Visualization of the microbody division inCyanidioschyzon merolae with the fluorochrome brilliant sulfoflavin

Summary A novel procedure is described for fluorescence staining of microbodies, which can be applied quickly and easily. We developed this technique of microbody staining with the unicellular red algaCyanidioschyzon merolae. Cyanidioschyzon merolae only contains a single chloroplast, mitochondrion, and microbody per cell, and the mitotic cycle and the organelle division cycle are easily synchronized. Knowing that the concentration of H₂O₂ in the microbody is higher than it is in the cytosol and other cell components, we attempted to visualize the microbody by using fluorescence microscopy to detect H₂O₂. Brilliant sulfoflavin (BSF), used for detecting Fe²⁺ in analytical chemistry, fluoresces when it reacts with Fe²⁺ and H₂C₂. We were able to specifically stain microbodies with BSF, under acidic conditions (pH 3.0 or pH 2.5) with blue-light excitation. Using this procedure, we observed division of the microbody and the effect of aphidicolin on the microbody. We also discovered that microbody division is regulated by the cell nucleus and follows division of the cell nucleus.     

Identification of human ST2 protein in the sera of patients with autoimmune diseases.

Soluble human ST2 protein (IL1RL1-a) in the sera of patients with various autoimmune diseases was identified by a newly developed procedure using specific monoclonal antibodies. After immunoprecipitation and subsequent immunoblotting, a glycosylated protein of about 60 kDa was detected in the sera of SLE patients, but not in the sera of healthy controls. The experiments using gel filtration and SDS-PAGE under a nonreducing condition indicated the existence of the ST2 multimer in serum. The mobility of the natural protein was slower than that of the recombinant human ST2 protein produced by COS7 cells in SDS-PAGE, suggesting a difference of glycosylation between humans and monkeys. The identification of the natural human ST2 protein should be important both to fundamental researches and the further clarification of the clinical implications of the ST2 protein.     

Substitutions for glycine alpha 1-637 and glycine alpha 2-694 of type I procollagen in lethal osteogenesis imperfecta. The conformational strain on the triple helix introduced by a glycine substitution can be transmitted along the helix

Two substitutions for glycine in the triple-helical domain were found in type I procollagen synthesized by skin fibroblasts from two probands with lethal osteogenesis imperfecta. One was a substitution of valine for glycine alpha 1-637, and the other was a substitution of arginine for glycine alpha 2-694. The effects of the mutations on the zipper-like folding of the collagen triple helix were similar, since there was post-translational overmodification of the collagenase A fragments (amino acids 1-775) but not of more COOH-terminal fragments of the protein. The mutations differed markedly, however, on their effects on thermal unfolding of the triple helix. The collagenase A fragment from the collagen containing the arginine alpha 2-694 substitution was cleaved at about amino acid 700 when incubated with trypsin at 30-35 degrees C. Therefore, there was micro-unfolding of the triple helix at a site close to the glycine substitution. Surprisingly, however, the collagenase A fragment with the valine alpha 1-637 substitution was also cleaved at about amino acid 700 under the same conditions. The results, therefore, demonstrated that although most glycine substitutions delay folding of the triple helix in regions that are NH2-terminal to the site of the substitution, the effects on unfolding can be transmitted to regions that are COOH-terminal to the site of the glycine substitution.     

CD40 ligand-activated human monocytes amplify glomerular inflammatory responses through soluble and cell-to-cell contact-dependent mechanisms.

