Follistatin as an activin-binding protein expressed in arteriosclerotic lesions.
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Activins↗
Biomedical subjects
T Hosoi
Publications and source records attributed to T Hosoi.
Porcine brain neurofilament-H tail domain kinase: its identification as cdk5/p26 complex and comparison with cdc2/cyclin B kinase.
Using dephosphorylated neurofilament (NF) proteins as substrates, the kinase with a higher activity for the dephosphorylated NF-H than the phosphorylated form of NF-H was searched for in the porcine brain extract. Most NF-H kinase activity in the brain extract pelleted with microtubules. The NF-H kinase purified from a high salt extract of the microtubule pellets was composed of cdk5 and a 26 kDa protein, a fragment of the 35 kDa regulatory subunit of cdk5. In contrast to the association of the active kinase with microtubules, each of uncomplexed cdk5 and the 35 kDa regulatory subunit was differently distributed in the supernatant fraction and the pellet, respectively, by ultracentrifugation of the brain extract. Dephosphorylated forms of NF-H and NF-M became reactive to antibodies recognizing in vivo phosphorylation sites (SMI31, 34, and 36, JJ31 and 51) by phosphorylation with cdk5/p26. cdk5/p26 showed similar enzymatic properties to p34cdc2/cyclin B kinase; the substrate specificity and inhibition by a p34cdc2 kinase specific inhibitor, butyrolactone I. However, p34cdc2/cyclin B kinase was distinguished from cdk5/p26 by its binding to p13suc1 protein and by its reactivity to anti-p34cdc2 antibodies. In spite of similar enzymatic properties of cdk5/p26 and p34cdc2/cyclin B kinase, cdk5/p26 did not display M-phase promoting activity when assayed with a cell-free system of Xenopus egg extract.
4-Butyrolactone↗
Evidence for cdk5 as a major activity phosphorylating tau protein in porcine brain extract.
The major kinase capable of phosphorylating tau in a porcine brain extract was suggested to be a brain cdc2-like kinase, called cdk5. Tau protein components of microtubules assembled in the brain extract using ATP were phosphorylated to a higher level, and showed a slower electrophoretic mobility than those assembled with GTP. Most of this phosphorylation and electrophoretic mobility shift, that occurred in the brain extract incubated with ATP, were inhibited by butyrolactone I, a specific inhibitor of cdc2 kinase and cdk5. Further, butyrolactone I inhibited phosphorylation of tau exogenously added to the brain extract by approximately 70%. cdk5 purified from porcine brain decreased the electrophoretic mobility of dephosphorylated tau by in vitro phosphorylation of tau to the level present in microtubules polymerized with ATP. cdc2 kinase purified from starfish oocytes also phosphorylated tau and shifted its electrophoretic mobility to an extent greater than that obtained with cdk5. Western blot analysis showed that cdc2 kinase phosphorylated epitopes recognized by SMI31, 33, 34, and tau 1 antibodies in tau proteins , while cdk5 phosphorylated the site recognized by SMI33 (corresponding to phosphorylation at Ser235 in the longest human tau isoform) and partially phosphorylated the tau 1 site. Phosphorylation experiments performed on tau in brain extracts, in the presence of okadaic acid, suggested the presence of both okadaic acid-sensitive and -insensitive phosphatases acting on phosphorylated Ser235. Rat tau that was prepared immediately after decapitation showed a similar phosphorylation state to tau in microtubules polymerized with ATP, suggesting that tau is relatively phosphorylated in vivo.
4-Butyrolactone↗
Expression of immunoreactive and bioactive activin A protein in adult murine lung after bleomycin treatment.
Activin A is a homodimeric protein structurally and functionally related to transforming growth factor beta (TGF-beta), and the expression of activin A is modulated by TGF-beta. Here, we demonstrate the expression of activin A in normal and bleomycin (BLM)-treated murine lungs. ICR mice were treated with BLM intraperitoneally for 10 days, whereas saline vehicle was injected into control mice. Intra-alveolar fibrotic changes were observed in the lung tissue obtained from the mice at day 14 after the final BLM administration. Immunohistochemical studies using a polyclonal antibody to activin A revealed the presence of activin A in the bronchiolar epithelium and smooth muscle cells of veins in both control and BLM-treated mice. In the BLM-treated mice at days 7 and 14, the marked infiltration of immunoreactive alveolar macrophages was observed in the area of fibrotic changes. Bioactivity of activin A measured by erythroid differentiation factor assay in the conditioned medium of alveolar macrophages obtained from BLM-treated mice at day 14 was significantly increased. These findings indicate that alveolar macrophages are a potent source of activin A after BLM treatment. The present study demonstrates for the first time the abundant expression of activin A in murine lung tissues after BLM administration, suggesting that activin A may play a role in the pathogenesis of BLM-induced pulmonary fibrosis.
