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S Koyasu

Publications and source records attributed to S Koyasu.

At least 109 records · Page 6Linked to original sources
PubMed1986

Insulin-like growth factors, insulin, and epidermal growth factor cause rapid cytoskeletal reorganization in KB cells. Clarification of the roles of type I insulin-like growth factor receptors and insulin receptors.

Insulin-like growth factor (IGF) I (greater than or equal to 10(-10)M, insulin-like growth factor II (greater than or equal to 10(-9) M), insulin (greater than or equal to 10(-9) M, and epidermal growth factor (EGF, greater than or equal to 10(-11) M) caused rapid membrane ruffling in KB cells. The morphological change was observed within 1 min after the addition of these growth factors and was accompanied by microfilament reorganization, but not by microtubule reorganization. IGF-I, IGF-II, and insulin induced morphologically very similar or identical membrane ruffles with the order of potency IGF-I greater than IGF-II greater than insulin, whereas EGF-induced membrane ruffles were morphologically different. KB cells possessed EGF receptors, type I IGF receptors, and insulin receptors, but few or no type II IGF receptors. Monoclonal antibody against type I IGF receptors, which completely inhibited the binding of 125I-IGF-I to the cells but did not inhibit the binding of 125I-insulin, caused marked inhibition of IGF-I (10(-8) M)-stimulated membrane ruffling. IGF-II (10(-8) M)-stimulated membrane ruffling was partially inhibited in the presence of this antibody, but insulin (10(-7) M)-stimulated membrane ruffling was only slightly inhibited. In contrast, monoclonal antibody against insulin receptors blocked insulin (10(-7) M) stimulation, but not IGF-I (10(-8) M) stimulation, of membrane ruffling. Thus, this study provides evidence that IGF-I and insulin act mostly through their own (homologous) receptors and that IGF-II acts by cross-reacting with both type I IGF and insulin (heterologous) receptors in causing rapid alterations in cytoskeletal structure.

Antibodies, Monoclonal↗
PubMed1986

Calmodulin-regulated binding of the 90-kDa heat shock protein to actin filaments.

We have found that the 90-kDa heat shock protein (HSP90) prepared from a mouse lymphoma exists in homodimeric form under physiological conditions and has the ability to bind to F-actin (Koyasu, S., Nishida, E., Kadowaki, T., Matsuzaki, F., Iida, K., Harada, F., Kasuga, M., Sakai, H., and Yahara, I. (1986) Proc. Natl. Acad. Sci. U.S.A., in press). Here we show that calmodulin regulates the binding of HSP90 to F-actin in a Ca2+-dependent manner. The binding of HSP90 to F-actin occurred optimally under physiological solution conditions, i.e. in 2 mM MgCl2 + 100 mM KCl. The binding was saturable in a molar ratio of about 1 HSP90 (dimer) to 10 actins. HSP90 was dissociated from F-actin by the binding of tropomyosin to F-actin. Calmodulin was found to inhibit the binding of HSP90 to F-actin in a Ca2+-dependent manner. Moreover, the equilibrium gel filtration demonstrated that calmodulin binds to HSP90 in the presence of Ca2+, but not in the absence of Ca2+. These data indicate that HSP90 complexed with Ca2+-calmodulin is unable to bind to F-actin. Ca2+-dependent interaction of HSP90 with calmodulin as well as calmodulin-regulated binding of HSP90 to F-actin revealed here may provide new insight into the function of HSP90 and the regulation of actin structure in cells.

Actins↗
PubMed1986

Expression of interleukin 2 receptors on interleukin 3-dependent cell lines.

Several mouse IL 3-dependent cell lines, IC2, LT4, FDC-P2, and PB-3C, derived from spleen or bone marrow cells were shown to express low affinity receptors for IL 2 (Kd; 0.5 to 8 X 10(-8) M). High affinity receptors for IL 2 were not detected on the IL 3-dependent cells within the experimental limitation of this study. The clones did not respond to IL 2 at all at the concentration as high as 25 micrograms/ml. The number of the receptors expressed on those clones was estimated to be 0.2 to 2 X 10(5)/cell, which is comparable with the number of those on IL 2-dependent T cell clones. Expression of IL 2 receptor was confirmed in mRNA levels for both IC2 and LT4 cells. A relatively low level expression of one (4.5 Kb) of four IL 2 receptor mRNA species was observed with those IL 3-dependent clones compared with IL 2-dependent T cells. It seems that these low affinity receptors may be expressed on IL 3-dependent cells that undergo differentiation or maturation in mast cell and some myeloid cell lineages.

