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Biomedical subjects

G Eguchi

Publications and source records attributed to G Eguchi.

At least 19 recordsLinked to original sources
PubMed1996

Characterization of terminally differentiated cell state by categorizing cDNA clones derived from chicken lens fibers.

To characterize a terminally differentiated state of cells at the gene expression level, a cDNA library of chicken lens fibers was analyzed. The major population of the library consisted of cDNAs encoding delta-crystallin (about 35% of the recombinants) and other crystallins (alpha A-, alpha B-, beta A3/A1-, beta B1-, beta B2-), as well as cytoskeletal proteins (CP49, CP95), and membrane protein (MP28). These cDNA clones representing lens structural proteins known, accounted for about 60% of the library. When 96 clones were randomly selected from this library, 55 clones corresponded to the above-mentioned major class proteins. Analyses of the remaining clones indicated that many of them were expressed in a lens-specific manner at very low levels. The partial nucleotide sequence analysis of these clones revealed that two cDNAs corresponded to the genes encoding lens-type connexin, three cDNAs to the genes encoding housekeeping proteins, and some cDNAs to the genes encoding regulatory proteins. The mRNA composition in the chicken lens fiber cells indicated rather simple organization of mRNA species of this cell type, and gave scope to the possibility of full description of differentiated lens fiber cells at gene activity level.

Animals↗
PubMed1996

Isolation of a novel chick homolog of Serrate and its coexpression with C-Notch-1 in chick development.

Intercellular signaling mediated by the transmembrane proteins, Notch as receptor and its ligands, Delta and Serrate, plays essential roles in the developmental fate decision of many cell types in Drosophila. The Notch genes are highly conserved both in invertebrates and in vertebrates, suggesting that Notch pathway regulates cell fate decisions during vertebrates development. Notch, Delta and Serrate homologs in chicken have been cloned (Henrique et al., Nature 375: 787-790, 1995; Myat et al., Dev. Biol. 174: 233-247, 1996). We isolated a novel chick homolog of Drosophila Serrate, named C-Serrate-2, and examined its expression patterns during the early chick development using whole-mount in situ hybridization. C-Serrate-2 transcripts were detected in several tissues including the forebrain, the myotome and the apical ectodermal ridge (AER) of the limb bud of a 4-day-old chick embryo. In most of the regions where C-Serrate-2 was expressed, C-Notch-1 was also expressed. Our observations suggest that Serrate-2-Notch-1 signaling plays a role in a variety of morphogeneses during the chick development.

Amino Acid Sequence↗
PubMed1996

Genesis of newt sperm axial fiber: cDNA cloning and expression of a 29 kDa protein, a major component of the axial fiber, during spermatogenesis.

Newt sperm has a unique structure: the tail consists of axial fiber, undulating membrane and flagellum. The genesis and chemical composition of the axial fiber remain unknown. The axial fiber consists of about 10 major components, as evidenced by SDS-polyacrylamide gel electrophoresis. In order to clarify the biochemical properties of the components of the axial fiber and study the mechanism of axial fiber formation, we focused our attention on a 29 kDa protein, the major constituent of the axial fiber. Immunofluorescent antibody technique showed that the 29 kDa protein was first expressed in the cytoplasm of early round spermatids but was expressed on fibers in the periphery of the cyst in late round spermatids. Double staining with tubulin antibody and 29 kDa antibody showed that the fibers around the cysts in early round spermatids were flagella alone but those in late round spermatids consisted of flagella and 29 kDa protein. These results indicated that 29 kDa proteins are synthesizsed in the cytoplasm of round spermatids and enter the preformed flagella in late round and elongated spermatids. A cDNA clone for 29 kDA protein was isolated. A database search could not find any homologous clones, indicating that the 29 kDa protein is a new one. Northern blot with the cDNA showed that mRNA for 29 kDa protein was highly expressed in round spermatids but barely in primary spermatocytes, indicating that the mRNA for 29 kDa protein is haploid-expressed.

Amino Acid Sequence↗
PubMed1995

From lens regeneration in the newt to in-vitro transdifferentiation of vertebrate pigmented epithelial cells.

Through studies to clarify the cellular origin of lens regeneration in the newt, the pigmented epithelial cells of the iris and the retina of many vertebrate species have been shown to possess a dormant potency to transdifferentiate into the lens. The method of in-vitro culture of pigmented epithelial cells has been optimized to enable detailed studies of the transdifferentiation process by molecular techniques. Growth factors and extracellular matrix components are found to be important in the control of the transdifferentiation process. New systems for in-vitro culture are introduced, while prospects for renewed in-vivo studies using newts are given.

