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G Eguchi

Publications and source records attributed to G Eguchi.

At least 73 records · Page 4Linked to original sources
PubMed1989

[Gene expression during lens transdifferentiation from pigmented epithelial cells].

Pigmented epithelial cells of chicken and human dedifferentiate in the medium containing phenylthiourea and testicular hyaluronidase, and then trans-differentiate into lens cells in vitro. To understand the molecular mechanisms of transdifferentiation, gene expression during lens transdifferentiation was analyzed. As the first step, pigment cell and lens specific genes were isolated and expression of these gene was analyzed by Northern blotting . These results clearly shown that lens transdifferentiation proceeds via neutral cell state in which both pigment and lens specific genes are repressed. Oncogene expression was also analyzed. An elevated expression of the c-myc gene was observed during dedifferentiation process. It is expected that elevated expression of c-myc gene might prevent the cells from entering the G0 phase and thus lead to dedifferentiated state.

Animals↗
PubMed1988

Cloning and nucleotide sequence of cDNA for the plastid glycerol-3-phosphate acyltransferase from squash.

The partial amino acid sequence and amino acid composition of acyl-(acyl-carrier-protein):glycerol-3-phosphate acyltransferase purified from squash cotyledons were determined. cDNAs encoding this enzyme were isolated from lambda gt 11 cDNA libraries made from poly(A)+ RNA of squash cotyledons by immunological selection and cross-hybridization. One of the resultant clones contained a cDNA insert of 1426 base pairs and an open reading frame of 1188 base pairs. The amino acid sequence deduced from the nucleotide sequence matched the partial amino acid sequence determined for the enzyme. The results suggest that a precursor protein of 396 amino acid residues is processed to the mature enzyme of 368 amino acid residues, losing a leader peptide of 28 amino acid residues. Relative molecular masses of the precursor and mature proteins were calculated to be 43,838 and 40,929 Da, respectively.

Acyltransferases↗
PubMed1988

The expression of melanosomal matrix protein in the transdifferentiation of pigmented epithelial cells into lens cells.

A monoclonal antibody (MC/1) was constructed against melanosomes purified from the chicken pigmented epithelial cells (PECs) in order to characterize the differentiative phenotypes of PEC in the process of transdifferentiation into lens cells. Immunofluorescent studies revealed that MC/1 antibody specifically stains both retinal PECs in the eye and melanocytes in the skin, of chicken embryos. Immunoelectron microscopy showed that the antigen molecules are located on the peripheral region of the melanosomal matrix. A single protein band with an apparent molecular weight of 115,000 was labelled by MC/1 in Western blotting. The 115 kDa polypeptide identified by MC/1 is considered to be a member of the melanosomal matrix proteins. The maintenance of specificity of pigment cell nature is followed in the system of transdifferentiation of PEC into lens in vitro, utilizing 115 kDa protein as a marker. In the dedifferentiated PECs, this protein was undetectable.

Animals↗
PubMed1988

Expression of gene coding for a melanosomal matrix protein transcriptionally regulated in the transdifferentiation of chick embryo pigmented epithelial cells.

Chicken 115-kDa melanosomal matrix protein (MMP115) was purified from cultured pigmented epithelial cells (PECs), and mouse antiserum was raised to isolate cDNA clones. lambda gt11 expression library made from poly(A)+ RNA of the homogeneous population of PECs was screened with the antiserum. Nine positive clones were obtained from 5 X 10(5) independent phages, and inserts of them shared a common nucleotide sequence. The beta-galactosidase fusion protein from the longest insert (MM-2, 1.0 kb long) was recognized by the anti-MMP115 antiserum in immunoblotting, and the antibody, which was affinity-selected by the fusion protein, specifically reacted with the 115-kDa protein in PEC extracts. The RNA blot analysis with the MM-2 insert as a probe revealed that a transcript of 2.6 kb was expressed by the PEC in a tissue-specific manner. mRNA expressions in the process of in vitro transdifferentiation from PECs to lens cells were analyzed using the MM-2 insert. The transcripts were detected in neither transdifferentiating, transdifferentiated lens cells nor bipotent dedifferentiated PECs, although the 2.6 kb transcript was vigorously synthesized by redifferentiating into PECs.