Activins↗
[Serum concentration of vitamin K in elderly women with involutional osteoporosis].
Oral administration of vitamin K was reported to increase bone mineral density. However, the possible role of vitamin K in the pathogenesis of osteoporosis still remains unclear. Therefore, we measured the serum concentration of vitamin K1 and K2 (menaquinone-4, 7, 8) in 24 elderly women with osteoporotic vertebral compression fracture and in 36 elderly women without fracture. Major forms of vitamin K present in sera in this study were vitamin K1 and menaquinone-7. On the other hand, serum menaquinone-4 and -8 were undetectable in most women. Serum concentration of menaquinone-7 was significantly lower in women with fracture than in those without fracture (3.29 +/- 3.63 ng/ml vs 6.26 +/- 5.62, mean +/- SD, respectively), while no difference was found in serum vitamin K1 concentration (0.837 +/- 0.620 ng/ml vs 0.820 +/- 0.686, respectively). There was no difference between both groups in background data such as age, body height, body weight, and body mass index, as well as serum level of calcium, inorganic phosphate, creatinine, albumin, and alkaline phosphatase. These results suggest the possibility that deficiency of vitamin K, particularly that of menaquinone-7, is one of the risk factors for developing osteoporosis.
Aged↗
Localization of follistatin, an activin-binding protein, in bone tissues.
We have previously reported that activin-A has a mitogenic effect on osteoblast-like MC3T3-E1 cells and that these cells produce an activin-binding protein, follistatin (Hashimoto, M., Shoda, A., Inoue, S., Yamada, R., Kondo, T., Sakurai, T., Ueno. N., and Muramatsu, M. (1992) J. Biol. Chem. 267, 4999-5004). Activin-A was also detected abundantly in bone matrix tissues and was found to act as an osteoclast differentiation factor. Here, we studied the expression of follistatin in bone tissues by in situ hybridization with a follistatin cRNA probe. Follistatin transcript was detected in the osteoblasts and a fraction of osteocyte population in the developing mouse mandible. Immunohistochemistry using anti-follistatin antibody supported this observation at the protein level. Follistatin mRNA was also detected in the callus of repairing bone after fracture. These findings suggest that activin and follistatin may play significant roles in the bone system in vivo.
Animals↗
Vascular smooth muscle cells as target for estrogen.
ER mRNA was detected as 6.0 kb band by Northern blot analysis in vascular smooth muscle cells (VSMC) derived from rat aorta. The presence of ER mRNA in VSMC was confirmed by reverse transcriptase-polymerase chain reaction using specific primers for rat ER cDNA. In addition, the immunocytochemistry of ER was performed in VSMC using a monoclonal anti-ER antibody which recognizes DNA-binding domain of ER. The immunoreactivity was distributed in the cytoplasm as well as in the nuclei. Thus, the expression of ER in VSMC was demonstrated at both the protein and the mRNA level. Furthermore, the expression of c-fos mRNA in VSMC was found up-regulated by 17 beta-estradiol treatment within 30 min. The observation that VSMC possess ER and respond to estrogen supports the idea that estrogen may directly influence vascular cell system through the ER.
Animals↗
Vitamin K2 modulates proliferation and function of osteoblastic cells in vitro.
A human osteosarcoma cell line, HOS TE85 cells, and a mouse osteoblastic cell line, MC3T3-E1 cells, were cultured for 3 days in a medium containing various concentrations of menaquinone-4 (vitamin K2). As a result, the proliferation of HOS cells was suppressed by vitamin K2 in a dose dependent manner up to 56% of control by 10(-7)M of vitamin K2 and that of MC3T3-E1 cells was suppressed to 84% of control by 10(-6)M of vitamin K2. Vitamin K2 increased alkaline phosphatase activity in both kinds of cells. Warfarin counteracted the effect of vitamin K2 on osteoblastic cell proliferation. Our results show that vitamin K2 modulates proliferation and function of osteoblastic cells by some mechanisms including gamma-carboxylation system.
