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S Kurachi

Publications and source records attributed to S Kurachi.

At least 19 recordsLinked to original sources
PubMed1998

Sialic-acid-binding lectin from the slug Limax flavus--cloning, expression of the polypeptide, and tissue localization.

A cDNA library of Limax flavus was constructed and screened for sialic-acid-specific lectins. Complementary DNA clones were categorized into seven groups corresponding to closely related but different sequences. Group 1 clones contained an ORF encoding 199 amino acids including a sequence identical to the partial amino acid sequence obtained from the lectin protein. Within its 1074-bp 3' untranslated region, ten closely related 60-bp sequence repeats were found. Group 2 clones contained an ORF encoding a polypeptide chain of the same number of amino acid residues, with 89.1% overall identity to that of the group 1 and eight 60-bp repeat sequences in the 3' untranslated region. The remaining groups of clones contained ORF with highly similar full or partial sequences, with or without 60 bp repeats in the 3' untranslated region. The large number of closely related but different cDNA clones obtained indicated that the slug sialic-acid-specific lectin gene is a member of a multigene family. The lectin amino acid sequence showed significant similarity with the fibrinogen domain of human tenascin-C, with a human C-type serum lectin, and with pig ficolin. Immunostaining analysis of slug tissue for the lectin indicated that it is present primarily on the epidermal surface and in mucous glands. Recombinant slug lectin protein lacking the 20-amino-acid N-terminal signal sequence produced in a bacterial expression system from a group-1 clone accumulated as aggregates in inclusion bodies, suggesting that large-scale production of the active agglutinin may be possible.

Amino Acid Sequence↗
PubMed1997

The carboxyl-terminal region of factor IX is essential for its secretion.

The carboxyl-terminal region of factor IX (residues 403-415) contains several natural mutations which result in mild to severe forms of hemophilia B. A battery of factor IX minigene expression vectors carrying various mutations in the C-terminal region were constructed and studied by transient expression assay using HepG2 cells. Mutations included in this study are Y404P, I408N, T412N, T412S, T415G, T415S, T415L, and T415R as well as five selected naturally occurring mutations in the region, R403Q, R403W, Y404H, W407R, and T412K. In comparison to the normal factor IX, these mutations neither significantly affected the factor IX mRNA level nor affected the stability of the secreted factor IX in the culture medium but did decrease to various extents the intracellular and secreted factor IX protein levels as quantified by enzyme-linked immunosorbent assay. T415L, T415S, and T415R showed only minor reductions in the intracellular and minor to moderate reductions in the secreted factor IX levels. T415G showed only minor reduction in the intracellular factor IX level but substantial reduction in the secreted levels. R403Q, R403W, and T412S showed moderate reductions in both intracellular and secreted factor IX levels. Y404H, Y404P, W407R, I408N, T412K, and T412N also showed minor to moderate reductions in the intracellular factor IX levels but very severe reductions in the secreted factor IX level. In one stage clotting assays, secreted factor IX mutants showed specific activities very similar to that of the normal factor IX, suggesting that the carboxyl-terminal region is neither directly involved in the tenase complex formation required for factor X activation nor involved in the activation of factor IX itself. In comparison to the normal factor IX, secreted levels of factor IX mutants with mutations R403Q, Y404H, W407R, and T412K were also very similar to the plasma levels reported for the hemophilia B patients carrying the same mutations. Treatment of cells with proteasome inhibitors (ALLM and ALLN) added to the culture medium at 50 microM resulted in drastic increases of the intracellular mutant factor IX to the levels equivalent to that of the normal factor IX, while the secreted factor IX levels were little or only marginally affected. With a higher concentration of the inhibitors (100 microM), however, both the intracellular and secreted mutant factor IX were fully elevated to the normal factor IX levels. Intracellular and secreted levels of the normal factor IX were little affected by the low inhibitor concentration and only marginally, if at all, at the higher concentration (< or = 10%). Serine protease inhibitors did not significantly affect intracellular and secreted factor IX levels. These results indicate that the carboxyl-terminal region plays a critical role in the cellular secretion of factor IX and that the mutant factor IX proteins carrying specific mutations in this region are subjected to efficient elimination by the proteasome protein degradation mechanism. Furthermore, these results strongly support that HepG2 cells can be utilized as a robust in vitro assay system for studying factor IX biosynthesis, well mimicking the in vivo phenomenon.

