Isoproteins of myosin alkali light chain (LC) were co-expressed in cultured chicken cardiomyocytes and fibroblasts and their incorporation levels into myofibrils and stress fibers were compared among members of the LC isoform family. In order to distinguish each isoform from the other, cDNAs of LC isoforms were tagged with different epitopes. Expressed LCs were detected with antibodies to the tags and their distribution was analyzed by confocal microscopy. In cardiomyocytes, the incorporation level of LC into myofibrils was shown to increase in the order from nonmuscle isoform (LC3nm), to slow skeletal muscle isoform (LC1sa), to slow skeletal/ventricular muscle isoform (LC1sb), and to fast skeletal muscle isoforms (LC1f and LC3f). Thus, the hierarchal order of the LC affinity for the cardiac myosin heavy chain (MHC) is identical to that obtained in the rat (Komiyama et al., 1996. J. Cell Sci., 109: 2089-2099), suggesting that this order may be common for taxonomic animal classes. In fibroblasts, the affinity of LC for the nonmuscle MHC in stress fibers was found to increase in the order from LC3nm, to LC1sb, to LC1sa, and to LC1f and LC3f. This order for the nonmuscle MHC is partly different from that for the cardiac MHC. This indicates that the order of the affinity of LC isoproteins for MHC varies depending on the MHC isoform. Further, for both the cardiac and nonmuscle MHCs, the fast skeletal muscle LCs exhibited the highest affinity. This suggests that the fast skeletal muscle LCs may be evolved isoforms possessing the ability to associate tightly with a variety of MHC isoforms.

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Expression of novel NP95 (nuclear protein, 95 kDa), which contains a leucine zipper motif, a zinc finger motif, a putative cyclin A/E-cyclin-dependent protein kinase 2 phosphorylation site, and retinoblastoma protein-binding motifs, is associated with S-phase progression of mouse cells. It is suppressed during G1 and G2/M phases in normal thymocytes but expressed at a constantly high level irrespective of cell stage in mouse T cell lymphoma cells. NP95 was shown previously to be expressed strongly only in proliferative tissues and cells. In this immunohistochemical study, we demonstrate that NP95 is localized in S-phase nuclei as dot-like foci. Double immunostaining of NP95 and proliferating cell nuclear antigen (PCNA) showed that NP95 was co-localized with PCNA. Construction of three-dimensional images indicated that NP95 was localized with PCNA in replication sites in a somewhat distinct temporal manner. During meiosis, NP95 was present not only in proliferating spermatogonia but also in meiotic spermatocytes and differentiating spermatids which were not proliferating. The possible role of NP95 in mitotic and meiotic cells is discussed.

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Through observations of colloidal gold with silver enhancement, we have demonstrated that 2-nm colloidal gold labeled-testosterone-bovine serum albumin (BSA) conjugate or hydrocortisone-BSA conjugate injected intravenously enters the hormone-target cell nuclei of rats (Nishimura and Ichihara, 1997; Nishimura and Nakano, 1997, 1999). To confirm immunocytochemically whether the nature of BSA in the steroid hormone-BSA conjugates (steroid-BSAs) remains intact in the hormone-target cell nuclei, testosterone-BSA, hydrocortisone-BSA or corticosterone-BSA was injected into the vascular system of rats, then the liver and testes of rats killed 2 h postinjection were reacted with FITC-conjugated anti-BSA antibody, and examined under fluorescence microscopy and confocal laser scanning microscopy. In the liver of rat injected with testosterone-BSA, the fluorescence was observed in the nuclei of endothelial cells, but not in the nuclei of hepatocytes, hepatic stellate cells and Kupffer cells. In the liver of rat injected with hydrocortisone-BSA, intense fluorescence was seen in the nuclei of hepatic stellate cells, but did not seem to be present in the nuclei of the other three kinds of cells. In the liver of rat injected with corticosterone-BSA, the fluorescence seemed to be in a few nuclei of hepatic stellate cells, and appeared as speckles in a few nuclei of the hepatocytes and Kupffer cells. In some seminiferous tubules of rat injected with testosterone-BSA, fluorescence was observed in the nuclei of spermatocytes and spermatids. These results suggest that BSA conjugated with steroid hormone can enter the hormone-target cell nuclei with its antigenicity kept intact, and that the fate of steroid-BSAs is decided at the cell membrane level.