Animals↗
[The possibility of hereditary factors in the susceptibility to diabetic nephropathy in NIDDM].
Diabetic nephropathy is the most important complication of diabetes, because it is a major cause of morbidity and mortality for diabetic subjects. Since not all subjects with diabetes are at risk of developing this complication, we conducted a study to determine if heredity might be a possible risk factor for diabetic nephropathy in non-insulin dependent diabetes. Twenty-one factors including inheritance of nephropathy and hypertension were investigated in 109 individuals with NIDDM: 50 patients without proteinuria (Group I), 20 patients with intermittent proteinuria (Group II), and 39 patients with continuous proteinuria (Group III) matched for age and duration of diabetes. Of those patients, 55 patients with inheritance of diabetes were also divided into three groups: 29 patients without proteinuria (Group I), 9 patients with intermittent proteinuria (Group II), and 17 patients with continuous proteinuria (Group III). Individuals in Groups II and III has significantly higher frequency of inheritance of diabetic nephropathy than those in Group I, and also individuals with inheritance of diabetic nephropathy had significantly higher frequency of diabetic nephropathy than those without it. Frequency of hypertension, retinopathy and body mass index in the past were significantly higher in subjects in Groups II or Group III than in those in Group I. There were no significant differences between subjects in Groups II and III. These findings suggest that susceptibility to diabetic nephropathy in NIDDM may be hereditary, although hypertension and obesity may also be important risk factors for diabetic nephropathy.
Aged↗
Characteristics of steroid hormone receptors in cultured MC3T3-E1 osteoblastic cells and effect of steroid hormones on cell proliferation.
We examined the binding characteristics of three kinds of steroid hormones--estrogen, androgen, and glucocorticoid--in cultured MC3T3-E1 mouse osteoblastic cells by whole-cell binding assay. The binding studies revealed the presence of a single class of high-affinity binding sites for [3H]17 beta-estradiol, [3H]mibolerone (a synthetic androgen), and [3H]triamcinolone acetonide (a synthetic glucocorticoid). The numbers of binding sites for these steroid hormones were found to be 4534 +/- 819, 14312 +/- 1884, and 24898 +/- 655 sites/cell; and the Kd values were 8.57 +/- 0.62 x 10(-10) M, 1.12 +/- 0.19 x 10(-9) M, and 6.08 +/- 1.24 x 10(-10) M, respectively. We also examined the effects of steroid hormones on the proliferation of MC3T3-E1 cells. 17 beta-estradiol significantly stimulated the proliferation of the cells (130-150% of control). Dihydrotestosterone also significantly stimulated the proliferation of the cells (115% of control); the effect was, however, much less potent than that of 17 beta-estradiol, although the number of binding sites was approximately three times more than that of 17 beta estradiol. Triamcinolone acetonide and dexamethasone had no effect on cell proliferation. These results suggest that estrogen and androgen act directly on osteoblastic cells through a receptor-mediated mechanism, and that androgen is much less potent than estrogen in stimulating the proliferation of MC3T3-E1 osteoblastic cells.
Animals↗
Effect of parathyroid hormone on release of interleukin 1 and interleukin 6 from cultured mouse osteoblastic cells.
This study was undertaken to examine the possibility that parathyroid hormone promotes the production of interleukin 1 and 6 in MC3T3-E1 osteoblastic cells derived from mouse calvaria. The cells were incubated in serum-free medium with or without synthetic human parathyroid hormone(1-34). The concentrations of interleukin 1 and 6 in the culture medium were determined by using specific bioassay. The cells cultured without parathyroid hormone for 24 hr released both of interleukins, and parathyroid hormone stimulated the release in a dose-dependent manner. When the cells were cultured with 10(-6) M parathyroid hormone, the release of both interleukins from the cells remained higher than control up to 144 hr. These results suggest that interleukin 1 and 6 release stimulated by parathyroid hormone may be involved in the bone resorbing activity of the hormone.
Animals↗
Photoaffinity labeling of the erythropoietin receptor and its identification in a ligand-free form.
