originally found in Drosophila segmentation and homeotic genes. In Drosophila, homeotic genes are clustered in the Antennapedia and Bithorax complexes, collectively called the homeotic gene complex (HOM-C). In the mouse genome, about 40 homeobox genes (Hox) are clustered in four chromosomal regions (Hox A to D). The Hox genes are arranged in the same order and have the same anteroposterior pattern of expression as their structural homologue in the HOM-C, suggesting that they control mouse pattern formation in the same way that HOM-C members do in Drosophila. Homeobox gene products are believed to be transcription factors that regulate expression of target genes. A few candidate target genes have been identified in Drosophila by various approaches but the Hox gene targets are poorly understood, mostly because of limitations in the available approaches. Here we identify several candidate Hox gene targets, including a mouse homologue of the Drosophila tumour-suppressor gene l(2)gl, by immunopurification of DNA sequences bound to a Hox protein in native chromatin.

Amino Acid Sequence↗
PubMed1993

Murine chromosomal location of four class III POU transcription factors.

A family of mouse genes encoding class III POU transcription factors consists of four members: Oct-6, Brn-2, Brn-1, and Brn-4. In accordance with mouse nomenclature, these loci have been designated Otf-6, Otf-7, Otf-8, and Otf-9, respectively. While the four genes share a highly conserved class III POU domain, the remaining regions are considerably diverged from each other. The POU domains of these transcription factors recognize the octamer-motif sequence and possess a similar DNA-binding specificity. Chromosomal mapping has demonstrated that the four genes are unlinked: Otf-6 is located on the distal region of chromosome 4, Otf-7 on the proximal region of chromosome 4, Otf-8 on chromosome 1, and Otf-9 on the X chromosome.

Animals↗
PubMed1993

Organization and expression of mouse Hox3 cluster genes.

We determined the physical linkage of six mouse Hox3 homeobox sequences, including a new homeobox sequence (Hox3.5), by analysis of overlapping genomic clones. Additionally, we defined the locations of Hox1.7 and Hox1.8 in the Hox1 cluster. Analysis of the expression patterns of Hox3.6 and Hox3.5 during embryogenesis revealed that the relationship between relative position in the Hox3 cluster and expression domain along antero-posterior axis appears similar to that seen for members of the other Hox clusters.

Amino Acid Sequence↗
PubMed1993

Transition of localization of the N-Myc protein from nucleus to cytoplasm in differentiating neurons.

N-myc is a developmentally regulated proto-oncogene encoding a putative sequence-specific DNA-binding protein. Previous studies on tissue distribution of N-myc transcripts indicated that one of the major sites of N-myc expression is the CNS and neural crest derivatives in developing embryos. We investigated N-Myc protein expression in embryonic neural tissues and found that the protein was usually localized in the nucleus, but accumulated in the cytoplasm upon differentiation of specific classes of neurons, e.g., retinal ganglion cells, neurons of spinal ganglia, and Purkinje cells of the cerebellum. The change of localization of N-Myc from the nucleus to the cytoplasm indicates a novel feature of regulation of myc family proteins and suggests functions of N-myc in the cytoplasm of maturing neurons.

Amino Acid Sequence↗