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Biomedical subjects

K Imai

Publications and source records attributed to K Imai.

At least 595 records · Page 33Linked to original sources
PubMed1996

Disruption of phospholipase B gene, PLB1, increases the survival of baker's yeast Torulaspora delbrueckii.

An uracil auxotrophic mutant of baker's yeast Torulaspora delbrueckii, which is resistant to 5-fluoro-orotic acid, was complemented by transformation with YEp24 which harbors 2 microns origin and URA3 derived from Saccharomyces cerevisiae. The phospholipase B in T. delbrueckii cells is active in both acidic and alkaline conditions. However, activity of phospholipase B gene (PLB1) in cells of disruption mutant (plb1:: URA3) was lost in both conditions, which indicates that all phospholipase B activity is encoded by a single gene (or a single polypeptide) in these yeast cells. Over-expression of PLB1 with YEp plasmid vector in T. delbrueckii cells showed approximately 2.5-fold increase in phospholipase B activity, comparing with that in wild-type cells. Cells of plb1 delta mutant showed increased survival when cells of plb1 delta mutant and wild-type strain were incubated in water at 30 degrees C. Cells of PLB1-over-expressed strain died rapidly even during the cultivation period, indicating that phospholipase B activity may be a determinant for the survival of this yeast.

Bread↗
PubMed1996

Interleukin-2 (IL-2) upregulates BAG-1 gene expression through serine-rich region within IL-2 receptor beta c chain.

BAG-1 is a Bci-2-binding protein which functions in protection from apoptotic cell death. Here we provide evidence for interleukin-2 (IL-2)-mediated upregulation of BAG-1 expression. In hematopoietic cell line BAF-B03 F7 cells, gene transfer mediated expression of the IL-2R beta c chain is sufficient to confer proliferation and cell survival responses to IL-2. In these IL-2R beta c-expressing cells, BAG-1 mRNA was dramatically induced by IL-2. The IL-2-mediated induction of BAG-1 expression required the activation of tyrosine kinase(s) and was sensitive to rapamycin as the induction of bcl-2 expression was. Analysis of the transfectants which express mutant IL-2R beta c chains or mutant Janus family protein tyrosine kinase Jak3 lacking the kinase domain showed that the IL-2-mediated BAG-1 gene expression required the serinerich region within the IL-2R beta c chain, but Jak3 activation was dispensable. The signaling pathway for BAG-1 gene expression thus highly resembles that for bcl-2 gene expression, strongly suggesting that their induction shares the same signaling pathway. In addition, deletion of the serine-rich region led to loss of IL-2-mediated protection from apoptotic cell death. Taken together, these studies demonstrate that the serine-rich region of the IL-2R beta c chain mediates the coordinated expression of bcl-2 and BAG-1 genes, thereby contributing to suppression of apoptosis.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

MUC1-specific targeting immunotherapy with bispecific antibodies: inhibition of xenografted human bile duct carcinoma growth.

For the purpose of establishing a new adoptive immunotherapy for bile duct carcinoma (BDC), we synthesized two bispecific antibodies (BsAbs), MUC1 x CD3 BsAb constructed with MUSE11 (anti-MUC1 tumor antigen) and OKT-3 (anti-CD3), and MUC1 x CD28 BsAb constructed with MUSE11 and 15E8 (anti-CD28) antibodies. These two BsAbs reacted well with both MUC1-positive target tumor cells and effector lymphokine-activated killer (LAK) cells. Investigation of in vitro cytotoxicity [3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide assay] revealed that the MUC1 x CD3 BsAb could antigen-specifically enhance the cytotoxicity of LAK cells. Addition of the two BsAbs (MUC1 x CD3 BsAb plus MUC1 x CD28 BsAb) in vitro resulted in a 60% cytotoxicity, similar to that obtained with BsAb (MUC1 x CD3) alone. Interleukin 12-induced LAK cells demonstrated far greater cytotoxicity (50%) than their interleukin 2-induced counterparts (LAK cells), and this was also enhanced by the BsAbs. When 2 x 10(7) LAK cells sensitized with both kinds of BsAbs were administered four times i.v. to BDC-grafted severe combined immunodeficient mice (tumor size 5 mm in diameter), inhibition of tumor growth was observed. Thus, BsAb-LAK therapy for control of BDC warrants clinical trials.

Animals↗
PubMed1996

Functional comparison of the myoglobins reconstituted with symmetric deuterohemes.

