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I Iuchi

Publications and source records attributed to I Iuchi.

At least 19 recordsLinked to original sources
PubMed1995

Cloning of cDNAs for the precursor protein of a low-molecular-weight subunit of the inner layer of the egg envelope (chorion) of the fish Oryzias latipes.

cDNA clones for L-SF, the precursor of a low-molecular-weight subunit (ZI-3) of the inner layer of the Oryzias latipes egg envelope were isolated from Lambda ZAP cDNA libraries constructed from the poly(A)+ RNA of the liver of spawning female fish and estrogen-treated male fish. Among them, a clone, L-SF41, is 1473 bp long and contains an open reading frame encoding a signal peptide of 19 amino acids and L-SF protein of 420 amino acids. L-SF protein seems to be glycosylated, judging from the result of the glycanase digestion. L-SF protein contains a domain similar to ZP-domains in ZP3 of some mammalian species. Northern blot analysis employing XhoI-SmaI fragments of the cloned cDNA as probes revealed that expression of the L-SF gene occurred exclusively in the livers of spawning female fish and estrogen-treated male fish and that there was no mRNA encoding L-SF in the ovary of the spawning female fish.

Amino Acid Sequence↗
PubMed1995

Temporal and spatial patterns of gene expression for the hatching enzyme in the teleost embryo, Oryzias latipes.

The hatching enzyme of the medaka, Oryzias latipes, consists of two proteases, high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE). They are synthesized and accumulated in the same unicellular hatching glands and are secreted from them at the end of embryonic development to digest the egg envelope. Recently, these enzymes were purified, and their cDNA clones were isolated. In the present study, we examined temporal and spatial patterns of expression of the hatching enzyme genes during embryogenesis using cDNAs for HCE and LCE as probes. According to Northern blotting analysis, the expression of both genes started at the same time (stage 21-22 embryos: brain differentiation and lens formation) and the patterns of expression changed in parallel during development. In situ hybridization to whole embryo and the sections revealed that the expression of the HCE genes was detected first in the anterior end of the hypoblast layer in stage 16-17 (late gastrula) embryos. Distinct signals of the HCE gene expression were then detected in a group of cells located at the front of the head rudiment of embryos at stage 18-19 (1 somite). Treatment of the embryos with retinoic acid, which is known to affect the anterior differentiation of embryos, suppressed the hatching gland cell differentiation in accordance with the result of in situ hybridization. In stage 22 embryos, the HCE-positive cells dispersed in an ectodermal layer under the forebrain and optic vesicles. Thereafter, the hatching gland cells expressing the HCE mRNA were aligned along the branchial arches and finally rearranged to the inner wall of the pharyngeal cavity, following a marked elongation of the lower jaw. The results of in situ hybridization to whole embryos at consecutive developmental stages demonstrated that the hatching gland cells located at the most anterior portion of the hypoblast migrated posteriorward to endoderm (pharyngeal endoderm) by way of ectoderm, while they were expressing mRNA for the hatching enzyme. Retinoic acid treatment of embryos gave rise to aberrations in the final location of the hatching gland cells probably by disturbing their migration. Moreover, the number of hatching gland cells increased markedly during their migration. This fact strongly suggested a concurrence of gene expression and mitosis of a gland cell and/or a successive initiation of gene expression in maturing gland cells during migration.

Animals↗
PubMed1995

[Determination of gene mutation of silent serum cholinesterase and its epidemiologic characters in the Japanese].

We detected 121 individuals with silent type of serum cholinesterase from 36 families in Japan. DNA analysis totaling 37 members of eleven blood unrelated families were carried out by four useful methods, namely, 1) PCR-SSCP analysis, 2) dot blot hybridization (DBH) with the use of synthetic oligonucleotide probe, 3) restriction endonuclease analysis (REA) and 4) direct sequencing analysis. Their mutations were classified into four groups, namely, 1) a G-->C transversion at codon 365, 2) a frameshift mutation with insertion of an extra A at codon 315, 3) an A-->G transition at codon 128 and 4) a C-->A transition at codon 400. The three procedures including (PCR-SSCP, DBH, REA) without the use of radio labeled materials (non-RI) are recommendable for the analyses. However, the direct sequencing analysis of bases with RI might be, at present, necessary for the final identification.

