Biomedical subjects
Publications and source records attributed to T Sudo.
In this report, we have found that fatty acid-free bovine serum albumin stimulated the expression of a transfected foreign gene in Cos1 cells in serum-free medium and that its activity was as same as that of fetal calf serum. This will simplify the purification of the gene product from the culture medium.
Specific monoclonal antibodies (MoAbs) to Actinobacillus pleuropneumoniae (APP) serotypes 1 and 2 which recognized serotype-specific antigens were produced. It was revealed that the two serotype-specific MoAbs H1-18 and H22-7 recognized O polysaccharides of the lipopolysaccharide (LPS) from APP serotypes 1 and 2, respectively, in the results of antigen analysis by means of SDS-PAGE and Western blotting. Furthermore, ddY mice immunized passively with the above type-specific MoAbs were protected against challenge infection by the homologous serotype of APP at 24 or 48 hr later. However, H1-9 and H3-2 MoAbs recognizing serotype-common protein antigens of APP did not show signs of any protective effect. These results showed that LPS from APP bacterial cells was one of the structural substances in the serotype-specific antigens, and an important component as one of the antigens protecting against the homologous serotype strain.
The trans-activating and transforming capacities of the c-myb proto-oncogene product (c-Myb) are negatively regulated through a leucine zipper structure in its negative regulatory domain. We show here tht in cotransfection assays, maximal Myb-induced trans-activation occurs with relatively low amounts of wild-type c-Myb, while higher levels of c-Myb result in reduced Myb-induced trans-activation. By contrast, this apparent negative autoregulation is not observed with a c-Myb mutant containing an impaired leucine zipper. Data presented here suggest that this negative autoregulation of trans-activation by wild-type c-Myb is a consequence of homodimer formation by c-Myb through its leucine zipper and of the inability of c-Myb dimers to bind DNA. These findings point to a novel mechanism of regulation of a transcription factor.
A monoclonal antibody, A7R34, that recognizes the high-affinity interleukin 7 receptor (IL-7Ra) and blocks the binding between IL-7 and IL-7Ra has been produced. Cell surface staining with A7R34 demonstrated that IL-7Ra is expressed in both B- and T-cell lineages. In the bone marrow, immature B-lineage cells that do not express surface IgM were IL-7Ra+. In the thymus, IL-7Ra was detected in CD4-8- T cells and also in CD4 or CD8 single-positive cells but not in CD4+8+ double-positive cells. In the peripheral lymphoid tissues, both CD4 and CD8 single-positive cells were the major cell types that express IL-7Ra. Addition of A7R34 to a long-term B-precursor-cell culture inhibited proliferation of the B-lineage cells, indicating that IL-7 is an absolute requirement for in vitro B-cell genesis. Consistent with this in vitro result, continuous injection of A7R34 into an adult mouse resulted in a decrease of B-precursor cells and also of thymocytes, whereas a considerable fraction of mature B and T cells in the peripheral tissues persisted over 2 weeks of the experiment. When A7R34 injection is started from day 14 of gestation, it is possible to produce mice that lack B cells. These results indicate that IL-7 is an essential molecule for generation of both B and T cells in murine bone marrow and thymus, respectively. Moreover, IL-7Ra would be the sole receptor system regulating these processes.
Explore the source record for details and available documents.
The effect of diltiazem or cimetidine pretreatment on the inhibition of theophylline oxidative metabolism was investigated in nine healthy male nonsmokers. Diltiazem 60 mg, cimetidine 400 mg, or placebo was given orally three times daily for 3 days in a randomized three-way crossover manner. Both diltiazem and cimetidine pretreatment decreased the mean theophylline clearance (0.702 on placebo versus 0.641 on diltiazem, P < .05, and 0.542 mL/minute/kg on cimetidine, P < .01), resulting in prolonged mean theophylline half-life (7.58 on placebo versus 8.59 on diltiazem, P < .05, and 10.08 hours on cimetidine, P < .01) with no change in volume of distribution. The mean metabolic clearances for three major theophylline metabolites, 1-methyluric acid (1-MU), 1,3-dimethyluric acid (1,3-DMU), and 3-methylxanthine (3-MX), were reduced significantly by cimetidine (28%; P < .05, 32%; P < .01, and 33%; P < .01, respectively). Conversely, diltiazem significantly reduced only the mean metabolic clearance of 1,3-DMU by 21% (P < .05) without changes in that of 1-MU or 3-MX. These results suggest that cimetidine inhibited both N-demethylation and 8-hydroxylation of theophylline, whereas diltiazem exerted little influence on N-demethylation in spite of inhibition in 8-hydroxylation.
