Makoto Mochii

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.

A Caenorhabditis elegans TGF‐β, DBL‐1, controls the expression of LON‐1, a PR‐related protein, that regulates polyploidization and body length

Using cDNA‐based array analysis combined with double‐stranded RNA interference (dsRNAi), we have identified yk298h6 as a target gene of Caenorhabditis elegans TGF‐β signaling. Worms overexpressing dbl‐1, a TGF‐β ligand, are 16% longer than wild type. Array analysis shows yk298h6 to be one of several genes suppressed in such worms. Disruption of yk298h6 function by dsRNAi also resulted in long worms, suggesting that it is a negative regulator of body length. yk298h6 was then mapped to, and shown to be identical to, lon‐1, a known gene that affects body length. lon‐1 encodes a 312 amino acid protein with a motif sequence that is conserved from plants to humans. Expression studies confirm that LON‐1 is repressed by DBL‐1, suggesting that LON‐1 is a novel downstream component of the C.elegans TGF‐β growth regulation pathway. Consistent with this, LON‐1 is expressed mainly in the larval and adult hypodermis and has dose‐dependent effects on body length associated with changes in hypodermal ploidy, but not hypodermal cell proliferation.

Global gene expression profiling and cluster analysis in Xenopus laevis.

We have undertaken a large-scale microarray gene expression analysis using cDNAs corresponding to 21,000 Xenopus laevis ESTs. mRNAs from 37 samples, including embryos and adult organs, were profiled. Cluster analysis of embryos of different stages was carried out and revealed expected affinities between gastrulae and neurulae, as well as between advanced neurulae and tadpoles, while egg and feeding larvae were clearly separated. Cluster analysis of adult organs showed some unexpected tissue-relatedness, e.g. kidney is more related to endodermal than to mesodermal tissues and the brain is separated from other neuroectodermal derivatives. Cluster analysis of genes revealed major phases of co-ordinate gene expression between egg and adult stages. During the maternal-early embryonic phase, genes maintaining a rapidly dividing cell state are predominantly expressed (cell cycle regulators, chromatin proteins). Genes involved in protein biosynthesis are progressively induced from mid-embryogenesis onwards. The larval-adult phase is characterised by expression of genes involved in metabolism and terminal differentiation. Thirteen potential synexpression groups were identified, which encompass components of diverse molecular processes or supra-molecular structures, including chromatin, RNA processing and nucleolar function, cell cycle, respiratory chain/Krebs cycle, protein biosynthesis, endoplasmic reticulum, vesicle transport, synaptic vesicle, microtubule, intermediate filament, epithelial proteins and collagen. Data filtering identified genes with potential stage-, region- and organ-specific expression. The dataset was assembled in the iChip microarray database, , which allows user-defined queries. The study provides insights into the higher order of vertebrate gene expression, identifies synexpression groups and marker genes, and makes predictions for the biological role of numerous uncharacterized genes.

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.

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.

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.

Partial amino acid sequence of the major intrinsic protein (MIP) of the chicken lens deduced from the nucleotide sequence of a cDNA clone

A cDNA clone of the major intrinsic protein (MIP) of the chicken lens was isolated. This clone covers the C-terminal half of the coding region and 3-untranslated region including a polyadenylation signal. Comparison with the bovine MIP cDNA sequence revealed that: (1) the amphilphilic transmembrane helix in bovine MIP is highly hydrophobic in chicken MIP, and is thus unlikely to offer a hydrophilic lining of the transmembrane pore, and (2) the possible calmodulin binding site is conserved especially at amino acid residues which are postulated to be important in its binding with calmodulin. Northern blotting revealed the presence of transcripts of different lengths, two of which correspond closely to the transcripts of bovine MIP.

Role of integrins in differentiation of chick retinal pigmented epithelial cells in vitro

When retinal pigmented epithelial cells (PEC) of chick embryos are cultured under appropriate conditions, the phenotype changes to that of lens cells through a process known as transdifferentiation. The first half of the process, characterized by dedifferentiation of PEC, is accompanied by a marked decrease in adhesiveness of PEC to collagen type I‐ or type IV‐coated dishes. To understand the underlying mechanisms of this change, we analyzed the expression of integrins, which are major receptors for extracellular matrix components. Northern blot analysis with cDNA probes for chicken α3, α6, α8, αv, β1 and β5 integrin mRNA showed that the genes for all these integrins are transcribed at similar levels in PEC and dedifferentiated PEC (dePEC). Further analysis of β1 integrin, which is a major component of integrin heterodimers, showed that although the protein amount of β1 integrin was not changed, its localization at focal contacts seen in PEC was lost in dePEC. When anti‐β1 integrin antibody was added to the PEC culture medium, a decrease of cell‐substrate adhesiveness occurred, followed by a gradual change in both morphology and gene expression patterns to ones similar to those of dePEC. These findings suggest that an appropriate distribution of β1 integrin plays an essential role in maintaining the differentiated state of PEC through cell‐substrate adhesion.