Asian People↗
PubMed1994

HCE, a constituent of the hatching enzymes of Oryzias latipes embryos, releases unique proline-rich polypeptides from its natural substrate, the hardened chorion.

HCE, a constituent protease of the hatching enzymes of Oryzias latipes embryos [1,2], releases unique proline-rich polypeptides from its natural substrate, the hardened chorion. The polypeptides consist of repeats of Pro-X-Y, mainly Pro-Glx-X. In addition, the polypeptides contain abundant gamma-glutamyl epsilon-lysine isopeptides which are regarded to be responsible for chorion hardening. These findings suggest that HCE recognizes specific site(s) of the chorion, releases the proline-rich polypeptides from it, and makes the substrate accessible to LCE, another protease of the hatching enzymes.

Amino Acid Sequence↗
PubMed1994

Synchronous production of the low- and high-molecular-weight precursors of the egg envelope subunits, in response to estrogen administration in the teleost fish Oryzias latipes.

Estrogen stimulation of production and accumulation of L-SF and H-SF, the low- and high-molecular-weight precursors of the egg envelope subunits in Oryzias latipes, was analyzed by immunohistochemical and immunoblotting methods. Both SF substances were detected in the liver of the male fish as quickly as 16-24 hr after estrogen treatment, and their production and accumulation in the liver and blood plasma increased as estrogen treatment continued. Immunohistochemical analysis revealed that the number of cells producing both SF substances was increased in the liver as estrogen treatment continued. These results strongly suggest the occurrence of synchronous production of L-SF and H-SF in response to estrogen. In liver extracts, but not in blood plasma, an additional L-SF-like immunoreactive protein was detected on immunoblots. Preliminary data indicate parallel appearance of vitellogenin and SF substances in the liver of estrogenized fish.

Animals↗
PubMed1993

Isolation of H-SF substances, the high-molecular-weight precursors of egg envelope proteins, from the ascites accumulated in the oestrogen-treated fish, Oryzias latipes.

High- and low-molecular weight spawning-female-specific (H-SF and L-SF) substances are liver-derived putative precursors of the inner layer of egg envelope of the medaka Oryzias latipes, and are also found in the ascites which are accumulated in the male fish on prolonged treatment with oestrogen. In the present study, the H-SF substances purified from the ascites of the oestrogen-treated male medaka were found to consist of three proteins. Their molecular weights ranged from 74,000 to 76,000, which corresponds to those of ZI-1 and ZI-2, the major components of medaka egg envelope. The amino acid composition of the H-SF substances was characterised by high contents of proline (17.58 mol%) and glutamic acid/glutamine (13.34 mol%). The characteristics were almost identical to those of ZI-1 and ZI-2 but differed from those of L-SF substance, a precursor of ZI-3, another major component of the egg envelope. These results confirm our view that H-SF substances are the precursors of ZI-1 and ZI-2. The average amino acid compositions of H-SF substances and L-SF substance coincided with the amino acid composition of whole egg envelope. This result strongly suggests that an equal amount of H-SF substances and L-SF substance would construct the inner layer of egg envelope after a little modification.

Amino Acids↗
PubMed1992

Isolation of cDNAs for LCE and HCE, two constituent proteases of the hatching enzyme of Oryzias latipes, and concurrent expression of their mRNAs during development.

The hatching enzyme of medaka consists of two types of proteases (HCE, LCE). cDNA clones for LCE and HCE were isolated from a lambda gt11 cDNA library constructed with poly(A)+ RNA of Day 3 embryos. LCE cDNA is 936 bp long and contains an 813-bp open reading frame encoding a preproenzyme with a 20-amino-acid signal sequence, a 51-amino-acid propeptide, and a 200-amino-acid mature enzyme. For HCE, two distinct cDNAs (HCE21, HCE23) having nucleotide sequences with 92.8% similarity were obtained. These cDNAs contain open reading frames encoding preproenzymes of 279 and 270 amino acids, respectively. The mature enzyme forms of both consist of 200 amino acids, the similarity between them being 95.5%. On Northern blotting analysis, the transcripts of LCE and HCE genes were first detected coincidentally in Day 2 embryos shortly before the production of LCE and HCE, accumulated thereafter in parallel, and dramatically decreased after hatching. The amino acid sequence, the HExxH motif, which is known to constitute an active site in some Zn proteases, is also found in LCE and HCE. However, the sequence analyses strongly suggest that both the enzymes belong to the astacin (protease) family, being distinct from sea urchin hatching enzyme, which is reportedly similar to collagenase.

