Biomedical subjects

T Sudo

Publications and source records attributed to T Sudo.

At least 19 recordsLinked to original sources

PubMed1992

Enhancement of murine hematopoiesis by synergistic interactions between steel factor (ligand for c-kit), interleukin-11, and other early acting factors in culture.

Entry into the cell cycle of dormant hematopoietic progenitors appears to be regulated by multiple synergistic factors, including interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), IL-11, and the ligand for c-kit, which is also known as steel factor (SF). We have tested the effects of these and other hematopoietic factors on the proliferation of partially enriched dormant murine progenitors in the presence and absence of serum. In serum-containing cultures, SF and IL-11 interacted to support the formation of multilineage colonies; the level of colony formation was comparable with the colony formation supported by other effective two-factor combinations. In serum-free cultures, colony formation supported by two factors was significantly less than that in serum-containing culture and the most effective two-factor combination in serum-free culture was SF plus IL-3. In serum-free cultures, three-factor combinations consisting of SF, IL-3, and one of IL-6, G-CSF, or IL-11 yielded colony formation that was comparable with that seen in serum-containing cultures. These studies indicate that IL-11 belongs to a group of early-acting hematopoietic synergistic factors that now includes IL-6, G-CSF, and IL-11. In contrast, SF is unique among the synergistic factors in that it interacts either with growth factors such as IL-3 or GM-CSF or with synergistic factors such as IL-6, IL-11, or G-CSF.

Animals↗

PubMed1992

3-O-alkylascorbic acids as free radical quenchers. 3. Protective effect on coronary occlusion-reperfusion induced arrhythmias in anesthetized rats.

Structural modification of ascorbic acid by substitution of the 3-hydroxy group with lipophilic moieties has allowed the development of agents for treating reperfusion injury. These ascorbic acid derivatives inhibited lipid peroxidation, and some of them also reduced coronary reperfusion-induced arrhythmias in anesthetized rats. We found that 3-O-[(dodecylcarbonyl)methyl]ascorbic acid (8) was protective against reperfusion injury without directly influencing hemodynamics. 2-O-Octadecylascorbic acid (19) and 5,6-O-dodecylideneascorbic acid (15) also exhibited a marked effect on reperfusion injury, but significantly reduced the arterial blood pressure and heart rate in rats.

Anesthesia↗

PubMed1992

Identification of proliferating dendritic cell precursors in mouse blood.

While it has been known that dendritic cells arise from proliferating precursors in situ, it has been difficult to identify progenitors in culture. We find that aggregates of growing dendritic cells develop in cultures of mouse blood that are supplemented with granulocyte/macrophage colony-stimulating factor (GM-CSF) but not other CSFs. The dendritic cell precursor derives from the Ia-negative and nonadherent fraction. The aggregates of developing dendritic cells appear at about 1 wk of culture, with 100 or more such clusters being formed per 10(6) blood leukocytes. The aggregates can be dislodged and subcultured as expanding clusters that are covered with cells having the motile sheet-like processes ("veils") of dendritic cells. By about 2 wk, large numbers of single, major histocompatibility complex (MHC) class II-rich dendritic cells begin to be released into the medium. Combined immunoperoxidase and [3H]thymidine autoradiography show that the cells that proliferate within the aggregate lack certain antigenic markers that are found on mature dendritic cells. However, in pulse-chase protocols, the [3H]thymidine-labeled progeny exhibit many typical dendritic cell features, including abundant MHC class II and a cytoplasmic granular antigen identified by monoclonal antibody 2A1. The progeny dendritic cells are potent stimulators of the mixed leukocyte reaction and can home to the T-dependent areas of lymph node after injection into the footpads. We conclude that mouse blood contains GM-CSF-dependent, proliferating progenitors that give rise to large numbers of dendritic cells with characteristic morphology, mobility, phenotype, and strong T cell stimulatory function.

Animals↗

PubMed1992

Two types of murine CD34 mRNA generated by alternative splicing.

