Search PubMed⌕ Search

Biomedical subjects

K Agata

Publications and source records attributed to K Agata.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1991

Isolation of cDNA for a Xenopus sperm-specific basic nuclear protein (SP4) and evidence for expression of SP4 mRNA in primary spermatocytes.

A cDNA library was prepared in lambda gt 11 from poly(A)+ mRNA isolated from a pure population of Xenopus round spermatids and screened with an antibody against SP3-5 (sperm-specific proteins) of Xenopus sperm. Positive clones were sequenced and an arginine-rich clone, designated pXSP531, was obtained. The 473-nucleotide sequence of pXSP531 contained an open reading frame of 237 nucleotides which was preceded by a 5' untranslated region of 67 nucleotides. The 3' untranslated region contained 149 nucleotides, including a consensus polyadenylation signal (AAATAAAA). Twenty nucleotides of a poly(A) tail was contained in the pXSP531. SP3-5 were separated from each other by reverse-phase chromatography and sequenced. The amino acid sequence of the peptide fragments which were obtained by digestion of SP4 with V8 protease and separated by reverse-phase chromatography was identical to the sequence of the N-terminal 43 and C-terminal 15 amino acids deduced from the nucleotide sequence of pXSP531. This result demonstrates that pXSP531 encodes SP4. Northern hybridization of RNA extracted from primary spermatocytes and round spermatids on Days 0 and 6 with SP4 cDNA probe (pXSP531) showed that SP4 mRNA is present both in primary spermatocytes and in round spermatids as is protamine mRNA in the rainbow trout. The size of the SP4 mRNA in round spermatids on Day 0 was longer by 60 nucleotides compared to that in primary spermatocytes and that in spermatids on Day 6 was shorter by 30 nucleotides compared to that on Day 0. These size differences were due to differences in the length of the poly(A) tracts because digestion of poly(A) with ribonuclease H resulted in the shortening of mRNA to the same size for three stages.

Amino Acid Sequence↗
PubMed1991

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.

Cell Differentiation↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1991

Complete sequence and expression of a cDNA encoding a chicken 115-kDa melanosomal matrix protein.

A full-length cDNA clone encoding a 115-kDa melanosomal matrix protein (MMP115) was isolated from a cDNA library constructed from poly(A)+ RNA of the chicken pigmented epithelial cells. Sequence analysis showed that the cDNA encoded a polypeptide of 762 amino acids, including a hydrophobic signal peptide. There are no membrane-spanning regions, but there are five N-linked glycosylation signals. A cysteine- and histidine-rich domain is present near the C-terminus. A sequence of 24 amino acids is repeated three times in the polypeptide. A database search for homologies yielded no sequence similarities in other proteins. A plasmid containing the full-length cDNA was transferred into mouse cell lines by transfection. The transfected cells produced a protein that had the same size, 115 kDa, as the mature MMP115. When B16 mouse melanoma cells were transfected, the chicken MMP115 was expressed in the melanosomes. The presence of a specific sorting signal was suggested for localization of melanosomal proteins. Southern blot analysis has revealed that the homologues of the chicken MMP115 gene are found in many vertebrate genomes.

Amino Acid Sequence↗
PubMed1991

Embryonic expression of beta-actin-lacZ hybrid gene injected into the fertilized ovum of the domestic fowl.

An experiment was carried out to investigate the expression of cloned DNA injected into the germinal disc of the chick fertilized ovum. The beta-actin-lacZ hybrid gene, MiwZ, was injected, in the closed circular form, into the cytoplasm of the germinal disc at the single-cell stage. The embryos were cultured in vitro, then in recipient eggshells up to day 4 of incubation. The survival rate of the embryos at day 4 was 42% (55/130), and the rate of embryos expressing MiwZ was 64% (35/55). Twenty-two embryos expressed the MiwZ in both embryonic and extraembryonic tissues, while the remainder expressed the MiwZ in only extraembryonic tissues. Mosaic expression was observed in most of the embryos expressing MiwZ in embryonic tissues. Expression throughout all tissues of the embryo including blood cells occurred in one case. In this case, the injected DNA was assumed to have integrated at an earlier stage. The results indicate that it is now possible to investigate the promoter activities of introduced exogenous genes as well as the effect of introduced genes on embryogenesis in early chick embryos. This technique may also facilitate the production of transgenic chicks.

Actins↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1989

Ontogenesis of alpha 2-adrenoceptor coupling with GTP-binding proteins in the rat telencephalon.

The ontogenesis of alpha 2-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 (Gi alpha + Go alpha) were increased with age and reached the adult level at day 12, whereas the 46-kDa protein (Gs alpha) 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 alpha- and beta-subunit of GTP-binding proteins) demonstrated that the 39-kDa alpha-subunit of (Go alpha) and the 36-kDa beta-subunit of GTP-binding protein (beta 36) increased with postnatal age. In contrast, 35-kDa beta-subunit (beta 35) did not change. From these results, it is suggested that the coupling activity of alpha 2-adrenoceptor with GTP-binding protein gradually develops in a manner parallel with the increase of alpha 2-adrenoceptor and pertussis toxin sensitive GTP-binding proteins, Gi, and that alpha 39 beta 36 gamma may be related to the differentiation and/or growth of nerve cells in rat telencephalon.

Adenosine Diphosphate Ribose↗
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

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↗
PubMed1983

Gene coding for a lens-specific protein, delta-crystallin, is transcribed in nonlens tissues of chicken embryos.

The question of whether or not delta-crystallin gene (delta-gene) is transcribed in tissues of chicken embryos other than lens was examined by Northern blot analysis, using cloned delta-crystallin cDNA as a probe. delta-Crystallin mRNA was detected in neural retinas, brains, and limb buds of 3.5-day-old embryos. However, the distribution of delta-gene transcripts is limited to lens only in posthatched chickens. It was indicated that the ratio of large delta-crystallin RNA, probably precursor RNA, to mature delta-crystallin mRNA is always much higher in nonlens tissues.

Animals↗