Animals↗
PubMed1986

Two mammalian heat shock proteins, HSP90 and HSP100, are actin-binding proteins.

Two high molecular weight heat shock proteins, HSP90 (Mr, 90,000) and HSP100 (Mr, 100,000), were separately purified from extracts of cultured cells of a mouse lymphoma cell line, L5178Y. Both of the HSPs exist in homodimeric form under physiological conditions. Their physicochemical properties are quite similar to each other. Each of the purified HSPs was shown to coprecipitate with rabbit skeletal muscle actin under actin-polymerizing conditions. Both HSP90 and HSP100 increased the low-shear viscosity of filamentous actin solutions in a dose-dependent manner, which suggests that these HSPs cross-link actin filaments. Although some molecular properties and the effects described above on actin solution of HSP90 and HSP100 resemble those of alpha-actinin, the HSPs were distinguished from alpha-actinin by various means, including visualization of molecular shapes by electron microscopy with the aid of the low-angle rotary shadowing technique. Immunofluorescence staining by specific antisera against HSP90 revealed that HSP90 was localized in ruffling membranes in addition to the cytoplasmic space.

Actins↗
PubMed1986

A heat shock-resistant variant of Chinese hamster cell line constitutively expressing heat shock protein of Mr 90,000 at high level.

A heat shock-resistant variant of Chinese hamster cell line (CHO) was isolated from ethane methane sulfonate-treated CHO cells through selection by repeated exposures to elevated temperature. The variant, designated HR-01, was one to two order of magnitudes more resistant to lethal heat shock (46.0 degrees C) than the parental CHO strain (p-CHO). The heat shock resistance characteristic of this variant was stable. In addition, the HR-01 variant showed more elongated cell morphology, and was more adherent to substrate than p-CHO. When total proteins of p-CHO and HR-01 cells were compared in two-dimensional polyacrylamide gel electrophoresis, HSP90, a heat shock protein of Mr 90,000, was found to be the only protein that was expressed at a significantly higher level in HR-01 cells than in p-CHO cells. Because of the known intriguing molecular properties of HSP90, the HR-01 variant would be useful for further investigation of functions of HSP90 as well as the mechanism of acquiring heat shock resistance in mammalian cells.

Animals↗
PubMed1985

Production of interleukin 3 and gamma-interferon by an antigen-specific mouse suppressor T cell clone.

An interleukin 2 (IL 2)-dependent, keyhole limpet hemocyanin (KLH)-specific, mouse suppressor T cell clone, 3D10, was found to produce interleukin 3 (IL 3) and gamma-interferon (IFN-gamma) in response to T cell mitogens Con A and PHA. Different from KLH-specific suppressor factor (TsF) that was spontaneously released into the medium when cultured in IL 2-containing conditioned medium, the production of IL 3 and IFN-gamma was induced by mitogenic stimuli. IL 3, IFN-gamma, and TsF were separable by gel filtration through a Sephadex G-100 column, being recovered in fractions of m.w. 25,000 to 30,000, 45,000 to 50,000 and 60,000 to 70,000, respectively. On the other hand, minimum size of IL 3 and IFN-gamma were shown to be about 25,000 and 20,000, respectively, by determining the lymphokine activities contained in the extracts from slices of SDS gels. These results indicate that IFN-gamma was present as a homodimer or hetero-complex with another carrier protein(s), whereas IL 3 was present as a monomeric form. A highly positive correlation (a correlation coefficient r = 0.96) between the titers of IL 3 and IFN-gamma produced by seven subclones derived from 3D10 was obtained, suggesting that IL 3 and IFN-gamma are induced by a process with a common mechanism. 3D10 also produced IL 3 and IFN-gamma when cultured with its specific antigen, KLH, in the presence of antigen-presenting cells. When Con A-stimulated 3D10 cells were labeled with L-[35S]methionine, we found that at least three proteins, with m.w. of 35,000, 25,000, and 20,000, were specifically released into medium by the stimulation. The latter two may be IL 3 and IFN-gamma described above, respectively, because of the similarities in m.w.