To characterize and clarify the function of CD34 antigen experimentally, we isolated two types of CD34 mRNA from a cDNA library of murine stromal cell line, PA-6 stimulated with lipopolysaccharide (LPS) and 12-o-tetra-decanoylphorbol 13-acetate (TPA) using a human CD34 probe. In addition to the clone (open reading frame [ORF]:1149bp) reported by Brown et al, a novel clone (ORF:978 bp) was obtained. The difference between the two clones was in the cytoplasmic portion of CD34; the former has 73 amino acids, while the latter has 16. We investigated the genomic sequence of cytoplasmic portion and found conserved nucleotide sequences at the exon-intron junction (GT ... AG). Thus, it was concluded that alternative splicing gave two types of CD34 mRNA. A novel clone contains the longer cDNA, including a insert of 156 bp, but results in a shorter predicted coding sequence because of the introduction of an inframe stop codon. Northern blot analysis using a murine cDNA probe (HindIII fragment, 900 bp) showed that CD34 was highly expressed in the brain and testis, and moderately in the thymus, spleen, and bone marrow, but not in adult liver. However, day 12 to 14 fetal liver cells showed significant expression of CD34. Quantitative reverse transcription polymerase chain reaction showed that spleen, thymus, bone marrow, and testis RNA gave two bands of almost equal intensity, but in the brain a novel clone was expressed three times more than the other clone. Furthermore, Northern blot analysis using a probe (156 bp) specific for the spliced intracellular region confirmed the significant mRNA expression of a novel clone. Although the biologic significance of alternative splicing remains to be elucidated, it is suggested that a different carboxyterminal tail causes a change in signal transduction.

Amino Acid Sequence↗

PubMed1992

c-Jun represses the human insulin promoter activity that depends on multiple cAMP response elements.

Glucose is known to increase the cAMP concentration in pancreatic beta cells. To determine the mechanism by which cAMP augments insulin gene expression, we first identified the cAMP response elements (CREs) of the human insulin gene. In DNase I footprint analysis, the bacterially synthesized CRE-binding protein, CRE-BP1, protected four sites: two sites in the region upstream from the insulin core promoter, one site in the first exon, and one site in the first intron. To examine the roles of those four sites, we constructed a series of DNA plasmids in which the wild-type and mutant insulin promoters were linked to the chloramphenicol acetyl-transferase gene. Studies of the transcriptional activity of these plasmids after transfection into hamster insulinoma (HIT) cells showed that these four sites contributed additively to the cAMP inducibility of the insulin promoter. Surprisingly, the c-jun protooncogene product (c-Jun) repressed the cAMP-induced activity of the insulin promoter in a cotransfection assay with the c-Jun expression plasmid. Northern blot analysis demonstrated that the level of c-jun mRNA was dramatically increased by glucose deprivation in HIT cells. These results suggest that glucose may regulate expression of the human insulin gene through multiple CREs and c-Jun.

Animals↗

PubMed1992

Production of macrophage colony-stimulating factor by adult murine parenchymal liver cells (hepatocytes).

The activity of macrophage colony-stimulating factor (M-CSF) was found in the culture supernatant of mouse parenchymal liver cell fractions in a bone marrow colony-forming assay. The activity of an M-CSF-like substance purified by a four-step procedure was neutralized by goat anti-mouse M-CSF antiserum. M-CSF mRNA was detected in cellular RNA prepared from cultured parenchymal liver cell fractions by Northern blot analysis and also in cultured parenchymal liver cells by in situ hybridization. These results indicate that parenchymal liver cells have the capacity to produce M-CSF. We discuss the role of M-CSF in hematopoiesis, the immune response, and other biological phenomena.

Acrylic Resins↗

PubMed1992

Mapping of the human gene for the human immunodeficiency virus type 1 enhancer binding protein HIV-EP2 to chromosome 6q23-q24.

The human gene encoding the human immunodeficiency virus type 1 enhancer binding protein HIV-EP2 has been isolated. Using Southern analysis of human-rodent somatic cell hybrid DNA with a human HIV-EP2-specific cDNA probe, the HIV-EP2 gene was assigned to chromosome 6. The gene was further localized to the region 6q23-24 by fluorescence in situ hybridization.

Animals↗

PubMed1992

Gastric cytoprotective anti-ulcerogenic actions of hydroxychalcones in rats.