Amino Acid Sequence↗
PubMed1992

Two constituent proteases of a teleostean hatching enzyme: concurrent syntheses and packaging in the same secretory granules in discrete arrangement.

Formation, accumulation, and storage of two components of the Oryzias latipes hatching enzyme, high and low choriolytic enzymes (HCE and LCE), were examined by immunocytochemical and immunoblotting methods. Both of the enzymes were found to be formed specifically in the hatching gland cells at the stages of lens formation to eye pigmentation and their accumulation proceeded markedly and concurrently up to Day 5.5 embryos (the stage just before hatching). The amount of HCE formed was more abundant than that of LCE. In the hatching gland cells, HCE and LCE were found to be packaged in the same secretory granules but in distinct arrangement; HCE is localized to the inside of granules whereas LCE is situated at the periphery of the same granules. Their segregated arrangement is compatible with their relative quantities formed per embryo. The results provide not only the cellular and developmental basis for a view that this hatching enzyme is an enzyme system composed of HCE and LCE but also a clue to the regulatory mechanism of concurrent syntheses of two different specific proteins in the same embryonic cell.

Animals↗
PubMed1992

[Identification of two different genetic mutation associated with silent phenotypes for human serum cholinesterase in Japanese].

Two different gene mutations associated with the silent phenotype for human serum cholinesterase were demonstrated. DNA from five individuals with silent gene phenotype of three unrelated Japanese families was amplified by the polymerase chain reaction (PCR) and analyzed by direct sequencing. The first instance demonstrated a G----C transversion at codon 365 from GGA (Gly) to CGA (Arg), which was seen in three individuals of the two families. This mutation was resulted to create a new Taq 1 restriction site (TCGA). The second mutation was shown by a double heterozygous condition with two different silent gene mutations in two members of remaining one family. These mutations were as follows: 1) one type was a frameshift mutation, in which an extra A was inserted in codon 315 (ACC----AACC) to create a new stop codon at position 322 and 2) the other was the same point mutation at codon 365 as seen in the first instance. These results indicated that many silent variants can be distinguished by direct sequence analyses of genomic DNA.

Asian People↗
PubMed1990

Hb Fukutomi [alpha 126(H9)Asp----Val]: a new hemoglobin variant with high oxygen affinity.

Hb Fukutomi [alpha 126(H9)Asp----Val], a new hemoglobin variant with a high affinity for oxygen (log P50 = 0.201 at pH 7.0, normal = 1.087) was detected in a Japanese male during a screening survey. The amino acid substitution is located in the alpha 1 beta 1 contact of the tetramic molecule. Like other variants of the high oxygen affinity type Hb Fukutomi showed increased resistance to auto-oxidation. The tryptic peptide alpha T-13, that usually forms part of the trypsin-resistant core of the alpha chain, was cleaved off by trypsin in the new variant and released into the soluble fraction.

Amino Acid Sequence↗
PubMed1989

A unique proteolytic action of HCE, a constituent protease of a fish hatching enzyme: tight binding to its natural substrate, egg envelope.

High choriolytic enzyme (HCE), a constituent protease of the hatching enzyme of the teleost, Oryzias latipes, swells its natural substrate, egg envelope (chorion) by hydrolyzing it partially. This enzyme was found to be bound tightly to the chorion when it exerted catalytic action. This was evidenced by the experimental results showing (i) that the turnover of this enzyme seemed to be hindered by the chorion, (ii) that the enzyme bound to the chorion could be recovered by washing with an alkaline medium, and (iii) that the bound enzyme could be quantified by radioimmunological estimation. The bound enzyme sustained its original activity and the binding between the enzyme and the chorion seems to be stoichiometric.

