Search PubMed⌕ Search

Biomedical subjects

G Eguchi

Publications and source records attributed to G Eguchi.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1993

Evidence that the chick lens cytoskeletal protein CP 49 belongs to the family of intermediate filament proteins.

A partial cDNA sequence for chick lens beaded-filament protein CP 49 showed the greatest similarity to the sequence of acidic cytokeratins, especially human cytokeratin 18. The predicted amino acid sequence of chick CP 49 corresponded to the entire coil 1a region of the rod domain of human cytokeratin 18, spacer 1, coil 1b, spacer 2 and about half of coil 2. For this sequence of 242 amino acids, there was an overall 38% identity and 76.8% similarity between the chick CP 49 and human cytokeratin 18. This is further evidence that CP 49 belongs to the family of intermediate filament proteins.

Amino Acid Sequence↗
PubMed1993

A set of anti-crystallin monoclonal antibodies for detecting lens specificities: beta-crystallin as a specific marker for detecting lentoidogenesis in cultures of chicken lens epithelial cells.

Seven hybridoma lines which produced monoclonal antibodies against lens crystallins were established. They could detect alpha A-, alpha B-, gamma-, delta-, and several beta-crystallins of different species of animals with high avidities. Although immunohistological analysis of chicken and mouse lenses showed the typical distributions of each crystallin as have been reported so far, the ectopic expression of alpha B or delta-crystallin was observed in the brain or the kidney of chicken, respectively. Subsequently, crystallin production during the process of lentoidogenesis in cultures of chicken lens epithelial cells was examined with these antibodies. Western blot analysis revealed that beta-crystallins were not detected in cultured cells before lentoidogenesis, although all other crystallins were detected throughout the culture period. Immunostaining of cultures indicated clearly that expression of beta-crystallins was restricted to lentoid bodies. These data confirmed the lens fiber specificity of beta-crystallins as previously reported in the messenger RNA level. In addition, we found that small-size delta-crystallin (48 kDa) accumulated before the onset of lentoidogenesis. These results strongly suggest that the differentiated state of lens cells in vitro could be classified by examination of the expression pattern of crystallins. In addition, the anti-crystallin monoclonal antibodies produced in this study could be useful for detecting lens specificities.

Animals↗
PubMed1993

[Regional cerebral blood flow in the patients with closed-head injury using 123I-IMP SPECT and computed tomography].

123I-IMP SPECT and CT were evaluated in 20 patients with closed-head injury. The detected focal lesions by both examinations were 36. Twenty lesions (55.6%) were seen on both CT and SPECT, and SPECT revealed larger abnormality than CT. Sixteen (44.4%) of 36 lesions were seen only on SPECT. Regional cerebral blood flow (r-CBF) of thirty-one focal lesions were quantified by reference sampling method and compared to r-CBF of the patients without neurological problem. r-CBF of 18 localized lesions accordant with low density area (LDA) on CT were significantly lower than those of 13 lesions seen only on SPECT. And r-CBF of both lesions were significantly lower than that of control patients. Regional cerebral blood flow on the areas with visually normal RI uptake were also significantly lower than that of control group. Regardless of CT findings, some disturbance of r-CBF was considered in patients with head trauma. Therefore the quantification of r-CBF with 123I-IMP SPECT was a useful method for evaluating functional change in those patients.

Adult↗
PubMed1993

[A case of Turcot syndrome (glioma polyposis)].

We reported a case of Turcot Syndrome (glioma polyposis) in a 19-year-old woman with nonfamilial polyposis coli and adenocarcinoma of the colon, and grade 3 astrocytoma in the right parietal lobe. The patient was admitted with the complaint of general convulsion after colostomy for polyposis and adenocarcinoma of the colon. CT scans on admission showed a large parietal tumor in the right side. Total removal was performed successfully and histological examination showed astrocytoma grade 3. One year after the operation, the tumor recurred. Conservative treatment failed to improve her condition and she died one year later. Turcot Syndrome (glioma polyposis) is very rare and only 10 cases have been reported in Japan. In this report, the clinical characteristics of this syndrome were discussed.

Adenocarcinoma↗
PubMed1993

Basic fibroblast growth factor as one of the essential factors regulating lens transdifferentiation of pigmented epithelial cells.

In vitro transdifferentiation of retinal pigmented epithelial cells of the chick embryo into lens cells can be markedly enhanced by culture in the presence of testicular hyaluronidase and phenylthiourea. Since the commercial preparations of hyaluronidase that had previously been used were very crude, a search for the actual effective molecule(s) enhancing lens transdifferentiation was conducted. First, we purified the enzyme and tested the effect of the purified hyaluronidase. Highly purified hyaluronidase itself did not enhance lens transdifferentiation. The crude hyaluronidase was then separated according to affinity with heparin, considering the possibility that the fibroblast growth factor (FGF) is contained in the crude hyaluronidase. Transdifferentiation-enhancing activity was detected in the fraction which was bound to heparin and eluted with 2 M NaCl, where no hyaluronate-degrading activity existed. Analysis of the fraction by SDS-PAGE revealed the existence of an 18 kDa protein whose NH2-terminal sequence was identical to that of basic FGF. The basic FGF derived from bovine brain also enhanced lens transdifferentiation of pigmented epithelial cells. These findings suggest that basic FGF must play a major role in enhancing transdifferentiation of pigmented epithelial cells to lens cells.

Amino Acid Sequence↗
PubMed1992

Tissue-specific response of estrogen receptor gene expression to estrogen in chick.

We have developed a sensitive assay system by RT (reverse transcription)-PCR(polymerase chain reaction) to detect the low level of chicken estrogen receptor (cER) transcript. Using this system, the differential expression of cER gene in chick tissues was observed. Moreover, we found that the oviduct cER transcript levels in chick was not affected, albeit the remarkable growth of this tissue, by exogenous estrogen. In contrast, estrogen enhanced the hepatic cER transcript level several folds. Thus, the present study clearly showed a novel tissue-specific response of cER gene expression to estrogen in chick.

Animals↗