Deuterohemins III and XIII were coupled with apomyoglobin to examine the influence of the modified heme-globin contacts on the functions of the reconstituted holoproteins. Owing to the molecular symmetry of the prosthetic groups, the resultant proteins are free from the heme orientational disorder. The coordination structures of the two reconstituted myoglobins were found to be normal and closely similar to each other. The equilibrium ligand bindings also resembled with each other in both ferric and ferrous states. The results demonstrate that the different local heme-globin contacts affect the structure and function of the reconstituted myoglobins only slightly. The results therefore suggest that the two asymmetric deuteroheme IX isomers, which are inverted about the alpha, gamma-meso carbon axis of the heme, also exhibit very similar functions in myoglobin.

Animals↗
PubMed1996

Proton: a major factor for the racemization and the dehydration at the cyclization/cleavage stage in the Edman sequencing method.

The racemization of the liberated 7-[(N,N-dimethylamino)sulfonyl]-4-(2,1,3-benzoxadiazolyl)-thiazoli none (DBD-TZ) amino acid during the cyclization/cleavage reaction with trifluoroacetic acid (TFA) in the Edman sequencing procedure has been carefully investigated, and evidence is presented to show conclusively that the racemization is caused by the replacement of a hydrogen atom by TFA. The fluorescent reagent 7-[N,N-dimethylamino)sulfonyl]-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS) was used for amino acid sequencing, and DBD-TZ amino acid was used for sequence and configuration determination. DBD-thiocarbamoylated peptides were cyclized and cleaved with deuterated TFA, and the protonated pseudomolecular ions (M-d1 + H)+ of DBD-TZ amino acids were detected by LC/MS. Furthermore, in the reaction kinetics study, we confirmed that the replacement reaction by TFA correlated sufficiently with the racemization of DBD-TZ amino acids. For the purpose of retaining D/L-amino acid configuration in sequencing, we used an aprotic acid, i.e., the Lewis acid boron trifluoride (BF3), for the cyclization/cleavage reaction. When we used BF3, the derivatized DBD-TZ amino acid was scarcely racemized under cyclization/cleavage conditions. Using this method, amino acid sequencing of D-Phe-Met-Arg-Phe-amide could be performed, retaining the D/L-configuration of the amino acid residues.

Amino Acid Sequence↗
PubMed1996

Release of secretory phospholipase A2 from rat neuronal cells and its possible function in the regulation of catecholamine secretion.

Here we show that secretory phospholipase A2 (sPLA2) that is immunochemically indistinguishable from type II sPLA2 is (i) stored in neuroendocrine cells, (ii) released in response to neurotransmitters or depolarization, and (iii) involved in the regulation of catecholamine secretion by these cells. Rat brain synaptic vesicle fractions contained PLA2 activity, which was neutralized completely by an antibody raised against rat type II sPLA2. sPLA2 immunoreactive with anti-(type II sPLA2) antibody was released from synaptosomes in response to depolarization evoked by a high concentration of potassium in the presence of Ca2+. Rat pheochromocytoma PC12 cells, which differentiated into adherent cells similar to sympathetic neurons in response to nerve growth factor, were used for the detailed analysis of the dynamics and function of sPLA2 in neuronal cells. Antibody against rat type II sPLA2 precipitated approximately 80% of the PLA2 activity in PC12 cell lysates. Transcript for type II sPLA2 was detected in PC12 cells by reverse transcriptase-PCR. When neuronally differentiated PC12 cells were stimulated with carbamylcholine or potassium, sPLA2 was released into the medium and reached a maximal approximately 40% release by 15 min. Inhibitors specific to type II sPLA2 suppressed catecholamine secretion by PC12 cells which had been activated by carbamylcholine. Furthermore, treatment of PC12 cells with exogenous type II sPLA2 alone elicited catecholamine secretion. These observations indicate that sPLA2 released from neuronal cells may regulate the degranulation process leading to release of neurotransmitters and are compatible with our earlier finding that this enzyme is involved in the degranulation of rat mast cells.

Adrenal Gland Neoplasms↗
PubMed1996

Wheat germ agglutinin-reactive chains of giant hemoglobin from the polychaete Perinereis aibuhitensis.

