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Biomedical subjects

T Kitamura

Publications and source records attributed to T Kitamura.

At least 505 records · Page 28Linked to original sources
PubMed1991

Reconstitution of functional receptors for human granulocyte/macrophage colony-stimulating factor (GM-CSF): evidence that the protein encoded by the AIC2B cDNA is a subunit of the murine GM-CSF receptor.

The high-affinity receptor for human granulocyte/macrophage colony-stimulating factor (hGM-CSF) is composed of two subunits, alpha and beta. The alpha subunit binds GM-CSF with low affinity, whereas the beta subunit does not bind GM-CSF by itself. The alpha and beta subunits together form the high-affinity GM-CSF receptor. The beta subunit has extensive sequence homology with the mouse interleukin 3 (IL-3) receptor (AIC2A) and its homologue (AIC2B) that does not bind IL-3 or other cytokines including GM-CSF. To examine the function of these receptor components, we expressed the alpha subunit of the hGM-CSF receptor with the human beta subunit or the mouse AIC2A or AIC2B in a mouse IL-3-dependent pro-B-cell line, Ba/F3, and in a mouse IL-2-dependent T-cell line, CTLL2. Coexpression of the alpha and beta subunits in Ba/F3 and CTLL2 cells resulted in high-affinity hGM-CSF binding and growth response to low concentrations of hGM-CSF. Whereas Ba/F3 cells expressing the alpha subunit alone proliferated in response to high concentrations of hGM-CSF, CTLL2 cells expressing the alpha subunit alone did not respond to hGM-CSF at all. Since Ba/F3 cells express endogenous AIC2A and AIC2B whereas CTLL2 expresses neither of them, we examined the possibility that either AIC2A or AIC2B is involved in the formation of a functional GM-CSF receptor. The expression of the human alpha subunit with AIC2B, but not with AIC2A, in CTLL2 cells conferred a growth response to hGM-CSF. These results indicate that the beta subunit of the GM-CSF receptor is required for generation of growth signals and that AIC2B is likely the beta subunit of the mouse GM-CSF receptor.

Animals↗
PubMed1991

Structural and functional analyses of glycosylation on the distinct molecules of human GM-CSF receptors.

We have previously demonstrated that granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors are composed of at least two molecules of 80 and 135 kDa, which were denoted alpha- and beta-chains, respectively [Chiba, S., Shibuya, K., Piao, Y.-F., Tojo, A., Sasaki, N., Matsuki, S., Miyagawa, K., Miyazono, K. & Takaku, F. (1990) Cell Regul. 1, 327-335]. In this paper, we describe an investigation of the biochemical disparity noted between the alpha- and beta-chains of GM-CSF receptors using proteolytic and deglycosidic enzymes, and further demonstrate the potential importance of carbohydrate structures of the GM-CSF receptors using different lectins and glycoprotein synthesis inhibitors. Cross-linked alpha- and beta-chains with 125I-GM-CSF were digested by Staphylococcus aureus V8 protease and gave a different pattern. Furthermore, the size of the alpha-chain was reduced by 25 kDa by the removal of the N-linked oligosaccharides with peptidase: N-glycosidase F treatment, whereas that of the beta-chain remained unmodified by the enzyme. These results suggest that the alpha-chain of GM-CSF receptors agrees with the recently cloned low-affinity GM-CSF receptor [Gearing, D.P., King, J.A., Gough, N. M. & Nicola, N.A. (1989) EMBO J. 8, 3667-3676] having approximately 30% N-linked oligosaccharides and is biochemically different from the alpha beta-chain. By analyses using lectins, some of the oligosaccharides in the alpha-chain seem to be the complex-type and/or hybrid-type, because wheat germ agglutinin and leukoagglutinating phytohemagglutinin inhibited both GM-CSF-induced proliferation and GM-CSF binding to its receptors. Further analyses using glycoprotein synthesis inhibitors showed that N-linked processing of the alpha-chain, especially glucose removal by glucosidase I and II (whose activities are inhibited by deoxynojirimycin), appeared to be required for the expression onto the cell surface although the beta-chain expression was little affected by their inhibitors. Thus the beta-chain, probably located near the alpha-chain on the cell surface, was associated with a high-affinity class of GM-CSF receptors.

Cell Division↗
PubMed1991

Trajectory of spinocerebellar fibers passing through the inferior and superior cerebellar peduncles in the rat spinal cord: a study using horseradish peroxidase with pedunculotomy.