The preventive effects of hydroxychalcone derivatives on ulcer formation induced by severe necrotizing agents such as 60% ethanol in 150 mM HCl (HCl-ethanol) and 0.2 N NaOH in rats were examined. Among the compounds tested, 2',4'-dihydroxychalcone gave the strongest activity in both experimental models and protected the gastric mucosa from the insult of either necrotizing agent at oral doses ranging from 1 to 10 mg/kg, as evidenced by a dose-related reduction in the ulcer index. The mucosal protective activity of 2',4'-dihydroxychalcone was not affected by pretreatment with indomethacin (5 mg/kg, s.c.). To investigate the detailed mechanism of the mucosal protective action of 2',4'-dihydroxychalcone, its inhibitory effects on the decrease in the hexosamine content from the gastric mucus induced by HCl-ethanol were studied by using it in combination with a dye, Alcian blue. As a result, 2',4'-dihydroxychalcone at an oral dose of 10 mg/kg inhibited the decrease in the dye-recovery from the gastric mucus induced by HCl-ethanol. PGE2 at an oral dose of 0.1 mg/kg exhibited a similar action. These results established that 2',4'-dihydroxychalcone is a potent cytoprotective agent similar to PGE2 effectively preventing gastric ulcer formation induced by strong necrotizing agents and seems to suggest that this compound protects the stomach against its own peptic secretions by reinforcement of gastric resistances. In fact, 2',4'-dihydroxychalcone prevented ulcer formation induced by water-immersion stress in rats and also showed a marked enhancement of the healing of acetic acid-induced gastric ulcers in rats.

Animals↗

PubMed1992

Molecular cloning of feline interferon cDNA by direct expression.

A cDNA sequence coding for feline interferon has been cloned for the first time by screening a cDNA library constructed using Okayama-Berg vector and mRNA derived from the feline cells (LSA-I) induced by TPA (12-o-tetradecanoylphorbor 13-acetate) for the ability of transient expression to produce feline interferon in COS1 monkey cells. The amino acid sequence, deduced from the nucleotide sequence by comparing it with the sequences of other mammalian IFNs, consists of 171 amino acids with 6 cysteins and an N-glycosylation site at the amino acid position 79, and has about 60% homology to human IFN alpha 1. The interferon was partially purified through Blue Sepharose, and its molecular weight was estimated to be 2.4 x 10(4). The antiviral activity was acid stable, and glycosylation was suggested.

Amino Acid Sequence↗

PubMed1992

Transcriptional control by myb oncogene product.

Structure and function of two domains of c-Myb were analyzed. We show that a leucine zipper structure is a component of the negative regulatory domain, because its disruption markedly increases both the transactivating and transforming capacities of c-Myb. Our results suggest that an inhibitor which suppresses transactivation binds to c-Myb through the leucine zipper, and that c-Myb can be oncogenically activated by mis-sense mutation. We also proposed a model, the "tryptophan cluster", for the structure of the Myb DNA-binding domain, in which the three tryptophans form a cluster in the hydrophobic core in each repeat. The results of NMR analysis of repeat 3 revealed that the conserved tryptophans play a key role to make the hydrophobic core.

Animals↗

PubMed1992

The c-myb proto-oncogene product binds to but does not activate the promoter of the DNA polymerase alpha gene.

The myb proto oncogene product (c-Myb) is a transcriptional regulator and its expression and function are tightly linked to the cellular entry into S phase and DNA synthesis. It has been shown [Venturelli, D., Travali, S. & Calabretta, B. (1990). Proc. Natl. Acad. Sci. USA, 87, 5963-5967] that inhibition of T-cell proliferation by a myb antisense oligomer is accompanied by down-regulation of DNA polymerase alpha expression. To examine whether the transcription of the DNA polymerase alpha gene is directly regulated by c-Myb, we have identified and characterized the 5' regulatory region of the human DNA polymerase alpha gene. Two major and several minor transcription start sites were identified by nuclease S1 mapping. DNA sequence analysis showed that the promoter region contains no TATA box, one CCAAT box and putative Ap-1, AP-2 and E2F binding sites. In DNAase I footprinting, the bacterially expressed c-Myb protected six sites in the 5' flanking region of the human DNA polymerase alpha gene. However, c-Myb did not activate the DNA polymerase alpha gene promoter in a co-transfection assay. Our results suggest that an unknown factor(s) is required for the c-Myb-induced activation of the DNA polymerase alpha gene promoter, or c-Myb does not directly activate this promoter.

Base Sequence↗

PubMed1992

[Molecular aspects of IL-7-IL-7 receptor system].