The effect of durations of diltiazem pretreatment on nifedipine kinetics was evaluated. Eighteen healthy male subjects were randomly allocated to three groups, 6 subjects each, for single doses of 60 mg diltiazem, and 3 days and 6 days with diltiazem 60 mg three times a day. All subjects received 20 mg nifedipine orally on two occasions using a double-blind cross-over, placebo-controlled method. No significant difference on pharmacokinetic parameters of nifedipine without diltiazem were observed among three groups. The single dose with 60 mg diltiazem significantly increased the area under the plasma concentration-time curve (AUC) for nifedipine compared with that in control an average of 35.1% (P < .05) and decreased the total body clearance (CL) an average of 24.0% (P < .05). Three days and 6 days pretreatment with diltiazem 60 mg three times a day significantly increased mean nifedipine AUC to 151.1% (P < .01), 188.0% (P < .05) of control values, and decreased CL to 58.2% (P < .01), 63.9% (P < .05) of control values, respectively. The elimination half-life (t1/2) of nifedipine were significantly prolonged both after 3 days' and after 6 days' pretreatment of diltiazem. These results suggest that diltiazem affects the nifedipine kinetics rapidly and pretreatment duration dependently. A clinically important drug interaction may occur when both drugs are administered simultaneously.
To evaluate the influence of nifedipine on the pharmacokinetics and the pharmacodynamics of diltiazem, five healthy subjects received 60 mg diltiazem orally on two occasions, diltiazem alone or after nifedipine pretreatment (10 mg three times daily for 3 days). After nifedipine pretreatment, the maximum concentration (Cmax) of diltiazem was increased and the time of Cmax was shortened, and the area under the concentration curve (AUC) tended to be increased. Although heart rate was increased, the corrected PQ interval tended to be prolonged after the nifedipine pretreatment. Both a decreased hepatic clearance and an increased bioavailability of diltiazem probably accounts for the increase in the Cmax and AUC of diltiazem after nifedipine pretreatment, and that might affect the pharmacodynamics of diltiazem.
A previously reported thymic nurse cell clone, TNC-R3.1 could form a unique complex with isolated adult mouse CD4-8- (DN) thymocytes and greatly sustained the cell viability of DN thymocytes in suspension culture. In addition, the TNC-R3.1 clone supported the differentiation of DN thymocytes into CD4+8+ (DP) thymocytes in a short-term culture. Addition of IL-7 into the coculture markedly enhanced DN thymocyte-TNC interaction and induced the proliferation and differentiation of DN thymocytes, though IL-7 alone did not induce the differentiation of DN thymocytes. Separation of DN thymocytes from TNC-R3.1 monolayer using a Millicell caused a great inhibition of the DN thymocyte differentiation, suggesting that direct contact between TNC-R3.1 cells and immature thymocytes was required for the differentiation of DN thymocytes. The kinetics study demonstrated that DN thymocytes started to differentiate into DP thymocytes through CD3-CD4+J11d+ intermediate cells 8-12 h after the initiation of the culture with TNC-R3.1 plus IL-7. The generation of DP thymocytes became maximal 20 h after coculture and gradually decreased thereafter. Furthermore, we demonstrated that TNC-R3.1 could support the differentiation of CD3+CD4+CD8- or CD3+CD4-CD8+ thymocytes from CD3-CD4-CD8- thymocytes in the presence of IL-7 and IL-2. These data indicate that our established in vitro culture system mimics the early stage of the intrathymic T cell developing pathway.
Immature hemopoietic cell lines were established by transforming fetal thymocytes in vitro with a ts mutant of Abelson murine leukemia virus. They are positive for c-kit and IL-2R alpha but negative for lineage specific markers. Their TCR and Ig heavy chain genes are in germline configuration, and are expressed as germline gene transcripts. When these cell lines were stimulated in vitro with IL-1 their morphology changed into that of typical macrophages (M phi). Subsequent analysis of a particular clone, which displayed the morphological change at the highest efficiency among established cell lines, indicated that the clone possesses the capacity to differentiate into I-A-M phi capable of secreting several cytokines, and supporting the proliferation of fetal and adult thymocytes in vitro. If their surface markers are considered, their normal counterparts would be present in a minor subset of CD4-CD8- double-negative cells in the thymus in early development. The results raise the possibility that the thymic organ at an early stage of development stores immature hemopoietic cells capable of differentiating into a non-T lineage constituting the thymic stromal elements.
c-fos and c-jun gene products form a heterodimeric complex (AP-1) that regulates target gene expression by binding to a specific DNA sequence motif. In order to study a role of AP-1 (Fos/Jun) in growth and differentiation of immature B lineage cells, we have established and mated two independent transgenic mice carrying the mouse c-fos gene or the viral v-jun gene fused to the H-2K promoter. IL-7 dependent bone marrow cell culture from doubly transgenic (H2-fos/jun) mice demonstrated severe delay of early B cell development. Proliferation of pre-B cells in the fresh bone marrow from H2-fos/jun mice to IL-7 stimulation was very low. These results suggest that the deregulated production of AP-1 perturbs IL-7 mediated proliferation and differentiation of immature B cells.