Amphibians↗
PubMed1995

Long-term histological evaluation of hydroxyapatite ceramics in humans.

We have investigated the interface between bone and hydroxyapatite (HA) chronically implanted in man. By light microscopy, HA appeared to bind directly to bone without intervening fibrous tissue. By transmission electron microscopy, two patterns were noted: (1) HA either bound directly to bone; or (2) electron-dense material intervened between HA and bone. The orientation of bone collagen fibres likewise showed two patterns: (1) collagen fibres were oriented parallel to the HA; or (2) the fibres were aligned perpendicularly. We have observed similar binding properties of HA to the jaw bone of humans in vivo and in vitro.

Adult↗
PubMed1995

A comparative study of 123I-IMP SPET and CT in the investigation of chronic-stage head trauma patients.

The aims of this study were (1) to compare N-isopropyl-p-[I-123]-iodoamphetamine (123I-IMP) SPET with computed tomography (CT) in chronic-stage head trauma patients with neurological abnormalities and (2) to quantify regional cerebral blood flow (rCBF) in patients with chronic-stage head trauma. 123I-IMP SPET and CT were performed in 23 patients with chronic-stage head trauma and 12 normal controls. Both types of imaging were evaluated visually. In addition for the SPET images, the rCBF in 12 regions (bilateral frontal cortex, temporal cortex, parietal cortex, occipital cortex, perilolandic area, cerebellum: total 276 regions) was analysed quantitatively using a reference sampling method. In total, 39 focal lesions related to neurological abnormalities were detected. Although 22 (56.4%) lesions were observed using both CT and SPET, another 17 (43.6%) were detected by SPET only. The mean rCBF in the localized lesions of each lobe, which correspond to low-density areas (LDAs), were lower on CT than those of each lobe seen only on SPET. These rCBF values were lower than the mean rCBF values in the lobes of the controls. The mean rCBF values in each lobe with visually normal tracer uptakes were also significantly lower in the 23 patients than in the controls. 123I-IMP SPET is useful for demonstrating brain dysfunction in morphologically intact brain regions and for providing objective evidence to account for the clinical presentation of patients presenting with chronic traumatic brain injury.

Accidents, Traffic↗
PubMed1995

Gene transfer into circulating primordial germ cells of quail embryos.

During early stages in avian embryogenesis primordial germ cells (PGCs) show a unique migration pathway toward the gonadal anlage through the circulation. In the present study, liposomes consisting of plasmid DNA (pMiwZ; containing lacZ as a reporter) and Lipofectin were injected into the marginal veins of quail embryos during the stages PGCs were circulating in the blood vessels. The lacZ expression was then histochemically detected in the gonads at later embryonic stages, indicating the expression of the injected DNA in PGCs.

Animals↗
PubMed1995

The predicted structure of chick lens CP49 and a variant thereof, CP49ins, the first vertebrate cytoplasmic intermediate filament protein with a lamin-like insertion in helix 1B.

The full length cDNA sequence for the lens-specific intermediate filament protein, CP49, from chicken is presented. The sequence contains features typical of the other intermediate filament proteins, including two major alpha-helical regions, helix I and II and appropriate linker regions. CP49 lacks a C-terminal non-alpha-helical domain and is only the second intermediate filament protein to be described missing this feature. Comparison to the bovine CP49 shows significant homology in all domains except the N-terminal non-alpha-helical domain. Besides bovine CP49, the other protein most homologous to chicken CP49 in the database was keratin 18, a type I keratin. A variant of CP49 is also described, called CP49ins. Of the 61 positive clones identified in the library, two encoded CP49ins, one of these being a full-length clone. The sequence differed to CP49 by the insertion of 49 amino acids in helix IB. This is the first chordate cytoplasmic intermediate filament protein sequence to be identified with an archetypal lamin-like insertion in this helical subdomain and represents a key discovery in tracing the evolutionary pathway of intermediate filament protein family.

Amino Acid Sequence↗
PubMed1995

Establishment and characterization of human fetal liver epithelial cell line transfected with SV40 T antigen.

The aim of the present study was to establish a stable cell line useful for the study of growth and differentiation of human fetal liver cells. We have established an immortalized human fetal liver cell line (designated NFL/T) by transfection with simian virus 40 (SV40) large T antigen, without any culture crisis. The cells showed growth properties similar to normal cells, including density-dependent cell growth. Electron microscopy demonstrated liver-specific differentiated morphological properties. Moreover, positive albumin and cytokeratin 7 production was detected immunologically. Thus, the NFL/T cell line is less transformed than the other fetal liver cell lines established by this method, and might be useful to study the growth and differentiation of human fetal liver cells.