Animals↗
PubMed1988

Cellular and molecular background of wolffian lens regeneration.

Based on studies of wolffian lens regeneration in the newt, in which the lens can be regenerated from the iris pigmented epithelium, we have shown by cell culture studies that the capacity of lens transdifferentiation is not limited to the newt cells, but widely conserved in pigmented epithelial cells (PECs) of chick and quail embryos and even of human fetuses. Recently, we have established a unique in vitro model system of chick embryonic PECs. In this culture system we are able to control each step of transdifferentiation from PECs into lens cells by regulating culture conditions and to produce a homogeneous cell population with potential for synchronous differentiation into either lens or pigment cell phenotype. These multipotent (at least bipotent) cells showed cellular characteristics resembling neoplastic cells in many ways. They did not express both lens and pigment cell specific genes analyzed so far, except delta-crystallin gene, which is expressed in developing lens of chick embryos. It has been proved by application of cell culture procedures of the system that PECs dissociated from fully-grown human eyes readily transdifferentiated into lens phenotypes in the manner observed in chick embryo PECs. In addition, we could predict that molecules detected in either cell surface or intercellular space stabilized the differentiated state of PECs in the newt and that the loss of these molecules might be one of the key steps of lens regeneration from the iris epithelium.

Aged↗
PubMed1988

Molecular cloning and the nucleotide sequence of cDNA for embryonic chicken pepsinogen: phylogenetic relationship with prochymosin.

Embryonic chicken pepsinogen is an aspartyl proteinase that is specifically secreted during the embryonic period in the chicken proventriculus (glandular stomach). To learn the phylogeny of this pepsinogen, we isolated a cDNA clone by screening a lambda gt11 library of embryonic proventricular cDNAs with an antiserum to the embryonic chicken pepsinogen. We obtained a 200-base pair cDNA clone which encoded 18 amino acids that had high sequence homology with the carboxyl termini of other pepsinogens. Northern blot analysis revealed that this cDNA clone hybridized to a mRNA of 1,600 bases in the embryonic proventriculus but not to the mRNA in the adult proventriculus. The almost complete nucleotide sequence of embryonic chicken pepsinogen-cDNA was determined by sequencing longer cDNAs obtained by screening the same library with the 200-base pair cDNA and primer extension with a synthetic primer. The cDNA consisted of 1,281 nucleotides and encoded 383 amino acids for prepepsinogen. The predicted amino acid sequence was compared with the sequences of other aspartyl proteinases: pepsinogen A of human, monkey, pig, and chicken, progastricsin of monkey and rat, and bovine prochymosin. The phylogenetic tree constructed for them indicates the possibility that embryonic chicken pepsinogen diverged from prochymosin, after prochymosin and pepsinogen A had diverged from each other.

Amino Acid Sequence↗
PubMed1988

An embryonic pineal body as a multipotent system in cell differentiation.

The differentiating potency of pineal cells from 8-day quail embryos was studied with cell culture. It was found that the differentiation of striated muscle fibres occurred abundantly in the pineal cells cultured in hypertonic culture conditions. Muscle nature of these fibres was confirmed by utilizing the antiserum against the striated muscle type creatine kinase (MM-CK). When CO2, NAHCO3, NaCl, KCl and MgCl2 were added in hypertonic concentrations, extensive myogenesis occurred in cultured pineal cells. Myogenesis in pineal cultures began as early as 2 days and, after 3 days in the medium with 75 mM additional NaCl, reached 100-fold when compared with that in the isotonic medium. Muscle fibres from pineal cells in culture were similar in morphology to the skeletal muscle fibres of mesodermal origin in situ. Myogenesis of pineal cells under hypertonic conditions was accompanied by the synthesis of a unique 56 x 10(3) Mr protein, which was not found in the intrinsic muscle cells. Clonal cell culture revealed that about 80% of clonable pineal cells were myogenic precursors. Pineal cells of 8-day quail embryos were not only myogenic but oculopotent (melanogenic and lentoidogenic) in cultures. This study examined whether multipotential progenitor cells with both potentials are present in the pineal or not. The results showed that at least 16% of all clonable pineal cells were multipotent precursors. The embryonic pineal is considered to be a typical multipotent system in parallel with the pigmented and neural retina, the neural crest and the teratocarcinoma.