Animals↗
PubMed1984

Caulobacter crescentus flagellar filament has a right-handed helical form.

Caulobacter crescentus flagellar filaments were examined for their shape and handedness. Contour length, wavelength and height of the helical filaments were 1.34 +/- 0.14 micron, 1.08 +/- 0.05 micron and 0.27 +/- 0.04 micron, respectively. Together with the value of the filament diameter, 14 +/- 1.5 nm, the parameters of the curvature (alpha) and twist (phi) were calculated as 3.9(%) for alpha and 0.026 (rad) for phi, which are similar to those of the curly I filament of Salmonella typhimurium. Dark-field light microscopic analysis revealed that the C. crescentus wild-type filament possesses a right-handed helical form. Given the result that C. crescentus cells normally swim forward, in the opposite direction to a polar flagellum, it is likely that C. crescentus swims by rotation of a right-handed curly shaped flagellum in a clockwise sense, whereas S. typhimurium and Escherichia coli swim by rotation of left-handed normal type flagella in a counterclockwise sense.

Biopolymers↗
PubMed1984

Alteration in penicillin-binding patterns during cell cycle of Caulobacter crescentus.

Alteration in penicillin-binding patterns during asymmetrical cell division in the cell cycle of Caulobacter crescentus was examined. Most penicillin-binding proteins (PBPs) were turned over slowly, and their penicillin-binding patterns did not alter appreciably during the cell cycle. One of the PBPs, PBP S2 in the soluble fraction, however, was turned over quickly and was detected only in swarmer cells, suggesting that PBP S2 is synthesized in swarmer cells and turned over and/or inactivated in the following stalked cell cycle.

Bacteria↗
PubMed1984

On flagellar formation in Caulobacter crescentus: novel flagellin synthesis in stub-forming non-motile mutants of C. crescentus.

Defective flagellar organella, stubs, and flagellin synthesis by L-[35S]methionine labeling of two non-motile mutants of C. crescentus CB15 were studied. CB15 wild type synthesized two flagellin species, flagellin A of molecular weight 26,000 and flagellin B of molecular weight 28,000. A DNA phage phi0CbK-resistant non-motile mutant, CB15 pdr-816 [Fukuda et al. (1981) J. Bacteriol. 145, 559-572] did not synthesize flagellin B but synthesized large amounts of flagellin A and a novel flagellin of smaller molecular weight 22,000 (22K flagellin), and formed stubs composed of both flagellin A and 22K flagellin. The other mutant, CB15 fla-801 synthesized only 22K flagellin and formed very short stubs composed of 22K flagellin. Motile revertants were isolated from these mutants. Full revertants were isolated from CB15 fla-801 at a frequency of ca. 10(-7). However, only partial revertants swimming slowly were obtained from CB15 pdr-816 at a similar frequency. Such partial revertants synthesized only flagellin A and formed short flagella in spite of the fact that flagellin A was still synthesized at a higher level than in the wild type. Given the previous finding that flagellin B first polymerizes onto the hook followed by addition of flagellin A onto flagellin B filament [Koyasu et al. (1981) J. Mol. Biol. 153, 471-475], flagellin B seems very important in the formation of normal flagella in C. crescentus. A small amount of 22K flagellin was detected even in wild type cell. Neither conversion nor alteration was observed in flagellin A or B or 22K flagellin by short-pulse and pulse-chase experiments. It was thus suggested that 22K flagellin was not a degradation product formed from other flagellins but was of different origin from flagellins A and B. C. crescentus flagellins consist of several subspecies of different isoelectric points. 22K flagellin consisted of at least three subspecies, in two-dimensional gel, with more basic isoelectric points than other flagellins. Heterogeneity in isoelectric point of polymerized and nascent flagellins was similar, indicating that each subspecies of different isoelectric point can polymerize into filament, and that conversion between subspecies is not needed to form flagellar filament.

Bacterial Proteins↗
PubMed1984

Characterization of an antigen-specific suppressive factor derived from a cloned suppressor effector T cell line.

