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Biomedical subjects

T Imai

Publications and source records attributed to T Imai.

At least 577 records · Page 32Linked to original sources
PubMed1994

Hematopoietic supportive function of human bone marrow stromal cell lines established by a recombinant SV40-adenovirus vector.

We have previously reported the establishment of a variety of human bone marrow stromal cell lines using a recombinant SV40-adenovirus vector. Using this vector, we obtained more clonal stromal cells. Here, we have characterized these cells and analyzed their capacity to support the proliferation and differentiation of hematopoietic cells. The stromal cells were cocultured with nonadherent human bone marrow cells used as hematopoietic cells. The total numbers of hematopoietic cells and CFU-GM in culture were counted every week. Two of the six stromal cell lines, AA101 and HAS303, supported the proliferation and differentiation of hematopoietic cells and CFU-GM for more than 9 weeks. Further, granulocytes, macrophages, and megakaryocytes were detected when cocultured with these cells. When hematopoietic cells were cocultured but separated from the two stromal cell lines by a 0.45-microns millipore membrane to prevent their attachment, almost all CFU-GM disappeared within 7 weeks. The supportive stromal cells produced GM-CSF and IL-6. However, other cell lines producing these humoral factors did not support hematopoietic cell proliferation for such a long time. These findings suggest that these established human bone marrow stromal cell lines will be useful, in that analysis of their supportive function in hematopoietic cell proliferation and differentiation through cell-to-cell interaction will shed some light on this area.

Adenoviridae↗
PubMed1994

Unilateral lung injury caused by ischemia without hypoxia in isolated rat lungs perfused with buffer solution.

It has not been clear whether or not pulmonary ischemia-reperfusion injury would occur while ischemic lungs were maintained in a well-oxygenated state. Our aim was to develop an ischemic lung model whose oxygen tension was maintained and to determine whether or not ischemia without hypoxia would injure the lung. Rat lungs isolated, ventilated, and perfused with Krebs-Henseleit solution (0.04 ml/gm body weight) were obtained by a standard technique, and the left and right pulmonary arteries were isolated to enable selective perfusion to either lung. After the left lung was exposed to ischemia for 60 minutes with ventilation maintained by a normoxic (20% O2, 5% CO2, 75% N2; normoxia) or a hyperoxic (95% O2, 5% CO2; hyperoxia) gas mixture, it was reperfused for 30 minutes either in a forced way (occluding the right pulmonary artery for 10 minutes followed by perfusion of both lungs for another 20 minutes) or in the natural way (distribution of flow dependent on vascular resistance) to compose three experimental groups (forced reperfusion-normoxia, natural reperfusion-normoxia, and forced reperfusion-hyperoxia).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

C33 antigen and M38 antigen recognized by monoclonal antibodies inhibitory to syncytium formation by human T cell leukemia virus type 1 are both members of the transmembrane 4 superfamily and associate with each other and with CD4 or CD8 in T cells.

C33 Ag and M38 Ag had been identified by mAb inhibitory to HTLV-1-induced syncytium formation. The cDNA encoding C33 Ag had revealed that it belongs to the newly defined transmembrane 4 superfamily (TM4SF). M38 Ag was detected on virtually all human cell lines and fresh leukocytes except for most granulocytes. It was also expressed on a mouse hybrid cell clone containing human chromosome 11q23-pter. Immunoprecipitation and immunoblot analyses identified a monomeric 26-kDa protein. The M38 epitope was dependent on S-S bonding. These characteristics were very similar to those reported for TAPA-1 (the target of antiproliferative antibody-1), which also belongs to TM4SF as C33 Ag. We therefore cloned the cDNA of human TAPA-1 and expressed it in COS cells. M38 indeed reacted with COS cells expressing human TAPA-1. We concluded that M38 Ag was identical to TAPA-1. To further investigate the biologic functions of C33 Ag and M38 Ag (TAPA-1) and their roles in HTLV-1-induced syncytium formation, molecules associated with these Ag were examined in T cells. Immunoprecipitation from surface-iodinated cell lysates revealed that proteins co-precipitated by C33 and M38 were mostly common including each other. Sequential immunoprecipitation-immunoblot experiments confirmed that C33 Ag and M38 Ag (TAPA-1) were associated with each other. The association was further confirmed in BHK cells doubly transfected with human cDNA for C33 Ag and TAPA-1. We extended similar analyses and found that C33 Ag and M38 Ag (TAPA-1) were regularly associated with CD4 or CD8. The association of these Ag on the cell surface was further supported by co-modulation of M38 Ag (TAPA-1), CD4 and CD8 with C33 Ag. This is the first time that a physical association between the members of TM4SF is demonstrated. Furthermore, the regular association of C33 Ag and M38 Ag (TAPA-1) with CD4 or CD8 might indicate that they play a role in expression and/or function of the CD4/CD8 co-receptor complex.