Monocytes/macrophages play a critical role in the initiation and progression of a variety of glomerulonephritides. We sought to define the interactions between physiologically activated human monocytes and glomerular mesangial cells (MC) by employing a cell culture system that permits the accurate assessment of the contribution of soluble factors and cell-to-cell contact. Human peripheral blood monocytes, primed with IFN-gamma and GM-CSF, were activated with CD40 ligand (CD40L) or TNF-alpha and cocultured with MC. CD40L-activated monocytes induced higher levels of IL-6, monocyte chemoattractant protein-1 (MCP-1) and ICAM-1 synthesis by MC. Separation of CD40L-activated monocytes from MC by a porous membrane decreased the mesangial synthesis of IL-6 by 80% and ICAM-1 by 45%, but had no effect on MCP-1. Neutralizing Abs against the beta 2 integrins, LFA-1 and Mac-1, decreased IL-6 production by 40 and 50%, respectively. Ligation of mesangial surface ICAM-1 directly enhanced IL-6, but not MCP-1, production. Simultaneous neutralization of soluble TNF-alpha and IL-1 beta decreased MCP-1 production by 55% in membrane-separated cocultures of MC/CD40L-activated monocytes. Paraformaldehyde-fixed CD40L-activated monocytes (to preserve membrane integrity but prevent secretory activity), cocultured with MC at various ratios, induced IL-6, MCP-1, and ICAM-1 synthesis by MC. Plasma membrane preparations from activated monocytes also induced mesangial IL-6 and MCP-1 synthesis. The addition of plasma membrane enhanced TNF-alpha-induced mesangial IL-6 production by approximately 4-fold. Together, these data suggest that the CD40/CD40L is essential for optimal effector function of monocytes, that CD40L-activated monocytes stimulate MC through both soluble factors and cell-to-cell contact mediated pathways, and that both pathways are essential for maximum stimulation of MC.     

Early changes in mitochondrial monoamine oxidase activity of rat liver during 2-acetylaminofluorene feeding

The activities of mitochondrial type A and B monoamine oxidase were determined in the liver of rats fed a diet containing 2-acetylaminofluorene (AAF). Three days after the initiation of AAF-feeding, there was a significant decrease of type B monoamine oxidase activity without affect on type A enzyme. The decreased activity of type B monoamine oxidase, which reached a minimum after three weeks, was sustained for as long as AAF-feeding was continued. Sex-related difference in response to AAF was seen in the rat with respect to the onset and the intensity of the decreased type B monoamine oxidase activity, male rats being more sensitive to the carcinogen than female rats. In contrast to the in vivo effect, AAF showed a potent inhibitory effect on type A monoamine oxidase, rather than on type B enzyme, when added in vitro. The pI₅₀ values were estimated to be 7.5 against type A monoamine oxidase and 4.1 against type B enzyme, respectively. The in vitro inhibition of both types of monoamine oxidase by AAF was competitive. The K_i values for AAF were calculated to be 9.51 · 10^?9 M for type A monoamine oxidase and 1.30 · 10^?5 M for type B enzyme, respectively. In accordance with the potent inhibitory effect of AAF on type A monoamine oxidase in vitro, a single administration of the carcinogen, at a dose of 50 mg/kg, resulted in a marked and temporal decrease of the enzyme activity in the mitochondria of male rat liver. Recovery of the decreased type B monoamine oxidase activity was slow, and the enzyme activity did not return to control levels, even if rats were fed the basal diet for 2 or 4 weeks after the cessation of AAF-feeding.     

Experimental Models for Prevention of Graft-versus-Host Reaction in Bone Marrow Transfusion

Induction and suppression of splenomegaly and cytotoxicity against C57BL/6 cells were studied in (AKR × C57BL/6) F1 hybrid adult mice after the transfer of AKR lymphoid and bone marrow cells. 1) Splenomegaly and cytotoxicity were dissociated in the developmental stages of the graft-versus-host reaction. When lymphoid and bone marrow cells of normal AKR mice were injected into F1 recipients, splenomegaly was prominent on days 5 and 7, but cytotoxicity of spleen cells was not detected. Splenomegaly became less prominent but the cytotoxicity became detectable on day 14 after the injection. 2) Cytotoxic activity of spleen cells of F1 recipients was suppressed by the treatment of AKR donors with C57BL/6 lymphoid cells in Freund's complete adjuvant. Splenomegaly, however, was substantially enhanced by such a treatment of the donors. On the other hand, induction of the cytotoxic activity was facilitated by the treatment of donors with C57BL/6 skin grafts. 3) F1 hybrid mice could be protected from the graft-versus-host reaction by the injection of AKR anti-C57BL/6 serum or pretreatment of AKR donors with sonicated cellular antigens of C57BL/6.     