Pure human recombinant erythropoietin (EP) was acylated through a primary amino residue with a cross-linking reagent, N-[[3-[[4-[(p-azido-m-[125I]iodophenyl)azo]benzoyl]amino] propanoyl]oxy]-succinimide (Denny-Jaffe reagent), which is photoreactive and cleavable at the azo residue. The resulting conjugated hormone (DJ-EP) was purified from unmodified EP by reverse-phase high-pressure liquid chromatography and maintained its capacity to bind to receptors for EP on erythroid progenitor cells. The receptor for EP was previously identified as two related proteins of 100 and 85 kDa molecular mass by chemical cross-linking to 125I-EP. Recently, D'Andrea and co-workers [(1989) Cell 57, 277-285] cloned a cDNA that codes for a protein of 55-66 kDa, which is thought to be the EP receptor. In this report, cross-linking to the receptor through the monofunctional DJ-EP labeled the same 140- and 125-kDa molecular mass bands (100- and 85-kDa proteins) cross-linked with 125I-EP and disuccinimidyl suberate. Furthermore, cleavage of the azo bond of the DJ-EP receptor complex by sodium dithionite (80 degrees C, 5 min) demonstrated that proteins of 105 and 90 kDa were labeled in ligand-free form by DJ-EP. This result demonstrates that artifactual cross-linking of multiple proteins or other artifacts of cross-linking do not explain the difference in molecular mass of the EP receptor identified by cross-linking and the receptor identified by expression cloning.
Acylation↗
Binding studies on the erythropoietin receptor.
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Animals↗
[Summary of the Educational Seminar at the 38th Congress of Japan Society of Blood Transfusion].
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Acquired Immunodeficiency Syndrome↗
[Emergency blood transfusion and massive blood transfusion].
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ABO Blood-Group System↗
Integration of Agrobacterium T-DNA into a tobacco chromosome: possible involvement of DNA homology between T-DNA and plant DNA.
We established tobacco tumour cell lines from crown galls induced by Agrobacterium. Restriction fragments containing T-DNA/plant DNA junctions were cloned from one of the cell lines, which has a single copy of the T-DNA in a unique region of its genome. We also isolated a DNA fragment that contained the integration target site from nontransformed tobacco cells. Nucleotide sequence analyses showed that the right and left breakpoints of the T-DNA mapped ca. 7.3 kb internal to the right 25 bp border and ca. 350 bp internal to the left border respectively. When the nucleotide sequences around these breakpoints were compared with the sequence of the target, significant homology was seen between the region adjacent to the integration target site and both external regions of the T-DNA breakpoints. In addition, a short stretch of plant DNA in the vicinity of the integration site was deleted. This deletion seems to have been promoted by homologous recombination between short repeated sequences that were present on both sides of the deleted stretch. Minor rearrangements, which included base substitutions, insertions and deletions, also took place around the integration site in the plant DNA. These results, together with previously reported results showing that in some cases sequences homologous to those in T-DNA are present in plant DNA regions adjacent to left recombinational junctions, indicate that sequence homology between the incoming T-DNA and the plant chromosomal DNA has an important function in T-DNA integration.(ABSTRACT TRUNCATED AT 250 WORDS)
Base Sequence↗
Structural analysis of the receptors for granulocyte colony-stimulating factor on neutrophils.
We investigated granulocyte colony-stimulating factor (G-CSF) receptors on neutrophils from three patients with chronic myelogenous leukemia (CML) in the chronic phase, in comparison with four normal volunteers. Because we experienced some difficulties in radioiodinating intact recombinant human G-CSF, we developed a new derivative of human G-CSF termed YPY-G-CSF. It was easy to iodinate this protein using the lactoperoxidase method because of two additional tyrosine residues, and its radioactivity was higher than that previously reported. The biological activity of YPY-G-CSF as G-CSF was fully retained. Scatchard analysis demonstrated that CML neutrophils had a single class of binding sites (1400 +/- 685/cell) with a dissociation constant (Kd) of 245 +/- 66 pM. The number of sites and Kd value of CML neutrophils were not significantly different from those of normal neutrophils (p greater than 0.9). Cross-linking studies revealed two specifically labeled bands of [125I]YPY-G-CSF-receptor complexes with apparent molecular masses of 160 and 110 kd on both normal and CML neutrophils. This is the first report describing two receptor proteins on neutrophils. According to the analyses of the proteolytic process of these cross-linked complexes and proteolytic mapping, we assume that alternative splicing or processing from a single gene may generate two distinct receptor proteins that bind specifically to G-CSF but have different fates in intracellular metabolism.
Adult↗