IL-7 is a multifunctional cytokine which acts on cells such as B cells, T cells and macrophages. As the IL-7 clearly plays many important roles the in control of growth and differentiation, it is important to understand how IL-7 and IL-7 receptor (IL-7R) are expressed. Recently, to understand the transcriptional regulation of IL-7 and IL-7R genes, a detailed analysis of the 5' flanking region of the IL-7 and IL-7R genes has been done. In this review recent progress on these topics is introduced and discussed.

Animals↗

PubMed1992

Effect of diltiazem on the pharmacokinetics of MPC-1304, a new calcium channel blocker.

The effect of diltiazem pretreatment on the pharmacokinetics and pharmacodynamics of MPC-1304, a new calcium channel blocker, were evaluated in six healthy male volunteers. Placebo or diltiazem 60 mg was given orally three times daily for 3 days in a double-blind crossover method. MPC-1304 10 mg was administered orally two times at an interval of four weeks as a washout period. Diltiazem significantly increased the maximum plasma level (Cmax) and the area under the plasma concentration-time curve (AUC) of MPC-1304 from control values (3.0 +/- 1.5 to 10.2 +/- 4.8 ng/ml, p less than 0.05, and 11.0 +/- 3.8 to 36.0 +/- 15.6 ng.h/ml, p less than 0.01, respectively) without any changes of t1/2. In the case of the major active metabolite, side chain reduced form (MI), Cmax, AUC, and t1/2 were significantly increased by diltiazem as follows: Cmax, 60.7 +/- 21.1 to 171.4 +/- 73.6 ng/ml, p less than 0.05; AUC, 317.6 +/- 62.6 to 1,334.9 +/- 563.6 ng.h/ml, p less than 0.01; t1/2, 2.8 +/- 0.7 to 3.9 +/- 0.6 h, p less than 0.01. Diltiazem pretreatment slightly, but not significantly decreased blood pressure after MPC-1304 dosing. The inhibition of hepatic metabolism on MPC-1304 may explain the significant changes in pharmacokinetics of MPC-1304 after diltiazem pretreatment.

Administration, Oral↗

PubMed1992

Purification of rat megakaryocyte colony-forming cells using a monoclonal antibody against rat platelet glycoprotein IIb/IIIa.

We recently reported the production and characterization of four monoclonal antibodies (MoAbs) against rat platelet glycoprotein IIb/IIIa (GPIIb/IIIa). In this study we developed a simple and efficient three-step procedure, based on positive selection by immunoadsorption (panning) using one MoAb, P55, to purify rat megakaryocyte colony-forming cells (megakaryocyte colony-forming units, CFU-MK) from normal bone marrow. Cells obtained after each step were assayed for their ability to form megakaryocyte colonies in the presence of Concanavalin A (Con A)-stimulated rat spleen cell-conditioned medium in soft agar cultures. Marrow cells were first separated on discontinuous Percoll gradients. Cells sedimented at densities between 1.063 and 1.082 g/ml were depleted of cells adherent to plastic tissue culture dishes. The nonadherent cells were further incubated on dishes coated with P55 MoAb. CFU-MK were enriched about 50-fold in the adsorbed cell fraction. This sequential fractionation procedure resulted in a 345-fold (range 276 to 412-fold) enrichment of rat CFU-MK over whole bone marrow cells. The average cloning efficiency of CFU-MK in the final fraction was about 7% (range 5%-9.2%) of the nucleated cells. The overall recovery of CFU-MK averaged 20% (range 9%-29%). The panning step provided a 46-fold enrichment of megakaryocyte burst-forming cells (megakaryocyte burst-forming units, BFU-MK), whose average cloning efficiency in the post-panning fraction was 0.14% (range 0.07%-0.2%). In addition, erythroid burst-forming cells (erythroid burst-forming units, BFU-E) were also significantly enriched by panning, but to a lesser degree than BFU-MK and CFU-MK. By contrast, granulocyte-macrophage colony-forming cells (granulocyte-macrophage colony-forming units, CFU-GM) and erythroid colony-forming cells (erythroid colony-forming units, CFU-E) were not enriched by panning. CFU-MK obtained after panning formed megakaryocyte colonies in the presence of recombinant rat interleukin 3 (rIL-3), mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF), or human erythropoietin (hEPO), as has been reported for murine CFU-MK in whole marrow cells. The highly enriched populations of rat CFU-MK should thus provide a basis for the further study of the regulation of megakaryocytopoiesis.

Animals↗

PubMed1991

Phosphorylation of cAMP response element-binding protein, CRE-BP1, by cAMP-dependent protein kinase and protein kinase C.

