Search PubMed⌕ Search

Biomedical subjects

K Itakura

Publications and source records attributed to K Itakura.

At least 37 records · Page 2Linked to original sources
PubMed1998

A novel DNA-binding motif shares structural homology to DNA replication and repair nucleases and polymerases.

A novel class of DNA-binding domains has been established from at least sixteen recently identified DNA-binding proteins. The three-dimensional structure of one of these domains, Mrf-2, has been solved using NMR methods. This structure is significantly different from known DNA-binding domain structures. The mechanism of DNA recognition by this motif has been suggested based on conserved residues, surface electrostatic potentials and chemical shift changes. This new DNA-binding motif shares structural homology with T4 RNase H, E. coli endonuclease III and Bacillus subtilis DNA polymerase I. The structural homology suggests a mechanism for substrate recognition by these enzymes.

Amino Acid Sequence↗
PubMed1997

Protein modification by lipid peroxidation products: formation of malondialdehyde-derived N(epsilon)-(2-propenol)lysine in proteins.

Malondialdehyde (MDA), a naturally occurring dialdehyde produced in the membrane lipid peroxidation, is known to react with lysine residues of proteins, but the MDA-lysine adducts generated in the proteins have not been characterized adequately. In the present study, we provide evidence that the enaminal-type MDA-lysine adduct, N(epsilon)-(2-propenal)lysine, is formed in human low-density lipoprotein (LDL) upon reaction with MDA or Cu2+. We found that the incubation of N(alpha)-acetyllysine with MDA generated N(alpha)-acetyl-N(epsilon)-(2-propenal)lysine as the predominant product. In addition, a polyclonal antiserum raised against the MDA-modified protein was found to contain antibody populations that could be purified by affinity gel prepared by covalent attachment of N(alpha)-acetyl-N(epsilon)-(2-propenal)lysine. It was concluded that the affinity-purified anti-N(epsilon)-(2-propenal) lysine antibody was highly specific to the enaminal derivative of both lysine residues and phosphatidylethanolamine, based on the observations that (i) MDA was the only aldehyde which generated immunoreactive materials in proteins; (ii) among structurally defined MDA-lysine adducts tested, the antibody recognized the enaminal adduct only; and (iii) immunoreactivity to N-(2-propenal)serine was still significant but much weaker than its reactivity to N-(2-propenal)ethanolamine. Furthermore, analysis of antibody recognition sites with a variety of N-(2-propenal)alkylamines revealed that the mono-specific antibody recognized the N-2-propenal-N-ethyl moiety [-(CH2)2-NH-CH=CH-CHO] of enaminal adducts. Determination by a competitive enzyme-linked immunosorbent assay demonstrated that N(epsilon)-(2-propenal)lysine accounted for 33.7 and 3.1% of the lysine residues that disappeared during in vitro incubation of LDL with MDA and Cu2+, respectively. These results suggest that N(epsilon)-(2-propenal)lysine represents a major form of MDA covalently attached to proteins.

Antibodies, Monoclonal↗
PubMed1997

MAZ, a Myc-associated zinc finger protein, is essential for the ME1a1-mediated expression of the c-myc gene during neuroectodermal differentiation of P19 cells.

To investigate whether MAZ (Myc-associated zinc finger protein) affects the expression of the c-myc gene during the retinoic acid-induced (RA-induced) neuroectodermal differentiation of P19 embryonal carcinoma (EC) cells, we introduced a CAT reporter construct, human c-myc promoter/CAT (pMyc2CAT), and a mutant CAT derivative that lacked an ME1a1 site (pMyc1CAT) into P19EC cells to monitor the promoter activity of the c-myc gene. The expression of CAT in pMyc2CAT-transformed cells declined fivefold after 24 h in the presence of RA, returned to the normal level within 48 h, and decreased again to below 20% of the normal level after 96 h. By contrast, the expression of CAT in pMyc1CAT-transformed cells did not return to the normal level after 48 h in the presence of RA. In addition, an electrophoretic mobility shift assay (EMSA) with ME1a1 DNA as probe demonstrated that the kinetics of the DNA-binding activity of MAZ were closely correlated with the changes in the expression of CAT from the c-myc promoter/CAT gene during the differentiation of P19EC cells. Taken together, these results suggest that MAZ plays a key role in the transient increase in the expression of the c-myc gene after 48 h of exposure to RA during the neuroectodermal differentiation of P19EC cells.

