Kiyokazu Agata

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.

Distinct structural domains in the planarian brain defined by the expression of evolutionarily conserved homeobox genes.

Abstract Homeobox genes such as orthodenticle in Drosophila and its mouse homologues, Otx1 and Otx2, are known to be essential for rostral brain development. To investigate the molecular basis of brain evolution, we searched for otd/Otx-related homeobox genes in the planarian Dugesia japonica, and identified two genes, DjotxA and B, whose expression appears to be restricted to the cephalic ganglion (brain). DjotxA was expressed more medially, in the region containing the termini of the visual axons, and in the visual cells, suggesting involvement in establishment of the visual system. DjotxB was expressed in a discrete region just lateral to the DjotxA-positive domain, but not in the more lateral branch structures, which in turn are characterized by the expression of Djotp, a planarian homeobox gene related to mouse Orthopedia (Otp). In transverse sections of planarians, DjotxA and B expression were observed only at the anterior ends of the stumps, corresponding to the regional pattern of the regenerating brain. Our findings suggest that the planarian brain is composed of structurally distinct and functionally diverse domains which are defined by the discrete expression of the three evolutionarily conserved homeobox genes.

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.

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.

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.

Immunological relationships among embryonic and adult chicken pepsinogens: a study with monoclonal and polyclonal antibodies

To investigate the immunological relationships of pepsinogen isozymes present in embryonic and adult chicken proventriculi, we obtained monoclonal and polyclonal antibodies to these pepsinogens. Zymograms and immunoblots demonstrated that monoclonal antibody Y37 reacted with both embryonic and slow‐migrating adult pepsinogens, while polyclonal antibodies against embryonic pepsinogen and fast‐migrating adult pepsinogen were specific for these respective antigens. Shift from embryonic to adult‐type pepsinogen occurred at about the time of hatching and the localizations of embryonic and adult‐type pepsinogens within proventricular gland cells were found to differ by the indirect immunofluorescence method. Results with these antibodies revealed the immunological relations of these pepsinogens and the unique properties of embryonic chicken pepsinogen.

Study of crystallin expression in human lens epithelial cells during differentiation in culture and in non-lenticular tissues.

Crystallin expression in human lens epithelial cells in culture and a number of non-lenticular tissues was studied by the technique of immunoblotting using monoclonal antibodies. The expression of alpha A, beta 5 and beta 6 crystallins per unit number of cells increased with passage number while alpha B appeared to be constant Lentoid bodies derived from cultured human lens epithelial cells not only expressed gamma-crystallin and MP26 as previously demonstrated, but also produced alpha A, alpha B, beta 5 and beta 6 crystallins. In human non-lenticular tissues including ciliary body, vitreous body, neural retina, cultured retinal pigment epithelial cells and scleral fibroblasts, alpha B-crystallin was detected, but was undetectable in cornea and iris. Alpha A was present only in the lens. These studies demonstrate that HLE cells maintain the ability to synthesize crystallins through several passages. Following differentiation, they not only synthesize gamma-crystallin and MP26 but continue to express alpha- and beta-crystallins similar to differentiated lens fiber cells in vivo. Consistent with previous observations, the expression of alpha B-crystallin does not appear to be specific for the lens.

Crystallin gene expression in the process of lentoidogenesis in cultures of chicken lens epithelial cells

One alpha B- and three different beta-crystallin cDNA clones were isolated from a chicken lens cDNA library by using anti-crystallin antibodies. The sequence of alpha B-crystallin cDNA showed more than 70% homology with exons of alpha B-crystallin genes of the human and hamster. Two beta-crystallin cDNAs showed almost identical sequences with previously reported chicken beta B1- and beta A3/A1-crystallin genes. The remainder showed 80% homology of sequence with bovine beta B2-crystalline cDNA. Using these newly cloned cDNAs, in addition to cDNAs of alpha A- and delta-crystallin, we examined the expression pattern of these crystallins in the process of lentoidogenesis of cultured lens epithelial cells of the chicken. All crystallins except beta-crystallins were expressed through the period of cell culture, but three beta-crystallins were expressed only after the confluent stage. These results suggest that: (1) alpha A-, alpha B- and delta-crystallin cDNAs can be used to detect differentiation of the lens epithelial cell; and (2) beta-crystallin cDNAs are superior in the detection of chicken lens fibre differentiation in vitro to delta-crystallin cDNA, which is ectopically expressed by various non-lenticular tissues.

Ontogenesis of α2-adrenoceptor coupling with GTP-binding proteins in the rat telencephalon

Abstract: The ontogenesis of α2‐adrenoceptors and GTP‐binding proteins and their coupling activity were investigated in telencephalon membranes of developing rats. The manganese‐induced elevation of [3H]clonidine binding was increased in an age‐dependent manner but the guanosine 5′‐O‐(3‐thio)triphosphate‐induced decrease in binding did not change. The extent of the binding of [3H]clonidine at 15 nM (saturable concentration) increased in an age‐dependent manner and reached the adult level at 4 days after birth. Cholera toxin and pertussis toxin catalyzed ADP‐ribosylation of proteins of 46 and 41/39 kilodaltons (kDa) in solubilized cholate extracts of the membranes. The 41/39‐kDa proteins ADP‐ribosylated by pertussis toxin (Giα+ Goα) were increased with age and reached the adult level at day 12, whereas the 46‐kDa protein (Gsα) reached its peak on day 12 and then decreased to the fetal level at the adult stage. The immunoblot experiments of the homogenates with antiserum (specific antibody against α‐ and β‐subunit of GTP‐binding proteins) demonstrated that the 39‐kDa α‐subunit of (Goα) and the 36‐kDa δ‐subunit of GTP‐binding protein (δ36) increased with postnatal age. In contrast, 35‐kDa δ‐subunit (δ35) did not change. From these results, it is suggested that the coupling activity of α‐adrenoceptor with GTP‐binding protein gradually develops in a manner parallel with the increase of α2‐adrenoceptor and pertussis toxin sensitive GTP‐binding proteins, Gi, and that α39β36γ may be related to the differentiation and/or growth of nerve cells in rat telencephalon.

Expression of the Retinal Pigmented Epithelial Cell‐Specific pP344 Gene during Development of the Chicken Eye and Identification of Its Product

In order to understand the transdifferentiation of the retinal pigmented epithelial cells (PECs) into the lens cells at the level of gene expression, a gene, tentatively called pP344 gene, was studied, because its expression appeared to be closely related with the differentiated state of PECs. We analyzed pP344 gene expression during chicken eye development by RT‐PCR and in situ hybridization and also characterized the pP344 protein using antipeptide antibodies. In addition to the previous observation that the transcript of pP344 gene is limited to the pigmented epithelium and not detected in the melanocytes, we show here that the transcript is limited to retinal PECs and is never observed in iris or ciliary PECs. The time course of expression level showed two peaks; the first peak occurred around the 10th day similarly to the expression of melanosome‐related genes, while the second peak occurred just after hatching when PECs had completely differentiated, suggesting that pP344 gene may be related to the function of fully differentiated PECs. Antisynthetic peptide antibodies detected pP344 protein in the culture medium of the PECs but not within the cells. Thus, we concluded that pP344 gene is specifically expressed by the retinal PEC and its product is a secreted protein.