Antibodies, Monoclonal↗
PubMed1993

5-Fluorouracil increases susceptibility of renal cell cancer cell lines to lymphokine-activated killer cells: evidence for alteration not at the level of recognition but at a post-binding stage of the lytic cycle.

To investigate the usefulness of 5-Fluorouracil (5FU) for combination with immunotherapy, we examined the effect of preincubation with 5FU on the susceptibility of a renal cell cancer (RCC) cell line, ACHN, to lymphokine-activated killer (LAK) cells. A 4-h 51Cr release assay showed a remarkable increase in the susceptibility of ACHN cells to LAK cells. Dose response experiments demonstrated that 5FU at concentrations as low as 0.002 microgram/ml increased susceptibility to LAK cells. Presence of 5FU at 2 micrograms/ml but not at 0.2 microgram/ml in media blocked LAK activity induction by IL-2. Furthermore, an adhesion assay showed that preincubation with 5FU did not alter the adhesion of LAK cells to tumor cells nor the expression of intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-associated antigen-3 (LFA-3) or major histocompatibility complex (MHC) class I molecules on tumor cells. Cold target competition did not show any difference between 5FU-treated and untreated competitors. These results suggest that increased susceptibility of RCC cells to LAK cells due to preincubation with 5FU might depend on changes in intrinsic lysability involving a post-binding stage of the lytic cycle.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1993

Study of interaction of carprofen and its enantiomers with human serum albumin--II. Stereoselective site-to-site displacement of carprofen by ibuprofen.

The site-to-site displacement of carprofen, a site II-specific drug, bound to human serum albumin (HSA) by ibuprofen, another site II-specific drug, was qualitatively and quantitatively studied by circular dichroism (CD) and equilibrium dialysis (ED). Carprofen gives rise to different CD spectra at lower (1:1) and higher (3:1) molar ratios to HSA, indicating different mechanisms for the binding of this drug to its high and low affinity sites on HSA. Ibuprofen at a 5:1 molar ratio to HSA displaces carprofen at a molar ratio of 1:1 to HSA from its high affinity binding site (site II) to its low affinity site (site I), as shown by production of the CD spectrum similar to that obtained in the case of the carprofen-HSA complex at a molar ratio 3:1. As revealed by the ED experiments, the free fraction of carprofen at a molar ratio of 1:2 to HSA (2 x 10(-5) M) was not initially increased by the addition of ibuprofen at a lower concentration, but at a higher concentration (6 x 10(-5) M), the free fraction was increased by only 90%. When site I was sufficiently blocked by a site I-specific drug like warfarin or phenylbutazone (6 x 10(-5) M), there was about a 4-fold increase in the free fraction of carprofen caused by ibuprofen. This site-to-site displacement demonstrated by carprofen was found to be stereospecific as indicated by the highest interaction between the S(+)-enantiomers of carprofen and ibuprofen. Moreover, the displacement of carprofen occurred at the azapropazone region rather than the warfarin region of site I on HSA.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1993

Enhanced expression of LFA-3 on human T-cell lines and leukemic cells carrying human T-cell-leukemia virus type 1.