Animals↗
PubMed1997

Oxidative modification of tryptophan residues exposed to peroxynitrite.

The aim of this study was to clarify the mechanism of loss of Trp residues in proteins exposed to peroxynitrite. The Trp residues in bovine serum albumin and collagen IV were decreased by peroxynitrite treatment. To identify the degradation products of the Trp residue by peroxynitrite, tert-butoxycarbonyl-L-tryptophan (Boc-Trp) was used as a model of the Trp residue in proteins, and the products formed from peroxynitrite-treated Boc-Trp were then isolated. Boc-Trp decreased with an increase in peroxynitrite concentration. N-Formylkynurenine, oxindole, and hydropyrroloindole were identified as major products. The formation of these products may account for the losses of Trp residues in proteins by peroxynitrite.

Animals↗
PubMed1996

Binding of THZif-1, a MAZ-like zinc finger protein to the nuclease-hypersensitive element in the promoter region of the c-MYC protooncogene.

A detailed analysis is reported of the binding of the zinc finger protein THZif-1 to the nuclease-hypersensitive element (NHE) in the promoter region of the c-MYC gene using the electrophoretic mobility shift assay and a series of mutants of a fusion protein composed of glutathione S-transferase and THZif-1. The THZif-1 protein bound specifically to the single-stranded (ss) pyrimidine-rich DNA of the NHE (ss c-myc NHE-C) with an apparent dissociation constant (Kd (app)) of 0.077 microM. By contrast, no binding to the single-stranded purine-rich DNA of the NHE (ss c-myc NHE-G) was detected. Moreover, the binding affinity of THZif-1 protein was 2-fold higher for the single-stranded 5-methyl-2'-deoxycytidine derivative of NHE (ss c-myc NHE-me5C) than for the unmethylated NHE. In the case of the binding of THZif-1 to methylated double-stranded (ds) NHE (ds c-myc NHE-me5CG), no significant binding to the DNA was observed. The decrease in binding to DNA of THZif-1 was significant in the case of mutated ds c-myc NHE, in which more than two sites of deoxycytidine residues were methylated. However, the binding affinity of THZif-1 protein for methylated and for unmethylated triple-helical DNA of the NHE was almost identical. Moreover, the domain of the THZif-1 protein that made the major contribution to binding to ss c-myc NHE-C or ss c-myc NHE-me5C corresponded to the amino-terminal second zinc finger motif. Taken together, the results indicate that the THZif-1 protein exhibits preferential DNA-binding activity with ss c-myc NHE-C, ds c-myc NHE-CG, and ts c-myc NHE but not with ss c-myc NHE-G and ds c-myc NHE-me5CG in vitro.

Chromosome Mapping↗
PubMed1996

Members of the MAZ family: a novel cDNA clone for MAZ from human pancreatic islet cells.

A human recombinant cDNA clone that encoded a zinc-finger protein (Myc-associated zinc-finger protein of human islet; MAZi) was cloned by screening a cDNA library prepared from human pancreatic islet cells. The encoded protein showed a high degree of homology to the Myc-associated zinc-finger protein MAZ (ZF87 or Pur-1). However, differences between the cDNAs for MAZi and MAZ were found in the length of the encoded polyalanine stretch and in the sequence of the 5'-end leader. MAZi transcripts were significantly more abundant in rat pancreatic islet carcinoma tissue than in normal rat islet cells. Moreover, MAZi protein bound specifically to the pyrimidine-rich strand of the CT-element of the c-myc gene in vitro and strongly induced the expression of chloramphenicol acetyltransferase (CAT) from a c-myc promoter/ CAT reporter construct in human pancreatic cells. Our results suggest that a distinct member of the MAZ family is expressed in human islet cells and enhances the transcriptional activity of the c-myc gene.

Amino Acid Sequence↗
PubMed1996

Repression by a differentiation-specific factor of the human cytomegalovirus enhancer.

