Goro Eguchi

The A5 antigen, a candidate for the neuronal recognition molecule, has homologies to complement components and coagulation factors

The A5 antigen is a neuronal cell surface protein of Xenopus presumed to be involved in the neuronal recognition between the optic nerve fibers and the visual centers. Analyses of cDNA clones revealed that the A5 antigen is a class I membrane protein containing two different internal repeats in the extracellular segment. The first repeat bears homology to domain III of complement components C1r and C1s, and the second repeat is homologous to the C1 and C2 domains of coagulation factors V and VIII. The mRNA for the A5 antigen was present in retinal ganglion cells and visual center neurons. Nonneuronal cells in the peripheral and central nervous systems did not express the mRNA for the A5 antigen.

Regenerative capacity in newts is not altered by repeated regeneration and ageing

The extent to which adult newts retain regenerative capability remains one of the greatest unanswered questions in the regeneration field. Here we report a long-term lens regeneration project spanning 16 years that was undertaken to address this question. Over that time, the lens was removed 18 times from the same animals, and by the time of the last tissue collection, specimens were at least 30 years old. Regenerated lens tissues number 18 and number 17, from the last and the second to the last extraction, respectively, were analysed structurally and in terms of gene expression. Both exhibited structural properties identical to lenses from younger animals that had never experienced lens regeneration. Expression of mRNAs encoding key lens structural proteins or transcription factors was very similar to that of controls. Thus, contrary to the belief that regeneration becomes less efficient with time or repetition, repeated regeneration, even at old age, does not alter newt regenerative capacity.

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 acyl‐transferase purified from squash cotyledons were determined. cDNAs encoding this enzyme were isolated from λgt11 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 43838 and 40929 Da, respectively.

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 (Eagles 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.

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.

Instability in cell commitment of vertebrate pigmented epithelial cells and their transdifferentiation into lens cells

Publisher Summary Although the cell type conversion of pigmented epithelial cells (PECs) can be observed in the process of Wolffian lens regeneration and of neural retina regeneration in a limited number of species, the results of cell culture works since 1973 permit to conclude that the population of PECs capable of transdifferentiation is widely conserved in vertebrates including humans, at least in the embryonic period. However, this multipotential property is well stabilized in physiological conditions in situ . The finding that close cell–cell contact may be one of the requisites for the stabilization of PECs is revealed by observations of a cohesive epithelial cell sheet supported by a continuous collagen substrate. This chapter establishes a culture system in which a pure population of dedifferentiated cells (dePECs) can be maintained by introducing phenylthiourea (PTU), hyaluronidase (HUase), and dialyzed fetal bovine serum in an appropriate way. With this system, the cellular properties of dePECs can be investigated in detail. Preliminary efforts to elucidate the mechanisms of transdifferentiation in terms of the regulation of gene expression have been started. It has been demonstrated that δ-crystallin mature mRNA can be detected in some nonlenticular tissues, such as pigment epithelium, neural retina, and others, in early chick embryos in situ . To further understand the multipotent dedifferentiated state of PECs at the level of gene expression, it is necessary to extend investigation to the transcription of genes that are specifically expressed in the melanogenic cell and to determine whether the transcripts are also present in dePECs.

Evaluation of a high-velocity flame-spraying technique for hydroxyapatite

Titanium alloy sprayed with hydroxyapatite (HA) was developed by a high-velocity flame-spraying technique (HVFST). Biocompatible responses of bony tissues to high-velocity flame-sprayed HA (HVFS-HA) implanted into tibias of adult male rabbits was investigated 4 and 18 months after implantation by light and electron microscopy. Both light and electron microscope features in histological sections showed that inflammatory responses of tissues in situ were completely cleared and the interface between the bone and the implant was filled with newly formed bony substrate. This suggests that the HVFS-HA was sufficiently biocompatible to be adapted to the bone in situ.

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.

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.

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.