Animals↗
PubMed1996

Age and sex dependent regulation of the factor IX gene in mice.

Plasma factor IX and liver factor IX mRNA levels in two normal mouse strains (B6D2F1 and BALB/CJNIA) were determined in relation to aging and sex of the animals. With male B6D2F1 mice, mean plasma factor IX activity levels for the 14 and 21 approximately 22 month-old animals were found to be 124% and 226%, respectively, of the 5 month-old group. Similarly, liver factor IX mRNA levels for the same age animal groups were 145% and 227%, respectively, of the reference group. Mean plasma factor IX levels for the same age female animals were 132% and 175%, respectively, and were accompanied by similarly elevated liver factor IX mRNA levels, 119 and 175%, respectively, of the 5 month-old female group. Factor IX activity and mRNA levels for the 5, 14 and 21 approximately 22 month-old female animal groups were lower than those of the corresponding male age groups by 25, 20 and 37%, and 20, 36 and 38%, respectively. With BALB/CJNIA mice, similar correlation was observed between the advancing age and substantial elevations in the factor IX mRNA level as well as on the unequal factor IX mRNA levels in females and males. These results indicate that the plasma factor IX level in both male and female mice is greatly elevated with aging, in general agreement with a similar phenomenon observed for human populations, and that this increase is due to a similar elevation in the factor IX mRNA level in the liver. In mice, both factor IX activity and mRNA levels are significantly higher in males than in females, which has not been described for humans.

Aging↗
PubMed1995

Role of intron I in expression of the human factor IX gene.

The first intron (intron I) of the human factor IX gene, which has been previously suggested of having an expression-augmenting activity, was systematically studied for its potential enhancer activity. When tested with the chloramphenicol acetyltransferase expression vector with a minimal factor IX promoter, subregions of intron I showed only marginal enhancing activities (1.7-1.9-fold enhancement at the highest). Smaller subregions encompassing nucleotides 5660-6350 of the intron sequence even showed some weak negative regulatory activities (approximately 50% suppression at the highest), while a cytomegalovirus enhancer sequence, which was used as the positive control, had a 7-fold enhancement. A set of three factor IX minigene expression vectors with the same factor IX promoter were then constructed: p-416FIXc which contained the factor IX cDNA, p-416FIXm1 which contained the factor IX cDNA with a largely truncated intron I, and p-416FIXm2 which contained the factor IX cDNA with the intron I sequence further truncated. The p-416FIXm1 and p-416FIXm2 constructs showed 7-9-fold higher expression activities than p-416FIXc. The elevated factor IX antigen levels agreed well with the grossly elevated factor IX clotting activity and mRNA levels. These results indicate that the expression enhancing activity of intron I is not due to specific enhancer elements present in the intron subsequences, but is due to functional splicing sequences present in the precursor mRNAs produced from the minigene constructs containing intron I. By being efficiently assembled into spliceosome complexes, transcripts with splicing sequences may be better protected in the nucleus from random degradations than those without such sequences.

Animals↗
PubMed1994

Regulatory mechanism of human factor IX gene: protein binding at the Leyden-specific region.