Animals↗
PubMed1995

Transforming growth factor-beta 1 induces morphological changes accompanied by extracellular matrix reconstitution in cultures of avian retinal pigmented epithelial cells.

The mechanisms of cell differentiation have been extensively studied with an avian retinal pigmented epithelial cell (PEC) culture system. Transforming growth factor-beta (TGF-beta) has been reported to have stimulatory or inhibitory effects on differentiation depending on tissue type. Although the mechanism by which TGF-beta exerts diverse effects is unknown, changes in the extracellular matrix (ECM) are believed to be related to the effects of TGF-beta. Therefore, the effects of TGF-beta 1 on the cellular morphology and the distribution of ECM components such as laminin, tenascin, fibronectin, and types I and IV collagens were investigated in confluent cultures of differentiated PECs during a period of 6 days. We found that differentiated PECs with polygonal morphology and pigmented granules changed to pleomorphic and less pigmented cells 4 days after the addition of TGF-beta 1. These changes were preceded by changes in the distribution of ECM components, particularly laminin and tenascin after 2 days. The effects of TGF-beta on the differentiation of PECs are discussed.

Animals↗
PubMed1994

A complete culture system for avian transgenesis, supporting quail embryos from the single-cell stage to hatching.

We report here a method to produce quail hatchlings by culture in vitro from the single-cell stage. The culture is composed of three steps. In the first step, the fertilized ovum surrounded by thick albumen obtained from the magnum is cultured for 24 hr at 41.5 degrees C in a tightly sealed 20-ml plastic cup with chicken thin albumen added to the equator level of the ovum (System Q1). In the second step, a quail egg shell, cut horizontally and emptied, is used as a bed shell. After the thick albumen is removed, the embryo with egg yolk is transferred to the bed shell and thin albumen from chicken eggs is added to fill the shell. Then, the embryo is cultured for an additional 52 hr at 37.5 degrees C while being rocked at an angle of 90 degrees at 30-min intervals (System Q2). The embryo is transferred again to a chicken bed shell and cultured at 37.5 degrees C with rocking at a 30-degree angle (System Q3). Just before hatching, the rocking of embryos is stopped. The procedure yielded a hatchability of 25%. For transgenesis, a plasmid construct containing a beta-actin-lacZ hybrid gene (pMiwZ) is microinjected into the ovum at the single-cell stage, which is cultured in vitro for 85-90 hr using Systems Q1 and Q2 consecutively. Seven out of 17 surviving embryos exhibited lacZ gene expression in embryonic tissues as detected by histochemistry. The procedure described here should be highly applicable for the production of transgenic birds.

Actins↗
PubMed1994

Gene regulation and differentiation in vertebrate ocular tissues.

Molecular biological techniques have contributed greatly to the study of vertebrate ocular tissues. The specification of ocular tissues has been shown to be closely related to the expression of transcription factors encoded by genes such as Pax6 and microphthalmia. Lens-specific expression of the delta 1-crystallin gene is controlled by factors, such as delta EF1, binding to its enhancer sequences. Retinal activity of the glucocorticoid hormone receptor is regulated by its binding with another transcription factor. Degeneration of photoreceptors in a retinal disease, retinitis pigmentosa, can be caused by the introduction of a mutated opsin gene into mice. In addition, the process of transdifferentiation in ocular tissues has been described at the level of gene expression.

Animals↗
PubMed1994

The loss of gap junctional cell-to-cell communication is coupled with dedifferentiation of retinal pigmented epithelial cells in the course of transdifferentiation into the lens.

Retinal pigmented epithelial cells (PECs) of the chick embryo can be cultured as a monolayer of melanized hexagonal cells. Modifications of the culture condition make the cells lose most of the phenotypes and further transdifferentiate into lentoid bodies within a few weeks. Ultrastructural observations showed that PECs and the lentoids have gap junctions with distinct morphology. Diffusion of a fluorescent dye confirmed the presence of gap junctions in both phenotypes. However, cells in the intermediate stage of transdifferentiation, which show neither the phenotype of the PEC nor that of the lentoid and are called dedifferentiated PECs here, have almost no gap junction structure. We propose the possibility that the dedifferentiation of PECs and the loss of cell-to-cell communication are tightly coupled events. This cell culture system is a suitable material for further studying this relationship by cellular and molecular approaches.