We detected a novel nuclear protein, MRF, that binds to multiple sites on the modulator which is located upstream of the human cytomegalovirus major immediate early gene enhancer. The expression of MRF is differentiation specific; the DNA binding activity is present in nuclear extracts from undifferentiated Tera-2 and THP-1 cells, but significantly reduced after these cells are induced to differentiate. In undifferentiated cells the enhancer activity is repressed by the modulator and upon differentiation the enhancer becomes active. Competitive binding assays demonstrate that MRF requires the presence of multiple A+T stretches for binding to DNA, rather than binding to a specific DNA sequence. Mutations of these stretches in the modulator reduce the binding activity of MRF, as well as the repressing activity on the enhancer. These results suggest that MRF may act as a repressor of enhancer function. We propose that MRF binds over the entire modulator and exerts repressor activity.

Base Composition↗
PubMed1996

[Heparin affinity high-performance liquid chromatography as an alternative to bioassay of CS23 mutein of recombinant human basic fibroblast growth factor].

We investigated whether a chemical assay by high-performance liquid chromatography (HPLC) as an alternative to the complicated and time-consuming bioassay for CS23 mutein of recombinant human basic fibroblast growth factor (rhbFGF-CS23) using the fetal bovine heart endothelial cell line ATCC CRL 1395. Physically, chemically or enzymatically denatured rhbFGF-CS23 was subjected to heparin affinity (HA)-HPLC and the bioassay. Good agreement was observed between the results obtained by these two methods. Moreover, HA-HPLC gave much more reproducible results (RSD = 1.9%, n = 6) than the bioassay (RSD = 7.4%, n = 18). HA-HPLC is therefore a simple, accurate and reproducible alternative to the bioassay for quality control and stability studies for rhbFGF-CS23 preparations. HA-HPLC is also considered to be applicable to assays for FGFs which have heparin affinity and biological activity similar to those of the CS23 mutein.

Animals↗
PubMed1995

Characterization of epitopes recognized by 4-hydroxy-2-nonenal specific antibodies.

In the present study, we have raised anti-peptide antibodies directed to the major membrane lipid peroxidation product 4-hydroxy-2-nonenal (HNE) attached covalently to histidine, and their specificities were compared with those of the polyclonal antibodies (anti-HNE-protein antibodies) raised against HNE-treated keyhole limpet hemocyanin (K. Uchida et al. (1993) Proc. Natl. Acad. Sci. USA 90, 8742-8746). The anti-HNE-histidyl peptide antibodies (anti-HNE-histidine antibodies) were prepared by immunizing rabbits with a HNE-conjugated heptapeptide (Gly3-His-Gly3 amide) coupled to the carrier protein. The antisera were purified on an affinity gel prepared by covalent attachment of a HNE-conjugated heptapeptide (Ala3-His-Ala3 amide). Among the structurally defined 4-hydroxy-2-alkenal-amino acid adducts tested, binding of anti-HNE-histidine antibodies to the HNE-treated protein was not only inhibited by HNE-histidine, HNE-cysteine, and HNE-lysine, but also by 4-hydroxy-2-octenal-histidine and 4-hydroxy-2-decenal-histidine adducts. Cross-reactivity studies revealed that both anti-HNE-protein antibodies had the highest affinity for the HNE-treated protein and that neither of the antibodies cross-reacted with the protein treated with aldehydes including malondialdehyde, 1-hexanal, 2-hexenal, or 2-nonenal. These results suggest that the dominant epitope recognized by antibodies is the 2-CH3(CH2)n-5-hydroxytetrahydrofuran (n > or = 3) moiety of the Michael adducts. The immunohistochemical analysis of atherosclerotic lesions of human aorta demonstrated that these antibodies reacted strongly with granular cytoplasmic elements of foam cells and weakly with elements in the surrounding sclerotic stroma.

Aged↗
PubMed1994

Human monoclonal antibody identified an immunoreactive tetrapeptide sequence (Lys-Tyr-Gln-Ile) in M(r) 43,000 protein of human melanoma.