Hemophilia B-Leyden is characterized by the gradual amelioration of bleeding after the onset of puberty. All Leyden phenotype mutations found to date lie within the Leyden-specific region, which spans roughly nt-40 to +20 in the 5' end of the human factor IX gene. With HepG2 cell nuclear extracts, the Leyden-specific region and its immediate neighboring region of the normal factor IX gene showed five DNase I footprints: FP-I (nt +4 to +19), FP-II (nt -16 to -3), FP-III (nt -27 to -19), FP-IV (nt -67 to -49), and FP-V (nt -99 to -77). Protein binding affinities of short oligonucleotides containing sequences of FP-I, FP-II, or FP-III were substantially reduced in the presence of Leyden phenotype mutations in these areas, correlating well with the negative effects of these mutations on factor IX gene expression. A Leyden phenotype mutation at nt -20 (T to A) caused a loss of both footprints FP-III and FP-II but generated a new footprint, FP-III' (nt -34 to -23), partially overlapping with FP-III, indicating mutation-dependent competitive protein binding at these sites. Although the FP-III' area contains an androgen responsive element-like sequence, the nuclear protein that binds at FP-III' is not androgen receptor. The protein was not recognized by anti-androgen receptor antibody and, furthermore, was present not only in liver but also in both androgen receptor-positive and androgen receptor-negative cells in electrophoretic mobility shift assays. The nuclear concentration of this protein increased significantly upon treatment of the HepG2 cells with testosterone. Its binding affinity to an oligonucleotide (-32sub) containing the FP-III' sequence was greatly reduced in the presence of exogenous androgen receptor, suggesting a possible interaction of this protein with androgen receptor. The affinities of both this protein and a protein which binds to FP-III (presumably HNF-4) to -32sub with a mutation at nt -26 were grossly lowered. These findings suggest that the amelioration of hemophilia B-Leyden with a mutation at nt -20 after puberty involves binding of a specific non-androgen receptor nuclear protein at FP-III' and it is able to substitute for the function of a protein bound at FP-III in the normal gene optimally through its elevated interaction with androgen receptor upon a surge of testosterone.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Biology of factor IX.

Within the past 20 years or so, factor IX has been at the centre of particularly intensive studies of its physiology, pathology and biochemistry as well as its molecular genetics and biology. With the complete nucleotide sequence of its human gene determined in 1985 and the molecular defects of over 600 abnormal human factor IX genes analysed to date, factor IX is among the few mammalian proteins which have been exhaustively studied in almost every aspect. The enormous amount of information we now have on this medium-sized plasma protein sheds light on how a gene and its protein evolve, how the protein carries out a highly regulated, specific and pivotal role in the delicately balanced blood coagulation reaction, and the correlation between clinical presentations and its highly diverse molecular mechanism of defects. This wealth of knowledge makes factor IX an excellent model for deeper study, such as truly quantitative analysis of its structure-function relationship and in vivo function and regulation. It will also provide a sound foundation which may lead to improved treatment of haemophilia B and perhaps to its cure. This paper attempts to review the recent progress in research on factor IX.

Amino Acid Sequence↗
PubMed1992

Deficiencies in factors IX and VIII: what is now known.

Genetic defects both gross and subtle have recently been identified in about 900 patients with hemophilia A or B. The defects, which include deletions, insertions, and point mutations, reveal much about genetic structure-function relationships in hemophilia. These insights will lead to improved treatment of bleeding disorders and perhaps to their cure.

Base Sequence↗
PubMed1992

Biology of factor IX.

Hemophilia B, one of two common hereditary bleeding disorders, is caused by a deficiency of factor IX in the circulation. Molecular mechanisms of hemophilia B are highly heterogeneous including gene deletions, insertions, complex rearrangements, and a large number of point mutations. Currently, hemophilia B is treated by plasma protein replacement therapy. This therapy is effective but exposes patients to possible side effects and complications such as infection of blood-borne pathogens including hepatitis viruses and HIV-1. Intensive efforts to develop alternative, safer therapies for hemophilia B, including somatic gene therapy, are now under way.

Amino Acid Sequence↗
PubMed1991

Expression of human factor IX in rat capillary endothelial cells: toward somatic gene therapy for hemophilia B.