The course of spinocerebellar fibers in the rat spinal cord was investigated by injecting horseradish peroxidase into the cerebellar anterior vermis after complete transection of the left inferior and right superior cerebellar peduncles. By this procedure, fibers passing via the inferior cerebellar peduncles (icp-fibers) were labeled retrogradely on the right side of the spinal cord, whereas fibers passing via the superior cerebellar peduncles (scp-fibers) were labeled on the left side. Crossed icp-fibers were located diffusely in the anterior and lateral funiculi in the sacral to lower lumbar segments. They gradually migrated laterally and dorsally in these funiculi and received many uncrossed icp-fibers moving laterally in the lateral funiculus from the gray substance in the upper lumbar to lower thoracic segments. These mixed fibers shifted more dorsally and laterally in the anterior and lateral funiculi to aggregate in the narrow peripheral zone of the lateral funiculus in the upper thoracic and lower cervical segments, and received many crossed fibers in the upper cervical segments. There were more icp-fibers than scp-fibers through the spinal cord. However, the extent of scp-fibers in the anterior and lateral funiculi was essentially the same as that for icp-fibers, except that a few scp-fibers were found in the dorsolateral marginal zone of the lateral funiculus. It has been generally accepted that the dorsal spinocerebellar tract ascends in the dorsal half of the lateral funiculus and enters the cerebellum via the inferior cerebellar peduncle, whereas the ventral spinocerebellar tract ascends in the ventral half of it and takes the superior cerebellar peduncle route. The results of this study suggest that it is necessary to revise this concept.

Animals↗
PubMed1991

The amino-terminal helix of GM-CSF and IL-5 governs high affinity binding to their receptors.

Transduction of the biological effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5) requires the interaction of each cytokine with at least two cell surface receptor components, one of which is shared between these two cytokines. A strategy is presented that allowed us to identify receptor binding determinants in GM-CSF and IL-5. Mixed species (human and mouse) receptors were used to locate unique receptor binding domains on a series of human-mouse hybrid GM-CSF and IL-5 cytokines. Results show that the interaction of these two cytokines with the shared subunit of their high affinity receptor complexes is governed by a very small part of their peptide chains. The presence of a few key residues in the amino-terminal alpha-helix of each ligand is sufficient to confer specificity to the interaction. Comparison with other cytokines suggests that the amino-terminal helix of many of these proteins may contain the recognition element for the formation of high affinity binding sites with the alpha subunit of their multi-component receptors.

Amino Acid Sequence↗
PubMed1991

Identification of the second subunit of the murine interleukin-5 receptor: interleukin-3 receptor-like protein, AIC2B is a component of the high affinity interleukin-5 receptor.

Murine interleukin-5 (IL-5) binds to its receptor with high and low affinity. It has been shown that the high affinity IL-5 receptor (IL-5-R) is composed of at least two membrane protein subunits and is responsible for IL-5-mediated signal transduction. One subunit of the high affinity IL-5-R is a 60 kDa membrane protein (p60 IL-5-R) whose cDNA was isolated using the anti-IL-5-R monoclonal antibody (mAb), H7. This subunit alone binds IL-5 with low affinity. The second subunit does not bind IL-5 by itself, and is expressed not only on IL-5-dependent cell lines but also on an IL-3-dependent cell line, FDC-P1. Expression of the p60 IL-5-R cDNA in FDC-P1 cells, which do not bind IL-5, reconstituted the high affinity IL-5-R. We have characterized the second subunit of the IL-5-R by using another anti-IL-5-R mAb, R52.120, and the anti-IL-3-R mAb, anti-Aic-2. The anti-Aic-2 mAb down-regulated binding of IL-5 to an IL-5-dependent cell line, Y16. Both R52.120 and anti-Aic-2 mAbs recognized membrane proteins of 130-140 kDa expressed on FDC-P1 and Y16 cells. The R52.120 mAb recognized both murine IL-3-R (AIC2A) and its homologue (AIC2B) expressed on L cells transfected with suitable cDNAs. The high affinity IL-5-R was reconstituted on an L cell transfectant co-expressing AIC2B and p60 IL-5-R, whereas only the low affinity IL-5-R was detected on a transfectant co-expressing AIC2A and p60 IL-5-R.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Ultrasonographic findings and management of intrahepatic biliary tract abnormalities after portoenterostomy.