The extended cytoplasmic processes from megakaryocytes (MK) are believed to be structural intermediates between MK and platelets. We could observe differentiation of purified rat CFU-MK toward mature MK to form extended cytoplasmic processes. Recombinant rat interleukin-3 (IL-3), human erythropoietin (Epo), and human interleukin-6 (IL-6) each was able to stimulate process formation although they varied somewhat in their potential. Electron microscopic observations showed that these processes were very similar to those from mature MK so far reported. The combination of IL-6 with IL-3 or Epo synergistically increased the number of MK forming processes without further increase in the number of total MK formed in the presence of IL-3 or Epo alone. In addition, IL-6 significantly increased the megakaryocytic diameter and DNA content of MK induced by IL-3 or Epo and shortened the MK transit time to hasten the process formation of MK. These findings suggest that IL-6 promotes further maturity in both the cytoplasm and ploidy of MK to form extended cytoplasmic processes. The ability of these factors to generate the process formation in vitro may be related to their thrombopoietic effects in vivo.
The protooncogene c-kit encodes a receptor type tyrosine kinase and is allelic with the W locus of mice. SLF, the c-Kit ligand which is encoded by the Sl locus, has growth promoting activity for hemopoietic stem cells. Previous studies demonstrated that c-Kit is functionally required for the proliferation of hemopoietic progenitor cells at various differentiation stages in adult bone marrow. However, the absence of functional SLF and c-Kit in fetuses with mutant alleles of Sl and W loci produces only minor effects on the myeloid and early erythroid progenitor cells in the fetal liver, although the level of the late erythroid progenitor cells is significantly affected. We used an anti-c-Kit monoclonal antibody to investigate the expression and function of c-Kit in murine fetal hemopoietic progenitor cells. Flow-cytometric analysis showed that hemopoiesis in the yolk sac and fetal liver started from cells that express c-Kit. The c-Kit expression decreased upon maturation into erythrocytes in each organ. By fluorescence activated cell sorting, the c-Kit+ cell population was enriched with the hemopoietic progenitor cells clonable in vitro (CFU-E, BFU-E and GM-CFC). To elucidate whether c-Kit functions in these progenitor cells in vivo, we took advantage of the antagonistic anti-c-Kit monoclonal antibody, ACK2, which can block the function of c-Kit. Administration of ACK2 after 12.5 days of gestation rapidly eliminated BFU-E and GM-CFC as well as CFU-E from the fetal liver. However, the number of these progenitor cells in the yolk sac and fetal liver was less affected when the fetuses were given ACK2 before 12.5 days of gestation. Our results provide evidence that there are two waves of hemopoiesis in murine embryos relative to c-Kit dependency. The c-Kit has an essential role on the growth of hemopoietic progenitor cells in the fetal liver after 12.5 days of gestation, whereas the progenitor cells in the liver and yolk sac of the earlier embryo do not depend on c-Kit and its ligand SLF.
SMXA recombinant inbred (RI) strains were derived from the F2 generation of a cross between two progenitor inbred strains, A/J and SM/J, which differ considerably with respect to many characters. In order to determine the morphometric profiles of the mandible of SMXA RI strains and to identify these strains, principal component and discriminant analyses (mandible analysis) were carried out using 11 mandible measurements. Principal component analysis revealed that the mandibles of each strain have their own characteristic size and shape. The RI strains, however, were roughly divided into 15 groups based on the morphometric profile of their mandibles. Discriminant analysis showed the probability of erroneous discrimination to be 6.49% (20/308) for the males and 8.06% (27/335) for the females. In both sexes, most of the erroneous discrimination (13 in the male, 17 in the female) occurred between RI strains having comparatively similar morphometric profiles. This suggested that all of the SMXA RI strains examined could be regarded as genetically homogeneous within each strain.
It is well known that laboratory rabbits are not controlled genetically like laboratory mice and rats. In order to test the usefulness of DNA fingerprinting in investigation of genetic uniformity of the laboratory rabbits strains and their relationships, we applied DNA fingerprinting using bacteriophage M13 probe to five strains (2 inbreds (JWY-NIBS and DuY-NIBS) and 3 outbreds (JW-NIBS, Icl:JW and WHHL)). DNA fingerprints of 2 inbred strains showed the same banding patterns within each strain but the strain-specific patterns. Although there were no rabbits showing the same banding patterns in 3 outbred strains, average percent differences (APD) were 13.7 to 18.6. A dendrogram based on APD of DNA fingerprints was constructed by 2 large clusters, JW group and DuY. The dendrogram was essentially similar to that based on rabbit mandible measurements. These results suggest that DNA fingerprinting is available not only for the genetic monitoring of the laboratory rabbit strains but also for the investigation of their genetic relationships.
Explore the source record for details and available documents.
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.
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.