In aiming to develop a gene therapy approach for hemophilia B, we expressed and characterized human factor IX in rat capillary endothelial cells (CECs). Moloney murine leukemia virus-derived retrovirus vectors that contain human factor IX cDNA linked to heterologous promoters and the neomycin-resistant gene were constructed and employed to prepare recombinant retroviruses. Rat CECs and NIH 3T3 cells infected with these viruses were selected with the neomycin analogue, G418 sulfate, and tested for expression of factor IX. A construct with the factor IX cDNA under direct control by long terminal repeat gave the highest level of expression (0.84 and 3.6 micrograms per 10(6) cells per day for CECs and NIH 3T3 cells, respectively) as quantitated by immunoassays as well as clotting activity assays. A single RNA transcript of 4.4 kilobases predicted by the construct and a recombinant factor IX of 68 kilodaltons identical to purified plasma factor IX were found. The recombinant human factor IX produced showed full clotting activity, demonstrating that CECs have an efficient mechanism for posttranslational modifications, including gamma-carboxylation, essential for its biological activity. These results, in addition to other properties of the endothelium, including large number of cells, accessibility, and direct contact with the circulating blood, suggest that CECs can serve as an efficient drug delivery vehicle producing factor IX in a somatic gene therapy for hemophilia B.

Animals↗
PubMed1991

Characterization of a mouse factor IX cDNA and developmental regulation of the factor IX gene expression in liver.

A mouse factor IX cDNA was isolated and characterized. The cDNA was 1,837 bp in length and contained the coding region as well as short 5' and 3' untranslated sequences. Northern blot analysis of liver RNA showed two mRNA species of 3.2 kb (major) and 2.2 kb (minor) for the mouse factor IX. An antisense RNA probe prepared from the mouse cDNA was employed to determine the steady state level of factor IX mRNA in mouse liver at various developmental stages. The factor IX mRNA level was very low (2-5% of the adult level) during the gestational period until day -3 (gestational day 17) followed by a rapid increase at day -2 through birth. This phase of rapid increase was followed by a gradual increase before it reached the adult level at around 20 to 24 days. At birth, the factor IX mRNA level was found to be at about 43% of that of the adult. The mRNA levels in mouse liver agreed well with the plasma factor IX activity levels. These results indicate that reduced factor IX activity in newborns is due to the low levels of factor IX mRNA available for translation.

Amino Acid Sequence↗
PubMed1989

Sumo15A: a lambda phasmid that permits easy selection for and against cloned inserts.

We report the construction of a phasmid vector, Sumo15A, designed for recombination-based screening of recombinant DNA libraries [Seed, Nucleic Acids Res. 11 (1983) 2427-2445]. This vector permits rapid selection in Escherichia coli for homology-mediated integration and excision between homologous DNA inserts cloned in a supF-carrying plasmid and in Sumo15A. The region available for recombination spans the homologous sequence shared by the plasmid and the phasmid. SupF is the selection tool that we used. Efficient selection for supF expression by Sumo15A requires recombination mediated by the lambda phage red gene, which promotes homologous recombination between phage and plasmid DNAs. Counterselection against supF expression by Sumo15A occurs because the presence of a pSC101-derived plasmid replicon in this phasmid permits the growth of Sumo15A as a plasmid in a specialized host, E. coli strain DK37. In strain DK37, Sumo15A cannot replicate as a phage, and the presence of a plasmid-carrying supF is lethal to cells plated on galactose plates. This scheme was developed to select for sequences that are transcribed from chromosomes of interest.

Bacteriophage lambda↗
PubMed1988

Surface antigenic profile and globin phenotype of two new human erythroleukemia lines: characterization and interpretations.

Detailed characterization of the composite phenotype of two newly established erythroleukemia lines (OCIM1, OCIM2) shows that these lines share many of their erythroid markers (ie, surface antigens and globin program) as well as several of their nonerythroid properties (myeloid/monocytic/megakaryocytic) with the two known erythroleukemia lines (K562, HEL). In addition, each displays novel and instructive features. We argue that the surface and globin phenotype of all erythroleukemia lines is nonrandom and that it may be of physiologic relevance; it could represent the most prevalent phenotype of cells transformed by leukemia in vivo, and it raises the possibility that cells with similar potentials exist transiently during normal hematopoietic differentiation before their irreversible commitment to a single lineage. As such, these cells demonstrate a greater phenotypic adaptability in vitro than do their single lineage-committed counterparts since they can differentiate toward more than one lineage.

Antigens, Surface↗
PubMed1987

Coexpression of embryonic, fetal, and adult globins in erythroid cells of human embryos: relevance to the cell-lineage models of globin switching.