The human monoclonal antibody (HuMAb) L92 reacts to an M(r) 43,000 protein associated with human melanoma. To identify the gene encoding its antigenic epitope, a complementary DNA expression library constructed from the human melanoma cell line UCLASO M14 was screened with HuMAb L92. DNA sequence analysis of the isolated clone revealed that the immunoreactive peptide was composed of 10 amino acids (QDLT-MKYQIF). The peptide was expressed in Escherichia coli with beta-galactosidase as a fused protein. There is no homology between the cloned sequence and other reported DNA sequences. Western blot analysis showed that the fused protein had specific binding to HuMAb L92. An antigen-encoding peptide with 10 amino acids was synthesized and tested for its immunoreactivity in vitro. HuMAb L92 reacted specifically to the 10-amino acid peptide in both an antibody-binding inhibition to the M(r) 43,000 protein and a solid-phase enzyme-linked immunosorbent assay. Using several truncated fusion proteins, we found the minimum number of amino acids required for the antibody binding to be 4 (KYQI). These results suggest that the identified peptide sequence encodes the antigenic epitope of the M(r) 43,000 protein.

Amino Acid Sequence↗
PubMed1994

Hepatic transcriptional up-regulator of the rat microsomal epoxide hydrolase gene.

Rat microsomal epoxide hydrolase (mEH) is one of the detoxification enzymes and selectively expressed in liver. A 350-bp DNA fragment of the proximal promoter was found to contain information sufficient to express the mEH gene in hepatoma cells, however not in nonhepatoma cells. We identified two cis-acting elements, epoxide hydrolase proximal element 1 (EHP1) and 2 (EHP2), in this promoter region by using transient transfection assays. Each element is a new cell-type-specific transcriptional up-regulator. The cell-type-specific activity of EHP1 correlates to the limited cell distribution of its cognate transacting factor(s). In the case of EHP2, a similar or possibly the same cognate factor(s) binding to EHP2 was detected by DNase I footprinting and gel retardation assays in both hepatoma and nonhepatoma cells. However, EHP2 functions as an up-regulator only in hepatoma cells. Our finding adds repertoire to a battery of cis-regulatory elements that are required for liver-specific transcription.

Animals↗
PubMed1994

Acute changes in liver gene expression in the N-nitrosodiethylamine-treated rat.

We constructed a complementary DNA (cDNA) library from mRNAs of rat liver induced by an initiating dose of a chemical carcinogen, N-nitrosodiethylamine (DEN). Using a differential hybridization with cDNA probes prepared from mRNAs of control and DEN-treated rat liver, eight cDNAs of which expression was altered by an acute single dose of DEN were cloned. Colony hybridization and nucleotide sequencing demonstrated six independent cDNA clones. These were known genes encoding liver-specific proteins such as microsomal epoxide hydrolase (mEH; epoxide hydrolase, EC 3.3.2.3), albumin, transthyretin, CYP2B7, CYP1A2 (microsomal cytochrome P450, EC 1.14.14.1) and argininosuccinate synthetase (EC 6.3.4.5). Quantitative Northern blot hybridization was carried out to measure the mRNA content of DEN-initiated rat liver at various times after DEN injection. We also analyzed the expression of glutathione transferase P (GST-P; glutathione transferase, EC 2.5.1.18), c-jun and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; glyceraldehyde-phosphate dehydrogenase, EC 1.2.1.12). A single injection of DEN increased the mRNA levels of mEH, beta-actin and c-jun markedly and those of GST-P and GAPDH moderately, but decreased the mRNA levels of CYP2B7, CYP1A2, albumin and argininosuccinate synthetase. Transthyretin mRNA content was not changed, indicating that it was a false-positive clone picked up by chance. These dramatic changes in liver gene expression after acute exposure to DEN are discussed in terms of acute reactions to the massive damage to the DNA and self-defense mechanisms against toxic xenobiotics.

Animals↗
PubMed1992

TGF-beta 1 inhibits DNA synthesis and phosphorylation of the retinoblastoma gene product in a rat liver epithelial cell line.