Animals↗
PubMed1987

Density dependent growth of corneal endothelial cells cultured in vitro.

Using the cornea of macaque monkey, we demonstrated the relationship between cell density and growth of endothelial cells in vitro. Corneal endothelial cells in a cell sheet grow most actively in regions with cell density of 1000 to 1800 cells/mm2, in explant cultures and cell sheets and in concentrated inocula dissociated cells. Cell morphology was well sustained in these cultures. Cells cultured at a higher cell density retained their potential to proliferate actively, showing clear contrast to cells cultured at a density lower than 200 cells/mm2. When dissociated cells were cultured at a low density and maintained for more than 4 weeks, they gradually lost their growth potential, altered into polymorphonuclear giant cells and eventually dedifferentiated. In addition, cells with no contact with each other did not express growth potential. Density dependent growth was confirmed by measuring the mitotic index against the cell density per square mm from the center to the peripheral regions in cultured explants. It is concluded that the growth pattern of corneal endothelial cells is closely related to cell density, and that growth of these cells might be regulated through intercellular communications.

Animals↗
PubMed1987

Cell lineage analysis of the primitive and visceral endoderm of mouse embryos cultured in vitro.

Cell lineages of the primitive endoderm and the visceral endoderm of mouse embryos were examined by culturing whole embryos in vitro. The primitive endoderm and visceral endoderm cells could be labelled by incubation of embryos in a medium containing horse radish peroxidase (HRP). HRP localization was chased throughout the culture period. The results show that the visceral endoderm derives from the primitive endoderm, and the visceral endoderm forms only the extra-embryonic endoderm (yolk sac endoderm) of the conceptus. The definitive endoderm which is probably derived from the head process, newly appears on the ventral surface of the embryo.

Animals↗
PubMed1986

In vitro analysis of cellular metaplasia from pigmented epithelial cells to lens phenotypes: a unique model system for studying cellular and molecular mechanisms of "transdifferentiation".

Pigmented epithelial cells (PECs) were dissociated from eyes of 8- to 9-day-old chick embryos and were cultured in EdF medium (Eagle's MEM supplemented with dialyzed fetal bovine serum) containing phenylthiourea (PTU) and testicular hyaluronidase (HUase). The PECs rapidly lost melanosomes as they proliferated and dedifferentiated in culture. These dedifferentiated PECs (dePECs) which did not manifest any identifiable specificity could be directed to one of two different differentiated phenotypes; viz., lens or pigment cells, depending upon subsequent culture conditions. Almost all dePECs began to synthesize melanin and redifferentiated to PECs by Day 10 of culture with EdF medium containing ascorbic acid (AsA). In contrast, the sister population of dePECs, when cultured at extremely high cell density with EdF medium containing PTU, HUase and AsA, synthesized delta-crystallin which is specific for lens. This transdifferentiation into lens cells occurred by Day 15 of culture. Using this culture system we are able to produce a homogeneous cell population with the potential for synchronous differentiation into either lens or pigment cell phenotype. The system is useful for studying mechanisms involved in cellular metaplasia.

Animals↗
PubMed1986

Studies on the mechanism of blastula formation in starfish embryos denuded of fertilization membrane.