Morphological study on the 'seams' in the multiroot formation of rat molar teeth.

'Seams' of the root furcation with multiroots in rat molar teeth, termed by Lester and Boyde, were investigated using transmitted light and scanning electron microscopy in the formation process. The seam was formed in the junctional line of Hertwig's epithelial root sheaths. The seam formation will be classified into three types in the initial stage based on the position of the epithelial root sheaths approaching each other: (1) the close junction or the very narrow slit or gap formed between them, (2) the clear gap containing mesenchymal cells and (3) the gap containing one blood vessel. When the roots were formed, the seam was formed as follows: (1) the slight ridge composed of the cellular cementum, (2) the proliferation or the depression in the dentine formation and (3) the accessory or lateral canal of the root. These structures were variously combined with each other into one seam, although the slight ridge was very common.     

Microbody proliferation and segregation cycle in the single-microbody alga Cyanidioschyzon merolae

The proliferation cycle of the microbody was studied in the primitive red alga Cyanidioschyzon merolae, which contains one microbody per cell. Cells were synchronized with a dark/light cycle, and the morphology of the microbody and its interaction with other organelles were observed three-dimensionally by fluorescence microscopy, transmission electron microscopy, and computer-assisted three-dimensional reconstruction of serial thin sections. The microbody in interphase cells is a sphere of 0.3 μm in diameter without a core. In M-phase, the microbody passes through a series of irregular shapes, in the order rod, worm, branched, H-shaped and dumbbell, and symmetric fission occurs just before cytokinesis. The microbody duplicates its volume in M-phase and three-dimensional quantitative analysis revealed that its surface area increases before its volume does. The microbody touches the mitochondrion and the chloroplast throughout its proliferation cycle, except briefly in interphase cells, winding around the divisional plane of the mitochondrion at one phase. Immunocytochemical labeling of catalase as a marker of matrix proteins of the microbody revealed that the duplication of catalase occurs in tandem with the volume increase. While no specific apparatus was identified in the microbody divisional areas, we identified an electron-dense apparatus about 30–50 nm in diameter between the microbody and the mitochondrion that may play a role in segregating the daughter microbodies. These results are the first characterization to show the morphological changes of one microbody in a one-microbody alga without proliferation-inducing substrates, which have been used in many studies, and clearly show that two daughter microbodies arise by binary fission of the pre-existing microbody. Received: 11 November 1998 / Accepted: 22 December 1998     

Investigation of telomere length dynamics in induced pluripotent stem cells using quantitative fluorescence in situ hybridization

Here we attempted to clarify telomere metabolism in parental cells and their derived clonal human induced pluripotent stem cells (iPSCs) at different passages using quantitative fluorescence in situ hybridization (Q-FISH). Our methodology involved estimation of the individual telomere lengths of chromosomal arms in individual cells within each clone in relation to telomere fluorescence units (TFUs) determined by Q-FISH. TFUs were very variable within the same metaphase spread and within the same cell. TFUs of the established iPSCs derived from human amnion (hAM933 iPSCs), expressed as mean values of the median TFUs of 20 karyotypes, were significantly longer than those of the parental cells, although the telomere extension rates varied quite significantly among the clones. Twenty metaphase spreads from hAM933 iPSCs demonstrated no chromosomal instability. The iPSCs established from fetal lung fibroblasts (MRC-5) did not exhibit telomere shortening and chromosomal instability as the number of passages increased. However, the telomeres of other iPSCs derived from MRC-5 became shorter as the number of passages increased, and one (5%) of 20 metaphase spreads showed chromosomal abnormalities including X trisomy at an early stage and all 20 showed abnormalities including X and 12 trisomies at the late stage.