Previously, we studied the surface expression of LFA-1 (CD11a/CD18) and ICAM-1 (CD54) in a panel of 16 human T-cell lines and found that those carrying HTLV-1 pro-viruses expressed high levels of ICAM-1. Enhanced expression of ICAM-1 was also seen in fresh leukemic cells from ATL patients. In the present study, we systematically examined the surface expression of 19 different cell-adhesion molecules (CAMs) (the immunoglobulin superfamily, the homing receptors, the integrins, etc.) in a panel of 20 human T-cell lines (6 HTLV-1-negative lines and 14HTLV-1-positive lines). The surface expression of LFA-3 (CD58) was constantly enhanced in HTLV-1-positive T-cell lines except for MT-1, an ATL-derived cell line, which was devoid of a number of CAMs including ICAM-1. LFA-3 proteins and mRNA were found at strongly increased levels in HTLV-1-positive T-cell lines.

Antigens, CD↗
PubMed1993

Expression and purification of functional human 17 alpha-hydroxylase/17,20-lyase (P450c17) in Escherichia coli. Use of this system for study of a novel form of combined 17 alpha-hydroxylase/17,20-lyase deficiency.

Enzymatically active human 17 alpha-hydroxylase cytochrome P450 (P450c17) has been expressed in and purified from Escherichia coli. The cDNA containing modifications within the amino-terminal eight codons which are favorable for expression in E. coli, as well as codons for 4 histidine residues at the carboxyl terminus, was placed in the pCWori+ expression vector. The modified human P450c17 was detected spectrophotometrically (400 nmol of P450c17/liter culture) and was found to be integrated into E. coli membranes. This previously inaccessible human P450 was purified to electrophoretic homogeneity (10.7 nmol of P450/mg) from solubilized bacterial membranes using two sequential chromatographic steps, nickel nitrilotriacetate followed by hydroxylapatite. The expected enzymatic activities of human P450c17 were reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase, giving turnover numbers of 8.0 nmol/min/nmol P450 for pregnenolone, 6.5 nmol/min/nmol P450 for progesterone, 0.06 nmol/min/nmol P450 for 17 alpha-hydroxypregnenolone, and no detectable activity for 17 alpha-hydroxyprogesterone. This system was utilized to study the molecular basis of a novel form of combined 17 alpha-hydroxylase, 17,20-lyase deficiency resulting from compound heterozygous mutations, a missense point mutation Tyr64(TAT)--> Ser (TCT), and an Ile112 duplication (ATCATC). Upon expression of these mutant proteins in E. coli, the Tyr64 mutant has 15% of the wild type 17 alpha-hydroxylase activity, whereas the Ile112 duplication shows no activity, results consistent with the observed clinical phenotype.

Adrenocorticotropic Hormone↗
PubMed1993

Up-regulation of ETB receptor subtype mRNA by angiotensin II in rat cardiomyocytes.

The effect of angiotensin (ANG) II on the expression of endothelin (ET) receptor subtype (ETA, ETB) mRNA in cultured neonatal rat cardiomyocytes was studied. ANG II (10(-7) M) increased steady-state mRNA levels of ETB receptor, but not ETA receptor, during 6-12 hr incubation, whose effect was dose-dependent (10(-9)-10(-7) M). ANG II receptor type-1 (AT1) antagonist (CV-11974) completely inhibited the ANG II-induced up-regulation of ETB receptor, whereas the inhibitory effect by ANG II receptor type-2 (AT2) antagonist (PD 123319) was incomplete. These results suggest that ANG II up-regulates cardiac ETB receptor, predominantly via AT1 receptor.

Angiotensin II↗
PubMed1993

Participation of poly(ADP-ribose) synthetase in the process of norepinephrine-induced inhibition of major histocompatibility complex class II antigen expression in human astrocytoma cells.

We previously demonstrated that the expression of a transfected poly(ADP-ribose) synthetase cDNA into macrophage tumor cells inhibited interferon-gamma-dependent induction of major histocompatibility complex(MHC) class II antigens. In the present study, we found that addition of norepinephrine to the cultured human astrocytoma STTG1 cells induced mRNA of poly(ADP-ribose) synthetase in 6-14h. Thus, we cultured the cells in the presence of norepinephrine for 24h, and then induced the MHC class II antigen by the addition of interferon-gamma. The expression of MHC class II antigen was inhibited, whereas it was not inhibited when norepinephrine and interferon-gamma were simultaneously added into the culture medium. These results suggest that an increase of poly(ADP-ribose) synthetase by norepinephrine cause the inhibition of interferon-gamma-mediated MHC class II antigen expression.