The cellular control of the switch from embryonic to fetal globin formation in man was investigated with studies of globin expression in erythroid cells of 35- to 56-day-old embryos. Analyses of globins synthesized in vivo and in cultures of erythroid progenitors (burst-forming units, BFUe) showed that cells of the yolk sac (primitive) erythropoiesis, in addition to embryonic chains, produced fetal and adult globins and that cells of the definitive (liver) erythropoiesis, in addition to fetal and adult globins, produce embryonic globins. That embryonic, fetal, and adult globins were coexpressed by cells of the same lineage was documented by analysis of globin chains in single BFUe colonies: all 67 yolk sac-origin BFUe colonies and 42 of 43 liver-origin BFUe colonies synthesized epsilon-, gamma-, and beta-chains. These data showed that during the switch from embryonic to adult globin formation, embryonic and definitive globin chains are coexpressed in the primitive, as well as in the definitive, erythroid cells. Such results are compatible with the postulate that the switch from embryonic to fetal globin synthesis represents a time-dependent change in programs of progenitor cells rather than a change in hemopoietic cell lineages.

Cells, Cultured↗
PubMed1987

Isolation and characterization of cDNAs encoding the heavy chain of human inter-alpha-trypsin inhibitor (I alpha TI): unambiguous evidence for multipolypeptide chain structure of I alpha TI.

Human inter-alpha-trypsin inhibitor (I alpha TI) is a plasma glycoprotein of Mr 180,000, which has been described as a single polypeptide chain. Recently, however, we proposed that I alpha TI might be composed of a heavy (H) chain (Mr = 95,000) and a light (L) chain (Mr = 40,000) synthesized by two separate mRNAs. In the present study we have characterized cDNAs for the H chain of I alpha TI. These cDNAs collectively covered two sequences (977 and 1450 base pairs in length) with single open reading frames. The deduced amino acid sequences were highly homologous to each other and well matched with partial amino acid sequences obtained from purified serum I alpha TI. RNA blot analyses of liver RNAs with H- or L-chain cDNAs as probes clearly identified two distinct mRNAs of 3.3 and 1.3 kilobases, which corresponded to H or L chain, respectively. Poly(A)+ RNAs hybrid-selected with H-chain cDNAs coded for polypeptide chains of Mr 90,000-95,000. These results unambiguously establish that I alpha TI is made of multipolypeptides, possibly including one H and two L chains. The H chain contains potential calcium-binding sites and also regions homologous to the proposed reactive site for thiol-proteinase inhibitors. These data indicate that I alpha TI is a complex, multifunctional protein. mRNAs for both the H and L chains were found only in liver.

Alpha-Globulins↗
PubMed1987

Analysis of the erythroid phenotype of HEL cells: clonal variation and the effect of inducers.

The erythroid phenotype of HEL cells, before and after the addition of a variety of inducers, was assessed at the cellular and biochemical level. Among 14 inducers used, delta-aminolevulinic acid (delta-ALA) was identified as the most optimal inducer of heme and globin synthesis in HEL cells. The relative synthesis of globin chains produced by HEL cells, mainly gamma and alpha chains with traces of epsilon and zeta chains, was not influenced by the majority of the inducers used. However, delta-ALA and bromodeoxyuridine did increase the relative synthesis of alpha and epsilon chains respectively. Subcloning experiments revealed heterogeneity in the constitutive expression of alpha globin; however, the latter was inducible in all clones by either hemin or delta-ALA. One rare clone of HEL cells was found to produce, in contrast to parental cells, significant amounts of epsilon globin. This clone differed from K562 cells by the absence of any zeta globin expression, thus demonstrating the independent regulation of the two embryonic chains, epsilon and zeta. Changes in the expression of several surface markers specific for erythroid cells were found to accompany the globin accumulation in these cells, and some of these changes appeared to be inducer specific. Thus, the unique globin and nonglobin phenotypic properties of HEL cells and their subclones make them valuable cellular models complementary to the existing K562 cells for studying regulatory aspects of erythroid-specific proteins.

Antigens, Surface↗