In the rat liver epithelial cell line, WB, the ability of TGF-beta 1 to inhibit DNA synthesis was shown to correlate with its ability to inhibit phosphorylation of the protein product of the retinoblastoma susceptibility gene, pRb. When WB cells were serum-starved, then refed with serum-containing medium, a peak of DNA synthesis occurred at about 18 h. Autoradiographs showed that 43.6% of cell nuclei could be labeled with 3H-thymidine at this time. When TGF-beta 1 was added simultaneously with serum, it blocked DNA synthesis and reduced the number of labeled nucleii to 6.3%. Cells treated with serum alone for 18 h also showed a pronounced increase in the highly phosphorylated form of pRb, as shown by mobility shifts in immunoblots, and in active phosphorylation of pRb, as shown by 32P incorporation. Simultaneous addition of TGF-beta 1 with serum abolished both 32P incorporation into pRb and its mobility shift on immunoblots. The effect of TGF-beta 1 on DNA synthesis measured at 18 h was sharply reduced if the cells were incubated with serum for 8 h (and thus allowed to enter S) before the addition of TGF-beta 1. If TGF-beta 1 was added after 8 h of serum treatment, its ability to inhibit pRb phosphorylation at 18 h was unchanged. If TGF-beta 1 was added after 13 h of serum treatment, its effects on pRb phosphorylation were reduced. Thus, as the cell population moved into S, the ability of TGF-beta 1 to inhibit both pRb phosphorylation and DNA synthesis was lost. In higher passages of WB cells the dose-response for inhibition of DNA synthesis by TGF-beta 1 was shifted to the right. Inhibition of pRb phosphorylation by TGF-beta 1 was also lost in higher passage WB cells. Thus, the passage-dependent loss of sensitivity to inhibition of DNA synthesis accompanied the loss of sensitivity to inhibition of pRb phosphorylation. Since the phosphorylation of pRb is believed to be required for the progression of cells from G1 to S, inhibition of pRb phosphorylation may be either a cause or a consequence of the G1 arrest of WB cells by TGF-beta 1.

Animals↗
PubMed1992

PCR-based quantitation of low levels of HIV-1 DNA by using an external standard.

Beginning with 10(3)-10(5) molecules of a purified HIV-1 target sequence as a starting template, we have examined the effects of starting template concentration and cycle number on the amplification efficiency of the polymerase chain reaction. An external standard DNA sequence has been designed that when added to a DNA sample enables a determination of the starting concentration of HIV-1 target sequence in that sample of DNA. Varying ratios of external standard and target DNA sequences were amplified for 22 cycles. When the starting concentration of the external standard was within 50-fold of the starting concentration of the target, the amplifications of both sequences were proportional. These same results were obtained when the two templates were amplified in the presence of an excess of heterogeneous genomic DNA. Using this quantitative method, the number of starting target molecules in a DNA sample can be calculated to within a two-fold range of accuracy.

Base Sequence↗
PubMed1992

Anterior capsule opacification: a cell culture model.

We cultured the human lens epithelial cells with attaching anterior lens capsule using surgically removed capsular flaps obtained in intraocular lens implantation. The capsular flap was placed with cell side down in a small culture well. In phase contrast microscopy, an anti-meniscus plate was placed on the bathing medium to avoid optical aberration. The human lens epithelial cells showed outgrowth which ceased after 3 to 4 weeks of incubation. The cells under the capsule lost distinctive cell margin. The cells outgrew from the capsular edge both onto the anterior capsular surface and the well bottom. The outgrowth length and Bromodeoxyuridine uptake indicated a low proliferative potency of the human lens epithelial cells.

Cataract↗
PubMed1992

A bipartite suppressor: conjunction of two distinct factor-binding sites is essential for down-regulation in rat epoxide hydrolase gene expression.

We describe a novel transcriptional suppressor element found in the control region of the gene that encodes rat microsomal epoxide hydrolase (mEH), an inducible xenobiotic metabolizing enzyme. This element consists of the juxtaposition of two distinct factor-binding regions. The first region is composed of a series of five tandemly repeated factor-binding sequences, and the second region is an unique AT-rich factor-binding sequence. Although each region binds its cognate factor(s) in vitro, a single region does not function as a suppressor independently of the other. Transcriptional suppression was observed only when the two regions were combined. Thus, we propose that this regulatory element is a bipartite suppressor, requiring two distinct factor-binding regions for its function. The element displayed position-independent but orientation-dependent suppressor activity. The level of suppressor activity was proportional to the number of repetitive sites in region 1. We speculate that this region could mediate the dose-response behavior of mEH gene expression induced by chemical carcinogens in vivo. A qualitative difference in the region 2 binding factor(s) was observed between normal liver cells and a hepatoma cell line or carcinogen-treated liver cells. The possible relationship between this observation and the deregulation of mEH gene expression during the course of hepatocarcinogenesis is discussed.

Animals↗