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Cytokeratin 8 (CK8) and cytokeratin 19 (CK19) is a specific cytoskeletal component of simple epithelia, including bronchial epithelial cells. We hypothesized that CK8 or CK19 released from epithelial cells may bind to and cause damage to extracellular matrixes through binding of anti-CK8 or anti-CK19 autoantibodies. In the present study, bindings of recombinant human CK8 and CK19 to laminin (both derived from mouse sarcoma cells and human), collagen, gelatin, and fibronectin were evaluated by a modified enzyme-linked immunosorbent assay (ELISA). In addition, binding of CK19 to laminin was also confirmed by inhibition assay. As a result, CK19 strongly bound to mouse laminin as well as human laminin. Pretreatment with laminin significantly reduced the binding of CK19 to laminin. However, binding of recombinant CK19 to laminin was not demonstrated by Western immunoblot, suggesting that SDS treatment of laminin diminished the binding. These results suggest that released CK19 from epithelial cells may have played a role in the damage of basement membrane by accumulation of an immune complex composed by CK19 and anti-CK19 autoantibody.

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The developmental relationship of myosin binding proteins (myomesin, connectin and C-protein) to myosin was studied in chicken cervical somites by immunofluorescence microscopy. Muscle and non-muscle myosins initially appeared as slender rods at the same sites, and then, fused to form non-striated fibrils. As muscle myosin formed striated structures (A bands), non-muscle myosin disappeared from this structure. Myomesin (reactive with monoclonal antibodies MyB4 and MyBB78) and connectin (carboxy terminal region, reactive with monoclonal antibody T51) were seen as dots in the center of these myosin rods. These proteins then formed characteristic mature striations on non-striated fibrils of myosin. Earlier alignment of these myosin binding proteins rather than myosin indicates that the correct assembly of these proteins seems to be related to the formation of initial myosin rods as well as subsequent linear and periodic alignment of myosin molecules to form early A bands. Connectin spots reactive with 9D10 were scattered around myosin rods/myomesin dots/connectin T51 dots. These spots may represent radiating connectin filaments from these rods/dots to link myosin rods to the I-Z-I structures of myofibrils to be incorporated. Since the slow isoform of C-protein formed its characteristic bands (“doublets”) prior to H zone formation within A bands by myosin, this isoform may help to precisely align myosin filaments within the A band region. The presence of the slow, then the slow and the cardiac, and finally the co-existence of the slow and the fast isoforms of C-protein may interfere with the incorporation and co-polymerization of non-adult isoforms into myofibrils.

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Oxygen-free radicals are thought to be a major cause of β-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H₂O₂ on cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and the activity of ATP-sensitive potassium (K⁺_ATP) channels in isolated rat pancreatic β-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H₂O₂ in the presence of 2.8 mM glucose increased [Ca²⁺]_i from 114.3±15.4 nM to 531.1±71.9 nM (n=6) and also increased frequency of K⁺_ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H₂O₂ inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H₂O₂, followed by a transient increase in [Ca²⁺]_i, the transient inhibition of K⁺_ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca²⁺, 0.1 mM H₂O₂ increased [Ca²⁺]_i from 88.8±7.2 nM to 134.6±8.3 nM. Magnitude of [Ca²⁺]_i increase induced by 0.1 mM H₂O ₂ was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca²⁺ pump (45.8±4.9 nM vs 15.0±4.8 nM). Small increase in [Ca²⁺]_i in response to an increase of external Ca²⁺ from zero to 2 mM was further facilitated by 0.1 mM H₂O₂ (330.5 ±122.7 nM). We concluded that H₂O₂ not only activates K⁺_ATP channels in association with metabolic inhibition, but also increases partly the Ca²⁺ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic β-cells.

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Clonal cell line ATDC5 enables the monitoring of the early- and late-phase chondrogenic differentiation in a single culture. Undifferentiated ATDC5 cells differentiate into type II collagen expressing chondrocytes through a cellular condensation stage (early-phase differentiation) and then to type X collagen-expressing hypertrophic chondrocytes (late-phase differentiation). Progression of cellular differentiation was accelerated by the activation of bone morphogenetic protein (BMP) signaling. ATDC5 cells expressed transcripts for at least four members of the BMP family. The BMP-4 transcripts were expressed in all stages of differentiation, as were transcripts for BMP type IA receptor (ALK-3) and BMP type II receptor. In contrast, transcripts for Growth/Differentiation factor-5 (GDF-5) were induced during a cellular condensation, and those for BMP-6 were induced during the formation of cartilage nodules, and declined as the differentiated ATDC5 cells became hypertrophic, and BMP-7 transcripts were only detected after cells became calcified. Exogenously added BMP-4 indeed promoted the early-phase differentiation. Late-phase differentiation of cells was also stimulated by BMP-4 and BMP-6. Thus, the cumulative increase in BMP signaling promoted the sequential transitions of differentiation steps of cells. These results indicate that the coordinated expressions of endogenous BMPs are involved in the progression of chondrogenic differentiation in ATDC5 cells.

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