Animals↗
PubMed1989

A glycoprotein from the liver constitutes the inner layer of the egg envelope (zona pellucida interna) of the fish, Oryzias latipes.

A glycoprotein from the liver, which shares epitopes with chorion (egg envelope or zona pellucida) glycoproteins, is present only in the spawning female fish, Oryzias latipes, under natural conditions. This spawning female-specific (SF) substance is distinct from vitellogenin but closely resembles a major glycoprotein component, ZI-3, of the inner layer (zona radiata interna) of the ovarian egg envelope with respect to some biochemical and immunochemical characteristics. Here we report that the [125I]SF substance, injected into the abdominal cavity of the spawning female fish, was rapidly transported by the blood circulation into the ovary and incorporated into the inner layer of egg envelope of the growing oocytes. The result strongly suggests that the SF substance from the liver is a precursor substance of the major component, ZI-3, of the inner layer of egg envelope in the fish.

Animals↗
PubMed1989

Purification and partial characterization of high choriolytic enzyme (HCE), a component of the hatching enzyme of the teleost, Oryzias latipes.

The hatching enzyme is an embryo-secreted enzyme(s) which digests the egg envelope, allowing the embryo to emerge at the time of hatching. The hatching enzyme of the fish, Oryzias latipes, has recently been found to consist of two kinds of proteases which may digest the inner layer of chorion (egg envelope) cooperatively [Yasumasu, S. et al. (1988) Zool. Sci. 5, 191-195]. In the present study, one of them, high choriolytic (egg envelope digesting) enzyme (HCE) was purified and some biochemical and enzymological properties were examined. The enzyme was a basic protein with a molecular weight of about 24 kDa, and exhibited choriolytic activity as well as proteolytic (caseinolytic) activity. The results of inhibitor studies and metal analyses strongly suggested that it was a zinc-protease. The purified HCE consisted of two probable isomers, HCE-1 and HCE-2. Both of them were markedly similar in amino acid composition, specific activities of choriolysis and proteolysis, and substrate specificity as determined using MCA-peptides. Moreover, they were not separable on SDS-PAGE, electrofocusing PAGE, or ultracentrifugal analysis, but were discriminated only on HPLC with a CM-300 column. Thus, the mixture of HCE-1 and HCE-2 could be regarded as almost a single enzyme, HCE. When it acted on an intact chorion, the purified HCE caused a remarkable swelling of its inner layer with concomitant release of peptides from it. Once the inner layer of chorion was swollen, the enzyme hardly digested it.

Amino Acid Sequence↗
PubMed1989

Isolation and some properties of low choriolytic enzyme (LCE), a component of the hatching enzyme of the teleost, Oryzias latipes.

One of the two component proteases of the hatching enzyme of the fish, Oryzias latipes, low choriolytic enzyme (LCE), was isolated from the hatching liquid and partly characterized. The enzyme was a basic protein with molecular weight of about 25.5 kDa. Like high choriolytic enzyme (HCE), the other component of the O. latipes hatching enzyme [Yasumasu, S. et al. (1989) J. Biochem. 105, 204-211], LCE was considered to be a zinc-protease from the results of inhibitor studies and metal analyses. However, LCE was found to be distinct from HCE not only in some biochemical characteristics such as molecular weight, amino acid composition, and isoelectric point, but also in some enzymological properties such as substrate specificity, heat stability, and mode of action toward their natural substrate, chorion (egg envelope). Although LCE was almost incapable of digesting the inner layer of intact chorion, it very efficiently digested the inner layer of chorion that had been swollen previously by the action of HCE. Taking account of the fact that HCE swells the inner layer of intact chorion by partial proteolysis but does not efficiently digest the swollen chorion any more [Yasumasu, S. et al. (1989) J. Biochem. 105, 204-211], the present results demonstrated an essential role of LCE in choriolysis, in cooperation with HCE.

Amino Acid Sequence↗