A starfish egg, denuded of the fertilization membrane and placed on a glass surface, becomes a cell monolayer after several cleavages. This sheet of cells folds and forms a hollow sphere resembling a normal blastula at the 2(9)-2(10)-cell stage ('closing movement'). A marked morphological change was observed in each cell, preceding the closing movement. The surface of each blastomere differentiated into two parts: one was smooth, whereas the other was rough with microvilli. The smooth surface was more adhesive and flexible than the rough surface, suggesting that the closing movement may be driven by a local increase in cell adhesiveness.

Animals↗
PubMed1986

Enhancement of expression of lens phenotype in cultures of pigmented epithelial cells by hyaluronidase in the presence of phenylthiourea.

Pigmented epithelial cells isolated from 8-9-day-old chick embryos can transdifferentiate into lens-like cells at the terminal period of the third generation of culture. However, efficiency of this transdifferentiation is usually rather low. Phenylthiourea, a potent inhibitor of melanin synthesis, effectively enhances transdifferentiation of pigmented epithelial cells into lens-like cells in vitro. Lentoid bodies began to appear in the multilayered region of primary cultures of pigmented epithelial cells maintained in medium containing phenylthiourea at concentrations between 0.5 and 1.0 mM. Furthermore, the enhancing effect of phenylthiourea can be amplified with testicular hyaluronidase. Under these conditions, pigmented epithelial cells grow vigorously and lose their differentiative properties, efficiently switching their phenotype into lens-like cells some 20 days after initiation of culture in the presence of both substances. Semiquantitative analysis revealed that testicular hyaluronidase amplified the effect of phenylthiourea more than 100-fold. It has been suggested that phenotypic expression of pigmented epithelial cells during transdifferentiation can be regulated by manipulating the microenvironment in which these cells reside.

Animals↗
PubMed1986

Production of crystallins and lens-like structures in differentiation-induced neoplastic pigment cells (goldfish erythrophoroma cells) in vitro.

We examined the crystallins present in lens-like cell aggregates produced by goldfish erythrophoroma (tumors of integumental erythrophores) cells in vitro using a combination of Sephadex-G-200 gel filtration, one- and two-dimensional sodium-dodecyl-sulfate/polyacryl-amide gel electrophoresis, immunoblotting, and indirect immunofluorescence assays. The two studied neoplastic pigment cell lines, GEM 81 and GEM 218, formed small, spherical, transparent cell aggregates, resembling lentoid bodies, within the cell mounds of monolayer cultures after treatment with dimethylsulfoxide (DMSO) and autologous serum. Partial purification of a water-soluble extract of such lens-like cell aggregates and subsequent immunoblotting using antibodies (polyclonal) against newt whole lens proteins revealed the presence of about 20 unequivocally conjugated peptides with molecular masses of 19-27 kilodaltons. From their antigenicity and their behavior during gel filtration and electrophoresis, most of these peptides were identified as either alpha- or beta-form crystallins. Immunofluorescence microscopy using antibodies to newt whole lens proteins revealed intense fluorescence in the lens-like cell aggregates formed by these erythrophoroma cells, whereas the cell mounds in cultures of the same cell lines that had not been subjected to differentiation induction were almost unlabeled. Thus, goldfish erythrophoroma cells appear to be capable of crystallin production as well as the formation of lens-like cell aggregates upon the induction of differentiation. There is little available information indicating that normal pigment cells are capable of lens formation and crystallin synthesis during vertebrate ontogeny, and thus it is possible that neoplastic transformation of pigment cells is associated with the acquisition of the ability to produce crystallins.

Animals↗
PubMed1986

In vitro system of corneal endothelium.

A method for preparation of highly ordered monolayer cultures of the corneal endothelium was described. The technique of agar and palladium double-coating of culture plates was utilized for demarcation of the haptotactic area. The culture plate was prepared by precoating with agar and subsequent coating with palladium to define areas where growth of corneal endothelial cells was allowed. A micro-mass culture of corneal endothelial cells on the area defined by the palladium coating demonstrated their active growth and their reconstitution of the typical cellular pattern of the corneal endothelium.

Animals↗