Wheat germ agglutinin-reactive chains of multisubunit extracellular hemoglobin from the polychaete Perinereis aibuhitensis were identified to clarify the carbohydrate gluing which is the carbohydrate-dependent supramolecular architecture of the hemoglobin (Ebina S. et al. (1995) Proc. Natl. Acad. Sci. USA 92, 7367-7371). Electron microscope micrographs of Perinereis hemoglobin showed a characteristic shape of two-tiered hexagonal rings whose diameter and height were determined to be 29.4 +/- 1.7 nm and 20.0 +/- 1.8 nm, respectively. Four types of globins and two types of linkers were isolated from the giant hemoglobin by reverse-phase chromatography and SDS-PAGE. These constituents showed similar NH2-terminal sequences as those previously reported for corresponding chains of Tylorrhynchus hemoglobin (Suzuki T. and Gotoh T. (1986) J. Biol. Chem. 261, 9257-9267; Suzuki T. et al. (1990) J. Biol. Chem. 265, 12168-12177). Thus, each globin of Perinereis hemoglobin was identified in terms of amino acid sequence homology and designated using names common to Tylorrhynchus hemoglobin, namely, a, A, b, and B. The linkers were stained by horseradish peroxidase (HRP)-lectins and PAS staining kits, indicating the presence of carbohydrate oligomers. Lectin staining was also significantly positive to globins a and A, which belong to strain A, but negative to globins b and B, which belong to strain B. Results showed that linkers and globins of strain A had a site in a carbohydrate oligomer to which wheat germ agglutinin (WGA) could bind. On the other hand, an alignment between known amino acid sequences of annelid globins and linkers and the sequences of lectins revealed that only the domain of the cysteine-rich motif in linkers has a homology with WGA-type lectins. The results of this study clarify the structuring mechanism of a supramolecule by lectin-like binding, called carbohydrate gluing.

Amino Acid Sequence↗
PubMed1996

Inverse association of cell adhesion regulator messenger RNA expression with metastasis in human colorectal cancer.

Alterations in several classes of adhesion molecules have been implicated in the progression of colorectal cancer. Cell adhesion regulator (CAR) has been identified as a regulator molecule of integrin-dependent cell adhesion. We have explored a possible involvement of the CAR gene in colorectal cancer. Reverse transcription-PCR revealed that CAR expression was detected in normal colonic cells, whereas it was decreased or undetectable in 6 of 13 (46.2%) human colon cancer cell lines. To further study the biological significance of CAR expression in colon cancer cells, a CAR expression vector was introduced into HT-29 cells, in which CAR is not expressed. Adhesion of HT-29 cells to extracellular matrix components was up-regulated by the introduction of CAR. In spite of similar growth properties with the controls, CAR-transfected HT-29 cells showed a significantly reduced spontaneous metastatic potential in nude mice. To determine whether these experimental results are of relevance with respect to actual human tumors, we investigated CAR expression in 30 surgical specimen pairs of human colorectal cancer and adjacent noncancerous tissue using semiquantitative reverse transcription-PCR. In 14 of 30 cases (46.7%), CAR expression in cancer was less than one-tenth of that in matched noncancerous tissue. The tumor:normal ratio of CAR expression was significantly lower in patients with lymph node metastasis than in those without it (P < 0.01) and in patients with distant metastasis than in those without it (P < 0.05). CAR expression was significantly lower in more advanced Dukes' stage tumors (P < 0.05). Our results suggest that down-regulation of CAR expression may play an important role in the progression and metastasis of colorectal cancer.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1996

Convergent evolution. The gene structure of Sulculus 41 kDa myoglobin is homologous with that of human indoleamine dioxygenase.

The abalone Sulculus diversicolor contains abundant myoglobin in its buccal mass. The myoglobin consists of 377 amino acid residues and has a molecular mass of 41 000 Da, 2.5 times larger than that of other myoglobins. Sulculus myoglobin can bind oxygen reversibly, and the P50 was determined to be 3.8 mmHg at 20 degrees C and pH 7.4, showing that the oxygen affinity of Sulculus myoglobin is lower than those of vertebrate and invertebrate myoglobins. The cDNA-derived amino acid sequence showed no significant homology with those of any other invertebrate myoglobins and hemoglobins, but surprisingly showed 35% homology with a vertebrate tryptophan-degrading enzyme, indoleamine dioxygenase (IDO). The structure of the Sulculus myoglobin gene has been determined to consist of 14 exons and 13 introns (15.3 kbp). Compared with the gene of human IDO (10 exon-9 intron structure), the splice junctions of 7 introns were exactly conserved between the two genes, suggesting that these introns have been conserved for at least 600 million years. The Sulculus gene has 5 additional introns, one of which is located outside the coding region. From these results we conclude that Sulculus myoglobin evolved from an IDO gene and represents a typical case of functional convergence. Comparison of the amino acid sequence of each exon of Sulculus myoglobin with those of usual globin sequences showed that there is no significant evolutionary relationship between them. The IDO-like myoglobin is unexpectedly widely distributed among gastropodic molluscs, such as Sulculus, Nordotis, Battilus, Omphalius and Chlorostoma.