Ultrasonographic examinations was made in 24 children who had undergone a portenterostomy to correct extrahepatic biliary atresia. Abnormalities were observed in six patients. These were the result of ongoing inflammatory reactions because all had been suffering from cholangitis after surgery, and the size of the biliary tract structure changed in accordance with the occurrence and subsidence of the cholangitis. Ultrasonographic examination showed dilation of the intrahepatic bile duct in one patient and cystic lesions in five patients. Treatment included percutaneous transhepatic bile drainage for dilated bile ducts, alcohol injections for intrahepatic cysts, and reoperation for cysts in the porta hepatis. Treatment was not required for cysts in controllable cholangitis. The results of these approaches were excellent, indicating that they were of benefit in treatment intrahepatic abnormalities occurring after portoenterostomy.

Bile Ducts, Intrahepatic↗
PubMed1991

The domain of Epstein-Barr virus nuclear antigen 1 essential for binding to oriP region has a sequence fitted for the hypothetical basic-helix-loop-helix structure.

The domain of Epstein-Barr virus nuclear antigen 1 (EBNA-1) which is essential for binding to a region containing oriP, an episomal replication origin of EBV DNA, was analyzed by DNA binding assay with beta-galactosidase-EBNA-1 fusion proteins. It was revealed that a 159-amino acid (aa) domain, 460-618 aa, of EBNA-1 retained the oriP-binding activity and the domain's activity was abolished by a deletion of 29 aa from its amino-terminal end and by a 38 aa deletion from its carboxyl-end as well. One of five monoclonal antibodies against EBNA-1 specifically inhibited the binding of the beta-galactosidase-EBNA-1 fusion protein to the oriP region. The epitope recognized by the monoclonal antibody was mapped in the crucial 29 aa region. An analysis of the domain's putative secondary structure and a computer search of amino acid sequence homology indicated that the 159-aa domain contains the hypothetical basic-helix-loop-helix structure which is considered to be a common characteristic structure of a family of DNA binding proteins. Examinations of DNA binding activity of the other EBNA polypeptides with a series of fusion proteins and similar structural analyses of their amino acid sequences were also performed. This study suggests that EBNA-1 is a constituent of the family of DNA binding proteins which are involved in transcriptional regulation critical for cell differentiation or cell-type determination.

Amino Acid Sequence↗
PubMed1991

Extracellular ATP, intracellular calcium and canalicular contraction in rat hepatocyte doublets.

Bile-canaliculus contraction in rat hepatocyte doublets is postulated to involve activation of an actin-myosin system. We examined this hypothesis by determining the relationship between canalicular contraction and cystolic free Ca2+ ([Ca2+]i) concentration after extracellular addition of ATP or microdialysis of myosin light chain kinase or its Ca(2+)-independent fragment, which retains catalytic activity. After incubation of doublets with 200 mumol/L ATP in the absence of extracellular Ca2+, [Ca2+]i peaked at 40 sec and 71% of canaliculi contracted within 4 min. Decreasing effects were observed with equimolar ADP, AMP and nonhydrolyzable ATP, but no effect was observed with adenosine. The effect of extracellular ATP on [Ca2+]i and canalicular contraction was dose dependent. Addition of extracellular Ca2+ and ATP resulted in a plateau level of [Ca2+]i. Cytochalasin D, which depolymerizes actin filaments, inhibited ATP-induced canalicular contraction, but not the increase in [Ca2+]i. Microdialysis of myosin light chain kinase and its Ca(2+)-independent fragment (but not the heat-denatured fragment, albumin, trypsin plus soybean inhibitor or buffer) into one hepatocyte of a doublet resulted in canalicular contraction in 86% of doublets. Injection of myosin light chain kinase or its Ca(2+)-independent fragment did not increase [Ca2+]i within 5 min. These results indicate that (a) the basolateral plasma membrane of hepatocytes has a P2Y-class purinoceptor, (b) increased [Ca2+]i after incubation with ATP is initially due to mobilization from internal sites and (c) canalicular contraction is directly related to [Ca2+]i and activation of an actin-myosin system. The physiological role of extracellular ATP in canalicular contraction is uncertain.

Adenosine Triphosphate↗
PubMed1991

Enhancing effect of ubenimex (bestatin) on proliferation and differentiation of hematopoietic progenitor cells, and the suppressive effect on proliferation of leukemic cell lines via peptidase regulation.