Astrocytoma↗
PubMed1993

[CO2US via an implantable port--drug distribution in intraarterial chemotherapy for hepatic tumors and evaluation of effect].

The distribution of blood flow was determined from the distribution of CO2 by US performed during the infusion of CO2 microbubbles via an implantable port (IP-CO2US) in intraarterial chemotherapy for hepatic tumor, and the usefulness of this method in determining tumor vascularity and evaluating the effects of therapy was investigated. A total of 16 patients, 12 of whom had metastatic liver tumor, two hepatocellular carcinoma, one gall bladder carcinoma, and one cholangiocellular carcinoma were studied. The enhanced areas in the liver in 16 patients in whom IP-CO2US was performed a total of 24 times were consistent in all cases with the enhanced areas demonstrated by IP-RI angiography performed a total of 10 times within 10 days, and were also consistent with one exception with the enhanced areas demonstrated by IP-CTA performed 14 times. The tumor detection rate was markedly higher with IP-CO2US than with plain US or IP-DSA, and was similar to that of IP-CTA. Evaluation of the vascularity of individual nodules by IP-CO2US surpassed that by IP-DSA, and was similar to that of IP-CTA. It was demonstrated that blood flow distribution (intrahepatic drug distribution) can be equally well grasped with IP-CO2US, which is a simple and convenient method, as with IP-RI angiography. It was also suggested that IP-CO2US is useful in the evaluation of tumor vascularity and the effect of therapy.

Antineoplastic Agents↗
PubMed1993

Tissue expression of mRNA of chloride channel from MDCK cells and its regulation by protein kinases.

A new chloride channel has recently been identified by expression cloning (Paulmichl, M., et al. Nature 356: 238). To date there is no information available on the distribution of this channel in mammalian tissues. We cloned rat homologue of this Cl channel and found 92% identity in deduced amino-acid sequence. We studied the tissue distribution of its mRNA and regulation by protein kinases. Its mRNA was expressed in all 17 bovine tissues we studied, most abundantly in the brain. In the kidney, it was expressed more in the medulla than cortex. Dehydration up to 5 days did not change its mRNA level in the rat kidney. Both phorbol myristate acetate (PMA) and forskolin down regulated its expression in MDCK cells, suggesting that both PKC and PKA modulated its expression. The physiological role of this chloride channel remains to be clarified.

Amino Acid Sequence↗
PubMed1993

Binding of suprofen to human serum albumin. Role of the suprofen carboxyl group.

The binding of suprofen (SP), a non-steroidal anti-inflammatory drug of the arylpropionic acid class, and its methyl ester derivative (SPM) to human serum albumin (HSA) was studied by dialysis and spectroscopic techniques. In spite of the remarkable differences in the physicochemical properties of SP and SPM, the binding of each molecule to HSA was quantitatively very similar. Thermodynamic analysis suggests that the interaction of SP with HSA may be caused by electrostatic as well as hydrophobic forces, whereas the interactions with SPM may be explained by hydrophobic and van der Waals forces. Similarities in the difference UV absorption spectra between ligand-detergent micelle and -HSA systems indicate that the SP and SPM molecules are inserted into a hydrophobic crevice on HSA. The same studies suggest that the carboxyl group of SP interacts with a cationic sub-site which is closely associated with the SP binding site. Proton relaxation rate measurements indicate that the thiophen ring and propanoate portion of the SP molecule is the major binding site for HSA. The locations of SP and SPM binding sites were identified by using fluorescence probes which bind to a known site on HSA. The displacement data implied that SP primarily binds to Site II, while the high affinity site of SPM as well as low affinity site of SP are at the warfarin binding site in the Site I area. From binding data with chemically modified HSA derivatives, it is likely that highly reactive tyrosine (Tyr) and lysine (Lys) residues, which may be Tyr-411 and Lys-195, are specifically involved in SP binding. In contrast, these two residues are clearly separated from the SPM binding site. The binding of SP and SPM is independent of conformational changes on HSA that accompany N-B transition. There is evidence that the carboxyl group may play a crucial role in the high affinity binding processes of SP to HSA.