The human recombinant CRE-BP1 was phosphorylated by cAMP-dependent protein kinase and protein kinase C, in vitro. These two protein kinases modified distinct serine residues of CRE-BP1. Ser-62 downstream of a putative metal finger structure of CRE-BP1 was the phosphorylation site by cAMP-dependent protein kinase, whereas two serine residues, Ser-340 and Ser-367, located in the basic region of this protein were the major protein kinase C phosphorylation sites. It seems possible that transcriptional and DNA-binding activities of CRE-BP1 are regulated by phosphorylation with these protein kinases.

Amino Acid Sequence↗

PubMed1991

Synergistic interactions between interleukin-11 and interleukin-4 in support of proliferation of primitive hematopoietic progenitors of mice.

Interleukin-11 (IL-11) is a newly identified lymphohematopoietic cytokine originally derived from the primate bone marrow stromal cell line, PU-34. Separately, we reported that IL-11 augments IL-3-dependent proliferation of primitive murine hematopoietic progenitors in culture. We have now examined the synergistic interactions between IL-11 and IL-4 in support of colony formation from marrow cells of mice treated 2 days before with 150 mg/kg 5-fluorouracil. Neither recombinant human IL-11 nor murine IL-4 alone was effective in the support of colony formation. When the two factors were combined, there was major enhancement of colony formation, including that of multilineage colony-forming cells. Serial observations (mapping studies) of development of multipotential blast cell colonies indicated that the synergy between IL-11 and IL-4 is due in part to shortening of the dormant period of the stem cells, an effect very similar to that of IL-6 and granulocyte colony-stimulating factor. The combination of IL-11 and IL-4 may be useful in the stimulation of dormant hematopoietic stem cells in vivo.

Animals↗

PubMed1991

USF-related transcription factor, HIV-TF1, stimulates transcription of human immunodeficiency virus-1.

The transcription factor HIV-TF1, which binds to a region about 60 bp upstream from the enhancer of the human immunodeficiency virus-1 (HIV-1), was purified from human B cells. HIV-TF1 had a molecular weight of 39,000. Binding of HIV-TF1 to the HIV long terminal repeat (LTR) activated transcription from the HIV promoter in vitro. The HIV-TF1-binding site in HIV LTR was similar to the site recognized by upstream stimulatory factor (USF) in the adenovirus major late promoter. DNA-binding properties of HIV-TF1 suggested that HIV-TF1 might be identical or related to USF. Interestingly, treatment of purified HIV-TF1 by phosphatase greatly reduced its DNA-binding activity, suggesting that phosphorylation of HIV-TF1 was essential for DNA binding. The disruption of HIV-TF1-binding site induced a 60% decrease in the level of transcription from the HIV promoter in vivo. These results suggest that HIV-TF1 is involved in transcriptional regulation of HIV-1.

B-Lymphocytes↗

PubMed1991

Expression and function of c-kit in hemopoietic progenitor cells.

The expression and function of a receptor tyrosine kinase, c-kit, in the adult bone marrow of the mouse were investigated by using monoclonal antibodies (mAbs) against the extracellular domain of murine c-kit. In adult C57BL/6 mouse, 7.8% of total bone marrow cells express c-kit on their surface. Half of the c-kit+ cells do not express lineage markers including Mac-1, Gr-1, TER-119, and B220, while the remainder coexpress myeloid lineage markers such as Mac-1 and Gr-1. After c-kit+ cells were removed from the bone marrow cell preparation, hemopoietic progenitor cells reactive to IL-3, GM-CSF, or M-CSF and also those which give rise to spleen colonies in irradiated recipients disappeared almost completely. Thus, most hemopoietic progenitors in the adult bone marrow express c-kit. To investigate whether or not c-kit has any role in the hemopoiesis of adult bone marrow, we took the advantage of one of the anti-c-kit mAbs that can antagonize the function of c-kit. As early as two days after the injection of 1 milligram of an antagonistic antibody, ACK2, almost all hemopoietic progenitor cells disappeared from the bone marrow, which eventually resulted in the absence of mature myeloid and erythroid cells in the bone marrow. These results provide direct evidence that c-kit is an essential molecule for constitutive intramarrow hemopoiesis, especially for the self-renewal of hemopoietic progenitor cells at various stages of differentiation.

Animals↗