A cloned effector-type suppressor T cell line, 3D10, which is known to suppress the antibody response against dinitrophenylated keyhole limpet hemocyanin (KLH), produced a soluble KLH-specific factor (TsF) that can replace the function of parental T cell clones. High activity of TsF was released spontaneously into the culture supernatant when cultured in interleukin 2 (IL 2)-containing medium, requiring no antigenic stimulation. The culture supernatant of 3D10 was also capable of inhibiting the KLH-induced proliferative response of primed T cells in an antigen-specific manner. The direct target of TsF was found to be Lyt-1+2- T cells undergoing an early stage of antigen-specific proliferation. TsF was antigen binding but lacked any other serologic markers such as I-J and immunoglobulin heavy chain-linked allotypic determinants on T cells. No genetic restriction was found in its action on allogeneic T cells. The production of IL 2 in proliferative T cells by antigenic stimulation was not inhibited by TsF. These results indicate that the TsF described here is the legitimate mediator produced by the effector-type suppressor T cell that suppresses the antigen-specific responses of Lyt-1+2- T cells. The m.w. of TsF was approximately 75,000.

Animals↗
PubMed1981

Regulation of polar morphogenesis in Caulobacter crescentus.

Deoxyribonucleic acid (DNA) phage phi CbK-resistant nonmotile mutants of Caulobacter crescentus CB15 were examined for their formation of polar surface structures (a stalk, a single flagellum, pili, and DNA phage receptors). These mutants were devoid of pili and DNA phage receptors and simultaneously defective either in both stalk formation and flagellar activity (stalk-defective type) or in the formation of normal flagella (flagella-defective type). DNA phage phi Cr30-mediated transductions revealed that stalk-defective mutants were of a single genetic type, whereas flagella-defective mutants were grouped into two different genetic types, I and II. To investigate how membrane proteins change in the above morphology mutants, cell envelopes pulse-labeled with L-[35S]methionine were analyzed by two-dimensional gel electrophoresis. No gross change of membrane proteins was observed in the stalk-defective mutant CB15 pdr-803, except a 49,000-molecular-weight (49K) protein which was found reduced. However, a 27K, two 28.5K, and a 70.5K protein were missing from the membrane of the flagella-defective type I mutant CB15 pdr-813. These proteins are most likely to be flagella-related protein, flagellins A and B, and hook protein, respectively. In another flagella-defective type II mutant, CB15 pdr-816, the 27K and two 28.5K proteins were similarly absent but the 70.5K protein was consistently present in the membrane. The synthesis of flagellin was next assayed radioimmunologically in the above 35S-labeled mutants. Stalk-defective CB15 pdr-803 synthesized flagellin normally, compared to the wild-type strain. Flagellins A (26K) and B (28K) formed multiple spots in isoelectric focusing. A 29K protein was also detected in the flagellin-specific radioactivity from the cytoplasm. Flagella-defective type I CB15 pdr-813 synthesized flagellin only at a basal level. Thus transcription or translation of flagellin appeared to be repressed in this mutant. Another flagella-defective type II strain, CB15 pdr-816, however, synthesized flagellin at an apparently enhanced rate compared with the wild type. Flagellin synthesized in CB15 pdr-816 was flagellin A and a smaller 22K flagellin. Flagellin B was not synthesized in the mutant. It then follows that flagellin B is not a precursor of flagellin A and the 22K flagellin. Flagella-defective type II CB15 pdr-816, without flagellin B, formed a stub structure with a hook attached to one end instead of normal flagella. In the wild-type membrane, flagellin B was the major flagellin, whereas flagellin A was major in the cytoplasm and the flagellar filament. It is suggested from these results that flagellin B is important in the assembly of normal flagella.

Bacteria↗
PubMed1980

The penicillin-binding proteins of Caulobacter crescentus.

The proteins that specifically bind penicillin G were studied with Caulobacter crescentus. This organism possessed at least 5 penicillin-binding proteins (PBPs): PBP 1A (132 K), PBP 1Bs (98 K), PBP 2 (77 K), PBP 3 (64 K), and PBP 4 (50 K). As expected from the unique morphology, the C. crescentus PBPs were different from those of other sources in molecular weight and localization: C. crescentus did not possess PBPs of low molecular weight. PBP 4 was found only in the outer membrane, while the other PBPs were present mostly in the inner membrane.

Bacteria↗