Amino Acid Sequence↗
PubMed1996

Defective calcium influx in rat myelomonocytic leukemia cells which are resistant to differentiation-inducing effects of lipid A.

We compared the calcium mobilization in parent lipid A-sensitive leukemia cells (P2) and lipid A-resistant cells (LR) after treating them with lipid A in order to clarify the signal transduction involved in the differentiation induced by lipid A. Lipid A induced differentiation in P2 cells; however, LR cells were completely resistant to it. A dramatic elevation of intracellular free calcium ion concentration ([Ca2+]i) occurred in P2 cells, but only a slight elevation of [Ca2+]i in LR cells. Calcium ionophore in combination with lipid A induced differentiation in LR cells. An elevation of [Ca2+]i observed in P2 cells was abrogated by an addition of EGTA, which partially inhibited the differentiation of P2 cells stimulated by lipid A. Altogether, these data indicate that calcium influx is essential for the differentiation of P2 cells stimulated by lipid A and that defective calcium influx is responsible for the resistance to lipid A in LR cells.

Animals↗
PubMed1996

Membrane-type matrix metalloproteinase 1 is a gelatinolytic enzyme and is secreted in a complex with tissue inhibitor of metalloproteinases 2.

The processing mechanism and gelatinolytic activity of the membrane-type matrix metalloproteinase 1 (MT-MMP-1) were examined by expressing in COS-1 cells a deletion mutant of MT-MMP-1 lacking the trans-membrane domain (delta MT1) and its site-directed mutant with a furin-resistant sequence in the propeptide domain (mutant delta MT1). delta MT1, but not mutant delta MT1, was processed to an active form and exhibited gelatinolytic activity as seen using gelatin zymography. delta MT1 isolated in a complex form with tissue inhibitor of metalloproteinases 2 (TIMP-2) from the stable transfectants demonstrated the NH2-terminal sequence of Ala113-IIe-Gln-Leu, indicating cleavage at one amino acid down-stream from the furin recognition sequence. The delta MT1/TIMP-2 complex formed a ternary complex with proMMP-2 through the COOH termini of TIMP-2 and proMMP-2. A human breast carcinoma cell line (MDA-MB-231 cells) also secreted MT-MMP-1 into culture media, which was purified in a complex form with TIMP-2 and showed gelatinolytic activity as seen using zymography. These results demonstrate for the first time that MT-MMP-1 is a gelatinolytic enzyme and secreted from cells in a complex with TIMP-2, which can form a ternary complex of MT-MMP-1/TIMP2/proMMP-2.

Animals↗
PubMed1996

Cell surface binding and activation of gelatinase A induced by expression of membrane-type-1-matrix metalloproteinase (MT1-MMP).

Gelatinase A is secreted as a proenzyme (progelatinase A) which is activated and bound on the surface of tumor and normal cells. We have reported that the expression of a membrane-type-1-matrix metalloproteinase (MT1-MMP) induces activation of progelatinase A. Here we demonstrate that the expression of MT1-MMP in COS-1 cells induces cell-surface binding of progelatinase A which is consequently processed to an intermediate form. Processing from the intermediate to the fully active form is dependent on the gelatinase A concentration. These results suggest that the cell-surface binding concentrates the gelatinase A intermediate form locally to allow autoproteolytic processing to the fully active form.

Animals↗
PubMed1996

A rat genetic map constructed by representational difference analysis markers with suitability for large-scale typing.

Representational difference analysis (RDA) was applied to isolate chromosomal markers in the rat. Four series of RDA [restriction enzymes, BamHI and HindIII; subtraction of ACI/N (ACI) amplicon from BUF/Nac (BUF) amplicon and vice versa] yielded 131 polymorphic markers; 125 of these markers were mapped to all chromosomes except for chromosome X. This was done by using a mapping panel of 105 ACI x BUF F2 rats. To complement the relative paucity of chromosomal markers in the rat, genetically directed RDA, which allows isolation of polymorphic markers in the specific chromosomal region, was performed. By changing the F2 driver-DNA allele frequency around the region, four markers were isolated from the D1Ncc1 locus. Twenty-five of 27 RDA markers were informative regarding the dot blot analysis of amplicons, hybridizing only with tester amplicons. Dot blot analysis at a high density per unit of area made it possible to process a large number of samples. Quantitative trait loci can now be mapped in the rat genome by processing a large number of samples with RDA markers and then by isolating markers close to the loci of interest by genetically directed RDA.

Alleles↗