Ubenimex (Bestatin) significantly enhanced the G- and GM-CSF-induced colony formation of human bone marrow cells at concentrations of 0.001, 0.01, 0.1 and 1.0 microgram/ml (21-61% enhancement), but not at 10 micrograms/ml. Ubenimex did not influence the EPO-induced erythroid colony and burst formation between 0.0001-100 micrograms/ml. Against human and mouse leukemic cell lines, the growth-inhibitory activities of ubenimex were dose-dependently observed. Aminopeptidase activities on U937 and TF-1 cells were almost inhibited with 10 and 100 micrograms/ml of ubenimex, respectively. Cross-linking studies of 125I-GM-CSF binding to TF-1 cells demonstrated that the 150-kDa band of 2 major bands was enhanced after incubation with 0.01 microgram/ml ubenimex but decreased after that with 100 micrograms/ml, and that the 95-kDa band was not changed at any concentration of ubenimex. Change in density of the 150-kDa band on ubenimex-treated TF-1 cells was correlated with that in expression of CD10 (neutral endopeptidase) on them, whereas that in expression of CD13 (aminopeptidase N) was not changed at any concentration. These results suggest that one possible mechanism of ubenimex action in hematopoietic progenitor cells is the up-regulation of the high affinity receptor for GM-CSF and that in leukemic cell lines is suppression of amino acid incorporation via peptidase regulation.

Aminopeptidases↗
PubMed1991

IL-1 up-regulates the expression of cytokine receptors on a factor-dependent human hemopoietic cell line, TF-1.

A factor-dependent human hemopoietic cell line, TF-1, requires interleukin 3 (IL-3) or granulocyte/macrophage colony-stimulating factor (GM-CSF) for its long-term growth. We have found that IL-4, IL-5, and IL-6 also support the growth of TF-1 and that IL-1 enhances the proliferative effect of these cytokines. Augmentation by IL-1 is associated with up-regulation of the receptors for IL-3, IL-5, GM-CSF, and erythropoietin (Epo). IL-1 increased the number of binding sites for IL-3 and Epo without changing their affinities. In contrast, IL-1 increased the number of high affinity binding sites for GM-CSF and IL-5, whereas the total number of binding sites was unchanged. Chemical crosslinking experiments indicated that the receptors for IL-3, IL-5, and GM-CSF were composed of two components and that the molecular masses of the larger components of these cytokine receptors were quite similar (120 kd). The enhanced expression of the larger components of the IL-3, IL-5, and GM-CSF receptors by IL-1 may be responsible for IL-1-induced up-regulation of these receptors. These observations are consistent with the model that the receptors for IL-3 and GM-CSF share a common component.

Cell Line↗
PubMed1991

Different induction of 70,000-Da heat shock protein and metallothionein in HeLa cells by copper.

To clarify the physiological roles of heat shock proteins induced by copper, we studied the synthesis of these proteins and metallothionein, as well as the level and nature of copper incorporated into HeLa cells. Incubation in medium containing 200 microM cupric sulfate and above induced the synthesis of 70,000-Da heat shock protein (hsp70) in these cells. However, the synthesis of hsp70 did not increase in the presence of less than 200 microM cupric sulfate. On the other hand, the synthesis of metallothionein increased due to 100 microM cupric sulfate. The uptake of copper into the cells depended on the cupric sulfate concentration in the medium. To analyze the nature of the intracellular copper, cell extracts were separated by gel filtration chromatography into three fractions: the high molecular weight, metallothionein, and low molecular weight fractions. No copper was found in the low molecular weight fraction of control cells, but appeared distinctly at 200 microM cupric sulfate and above. Copper in the high molecular weight fraction also began to increase at 200 microM cupric sulfate and above, whereas in the metallothionein fraction it began to increase even at 50 to 100 microM cupric sulfate. Furthermore, inhibition of cell growth was also observed at 200 microM cupric sulfate and above but not at 100 microM and below.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Division↗
PubMed1991

Sensitive detection of viral antigens with a new method, "laser magnet immunoassay".

A new method, "laser magnet immunoassay" (LMIA), has been developed for sensitive detection of viral antigens. Target viruses captured on microbeads were made to react with antibodies labeled with magnetite particles. In a magnetic field, magnetically labeled antigens dispersed in water were attracted to and concentrated at one point on the surface, resulting in the lifting up of a small surface area. A laser beam which was incident on the point reflected, making an interference fringe. The intensity of the fringe indicates the amount of the magnetite conjugated with antigen. A very low concentration of antigens, such as 5 particles of influenza virus and 0.1 pg/ml of human immunodeficiency virus (HIV) p24 antigen in human serum, could be detected by this method. Application of this method to diagnoses of viral diseases in early stages is discussed.