2-Hydroxy-5-nitrobenzyl Bromide↗
PubMed1993

Molecular cloning of a chloride channel that is regulated by dehydration and expressed predominantly in kidney medulla.

Complementary DNA encoding a rat kidney chloride channel (CIC-K1) was isolated by a polymerase chain reaction (PCR) cloning strategy. We designed degenerate primers, based on the regions where previously cloned chloride channels (CIC-0, -1, and -2) possess significant amino acid identity, and performed reverse transcription PCR with whole kidney mRNA. The 686-amino acid protein encoded by CIC-K1 is about 40% identical to the previously cloned chloride channels and has a similar hydropathy profile. Expression of CIC-K1 in Xenopus oocytes induced Cl- currents that activate instantaneously upon hyperpolarization and depolarization, and displayed a slightly outwardly rectifying current-voltage relationship. The message for CIC-K1 was 2.4 kilobases and was found predominantly in kidney, especially in the inner medulla. Reverse transcription PCR technique using micro-dissected nephron segments revealed that the main site of expression in kidney was the thin ascending limb of Henle's loop, which has the highest Cl- permeability among the nephron segments and is thought to be involved in a counter-current system for urine concentration in the inner medulla. The abundance of CIC-K1 mRNA in kidney increased about 4-fold as rats became dehydrated by deprivation of water for 5 days. The site of expression and the regulation by dehydration suggest that CIC-K1 function may be important in urinary concentrating mechanisms.

Amino Acid Sequence↗
PubMed1993

Mapping the breakpoint of a constitutional translocation on chromosome 22 in a patient with NF2.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder characterized by development of bilateral acoustic neurinomas and increased incidence of meningiomas. Frequent losses of I allele of chromosome 22 in neurinomas and meningiomas has indicated that the gene responsible for NF2 functions as a tumor suppressor. Although the NF2 gene has been mapped within a 13 cM region between D22S1 and D22S28 by linkage analysis, its location with respect to D22S15 is uncertain. We previously reported an NF2 patient with a constitutional balanced translocation t(4;22)(q12;q12.2); the NF2 gene is probably disrupted at the breakpoint. To define the location of this breakpoint on chromosome 22, we performed fluorescence in situ hybridization (FISH) with DNA markers in the NF2 region and determined the physical order of 5 loci: D22S1-NF2-LIF-D22S15-D22S32.

Base Sequence↗
PubMed1993

Long-range mapping of the 11q23 region involved in chromosome aberrations in human tumors by pulsed-field gel electrophoresis with a yeast artificial chromosome.

We have previously demonstrated that the RCK gene involved in t(11;14)(q23;q32) and the more centromeric MLL/ALL1 gene involved in t(4;11)(q21;q23) and t(11;19)(q23;p13) are localized on different adjacent NotI fragments by using pulsed-field gel electrophoresis (PFGE) analysis with the yeast artificial chromosome (YAC) clone yB22B2. The PFGE analysis using the YACs of YTY17 containing the prophobilinogen deaminase (PBGD), CBL2 and THY1 genes and yB22B2 allowed the following ordering of genes and breakpoints from CD3 to THY1 on 11q23: cent-CD3-ALL/MLL1-RCK-PBGD-CBL2-THY1, and the establishment of a long-range restriction map covering these genes. In addition, we showed that the FLI1 region involved in the t(11;22)(q24;q12) in Ewing's sarcoma was more telomeric region that the THY1 gene by analyzing somatic cell hybrids carrying the 11q- and/or 14q+ chromosome of the t(11;14)(q23;q32) translocation, and by PFGE analysis of the YAC clone YTY17.

Acute Disease↗