Animals↗
PubMed1994

Predominant melanogenesis and lentoidogenesis in vitro from multipotent pineal cells by dimethyl sulfoxide and hexamethylene bisacetamide.

Pineal cells of the 8-day embryonic quail are multipotent cells which differentiate in vitro into skeletal muscle fibers, pigmented epithelial cells (PECs), lens cells and neurons. However, it was not yet clear whether precursor cells which gave such a wide repertoire of differentiation were single type or not. The present culture studies revealed that pineal cells were exclusively directed to ocular differentiation pathways by dimethyl sulfoxide (DMSO) and hexamethylene bisacetamide (HMBA), suggesting a single type of precursor cell in the pineal body. DMSO directed pineal cells to differentiate into PECs. Co-administration of basic fibroblast growth factor (bFGF) with DMSO partially inhibited PEC differentiation and promoted lens cell differentiation. Northern blot analysis using cDNAs specific to PEC and lens cell confirmed this morphological observation. HMBA completely inhibited pigmentation of cultured pineal cells and markedly promoted lens cell differentiation. Ocular differentiation of pineal cells was accompanied with the loss of myogenicity. We discuss three possible pathways of lens cell differentiation from pineal cells. The agents which affect pineal cell differentiation seemed to modulate the cell-substrate interaction. And the interaction was suggested to be one of the environmental cues in the differentiation.

Acetamides↗
PubMed1993

Retinal differentiation from multipotential pineal cells of the embryonic quail.

Pineal cells of the embryonic quail are multipotent stem cells which are able to differentiate in vitro into pigmented epithelial cells, lens cells and skeletal muscle fibers. Neuronal expression was added in this study in the repertory of differentiating potency of pineal cells. We used immunohistochemical methods to characterize neuronal properties with antibodies against serotonin, GABA, tyrosine hydroxylase and neuron-specific antigen (HPC-1) in addition to the enzyme histochemistry for acetylcholinesterase activity. Cells in the culture were found to be positively stained with these methods, suggesting that embryonic pineal cells are neuropotent to differentiate various types of neuronal cells. We have studied the culture conditions which favor increment of neuronal cells with extension of neuritic processes, and we have found that neuronal cells are maintained for quite a long period under suppressive conditions of DNA synthesis and under the effect of basic fibroblast growth factor (FGF). Suppression of DNA synthesis was achieved by the addition of aphidicolin, an inhibitor of DNA polymerase alpha, in the medium. Time lapse videograph revealed two different cell types participated in neurogenesis; a minor population of small round cells and a major one of flat epithelial cells. Since embryonic quail pineal cells have been shown to differentiate into two types of photoreceptors, the present results show wider retinal potency of cell differentiation by embryonic pineal cells. The cessation of DNA synthesis as well as growth factor(s) may be positively involved in the mechanisms of determination and differentiation of pineal neurons.

Animals↗
PubMed1993

Transdifferentiation.

Recent progress in studies of development and differentiation has greatly stimulated analysis of transdifferentiation, and more cell types capable of transdifferentiation have been documented. Growth factors must be essential, key factors in the regulation of the transdifferentiation process, in cooperation with components of the extracellular matrix, which helps to stabilize the differentiated state of tissues. Trials to induce transdifferentiation artificially by transfection of genes have also begun.

Animals↗
PubMed1993

Genetic characterization of the multipotent dedifferentiated state of pigmented epithelial cells in vitro.

Retinal pigmented epithelial cells (PECs) of chicken embryos extensively and almost synchronously transdifferentiate into lens cells in medium containing phenylthiourea and testicular hyaluronidase, passing through the bipotent dedifferentiated state. We have isolated genes that are expressed specifically by either pigment or lens cells and analyzed their expression in the transdifferentiation process. The expression of some proto-oncogenes was also studied. In the dedifferentiation process, expression of the c-myc gene was enhanced and the transcription of PEC-specific genes (MMP115, pP344) was completely repressed. However, transcription of lens-specific genes (alpha-, beta- and delta-crystallins genes) remained silent in dedifferentiated pigment cells. Activation of len- or PEC-specific genes occurred only in conditions permissive for lens or PEC differentiation, respectively. These results indicated that lens transdifferentiation from PECs proceeds through a multipotent (or at least bipotent) intermediate cell state in which the c-myc gene is activated, but neither PEC- nor lens-specific genes are expressed.

Animals↗