Antigens, Viral↗
PubMed1991

Effect of cacao husk extract on human immunodeficiency virus infection.

A sodium hydroxide extract from cacao husk inhibited the cytopathic effect of human immunodeficiency virus type 1 (HIV-1) against HTLV-1-transformed T-cell lines MT-2 and MT-4. It also inhibited syncytium formation between HIV-infected and uninfected lymphoblastoid T-cell line, MOLT-4. The anti-HIV activity was concentrated by membrane filter fractionation to a fraction with molecular weight of 100-300 KDa. Anti-HIV activity of the extract was attributable to interference with the virus adsorption, rather than to inhibition of the virus replication after adsorption.

Cacao↗
PubMed1991

Myosin in hepatocytes is essential for bile canalicular contraction.

Active dynamic contraction of bile canaliculi has been observed in cultured doublet hepatocytes using time-lapse cinephotomicrography. This contractile movement plays an important role in normal bile formation. The mechanism of bile canalicular contraction has been proved to involve the Ca(2+)-calmodulin system and pericanalicular actin filaments. However, the role of myosin in this system is still unknown. In this study, using the newly synthesized myosin light-chain kinase inhibitor ML-9, we found that the treatment of cultured doublet hepatocytes with ML-9 inhibited canalicular contraction. This inhibitory effect suggests that myosin is involved in this complex cellular function and that the integrity of the actin-myosin system, as well as the Ca(2+)-calmodulin system is essential for normal bile canalicular contraction.

Actins↗
PubMed1991

[Novel PCR methods for detection of HIV proviruses using digoxigenin labelled probes--Southern blot and dot blot].

Polymerase chain reaction (PCR) is useful for diagnosis of HIV infection in the silent stage, for HIV transmission from mother to child and for existence of HIV in blood and blood products. We developed a simple, rapid and safe PCR method by using digoxigenin labelled probes. Furthermore, we developed a semi-quantitative dot blot method. These methods will be helpful for diagnosis.

Blotting, Southern↗
PubMed1991

Typing of human polyomavirus JC virus on the basis of restriction fragment length polymorphisms.

JC virus DNA clones from the urine of nonimmunosuppressed Japanese individuals regularly contain an archetypal regulatory sequence which may have generated various regulatory sequences of JC virus isolates from patients with progressive multifocal leukoencephalopathy (PML). In this study, we established 15 new clones from the urine of Dutch, German, and Taiwanese healthy volunteers and patients. Most of these clones contained regulatory sequences essentially identical to the archetypal regulatory sequence. These clones, along with two representative urine-derived clones in Japan and five clones from the brains of PML patients (four established in the United States and one established in Japan), were analyzed with a number of restriction enzymes. We found nine restriction fragment length polymorphisms by which all clones were classified into either of the two types, A and B. Type A contained only clones from the West, while type B contained some from the West and all from eastern Asia. Each type contained both urine-derived and PML-derived clones. Furthermore, there was a close relationship between some urine-derived clones and some PML-derived clones in restriction site mapping analysis. These findings support the adaptation hypothesis which has been postulated to explain the genesis of PML-type JC viruses.

Base Sequence↗
PubMed1991

Sequence rearrangement in JC virus DNAs molecularly cloned from immunosuppressed renal transplant patients.

From nonimmunocompromised individuals, we have recently identified a possible archetypal JC virus DNA sequence from which various regulatory sequences of JC virus isolates derived from patients with progressive multifocal leukoencephalopathy (PML) could have evolved. In this study, we analyzed the regulatory sequences of JCV DNAs cloned from urine samples of a PML risk group (renal transplant patients on immunosuppressive therapy). A number of JC virus DNAs were molecularly cloned from virions excreted in the urine of eight patients. Furthermore, fragments containing the regulatory region were amplified by the polymerase chain reaction and subsequently molecularly cloned from cell-associated JC virus excreted in the urine of two patients. The regulatory regions in all clones were analyzed with restriction enzymes, and those in representative clones were sequenced. We found that clones with the archetypal regulatory sequence were predominant in all urine samples, but a few clones carried regulatory sequences that diverged from the archetypal sequence by deletion or duplication. The finding that sequence rearrangement in the archetypal regulatory region occurs in the course of infection in immunosuppressed hosts is consistent with the adaptation hypothesis which has been put forward to explain the divergence of the regulatory regions in PML-derived JC virus isolates.

Base Sequence↗