The osteogenic capability of cells derived from the bone marrow of young (4 week old) or adult (10 month old) male Wistar rats was investigated in vitro. The proliferation ratio of osteogenic cells from the young and adult animals increased until 9 days of culture, and then rapidly de creased. Alkaline phosphatase (ALP) activity in both types of cells increased in intensity from 5 days and peaked at ll days, and ALP activity in the young cells was significantly higher than in the adult cells at all time periods. Calcified nodules were first detected after 7 days in both types of cells. The Ca/P ratio of calcified nodules increased until 13 days in both types of cells, and was sig nificantly higher in the young cells than that in the adult cells at all time periods, except at 7 days. Connexin 43-immunoreactive spots were detected at cell-to-cell junctions at early times in the young cells, but in the adult cells, only a few connexin 43 immunoreactive spots were observed at early times, and these gradually increased at later times. These results suggest that cells derived from the bone marrow of young rats differentiate into osteogenic cells and synthesize calcified nodules at earlier time periods in vitro compared with cells derived from adult rats.
The temperature dependence of the initial burst in the NADH-dependent reduction of rat liver microsomal cytochrome b5 in sin: in the membrane was examined by means of stopped-flow spectrophotometry. The rate of cytochronte b5 reduction increased only moderately with increasing temperature, in contrast to the larger increase in a pure, delipidated reconstituted system in free solution. Treattnent with a low concentration (0.05%) of sodium deoxycholate, which perturbs the membrane integrity without causing disassembly, abolished the characteristically weak temperature dependence of cytochrome b5 reduction observed in intact microsomes. Despite the striking difference in the behavior of intact and deoxycholate-treated microsomes as regards ternperature dependence, the microviscosity in these preparations was found to be essentially the same by means of fluorescence polarization studies. The microsomal NADH-ferricyanide reductase and NADH-cytochrome c reductase activities with a water-soluble electron acceptor showed a usual linear dependency on temperature, even in deoxycholate-treated microsomes. It is concluded that the microenvironment (membrane structure) of the microsomal enzyme system strongly influences the electron transfer from the reductase to cytoclnome b5. A model explaining the results is proposed and discussed.
Hepatocyte growth factor (HGF)/c-met system has been thought to play an important role in the epithelial-mesenchymal interaction during the initial phase of gastric ulcer healing. However, in chronic course of gastric ulcer healing, the role of this factor remains largely unknown. In this study, we examined temporal changes of HGF/c-met expression by immunohistochemistry and in situ hybridization in an experimental ulcer induced by the application of acetic acid to the rat stomach. The rat normal gastric mucosa expressed c-met but not HGF in the surface epithelium. The expression of c-met in regenerating epithelium was weak at the active stage, but it increased the strength with the healing of ulcer progressing. The expression of HGF in the granulation tissue was undetectable at the active stage, but it increased the strength in concert with the c-met expression during the ulcer healing. In addition, c-met expressing cells appeared in granulation tissues from the active stage and reached a peak number at the early healing stage, which preceded the appearance of HGF expressing cells. At the scarring stage, expressions of both c-met and HGF decreased the intensity to the same level as normal gastric mucosa. The results of this study suggested that HGF/c-met system may work for the differentiation and maturation of regenerating epithelium and granulation tissues during ulcer healing.
In this study, the interaction between the small heat shock protein Hsp27 and α/β tubulin heterodimer was investigated. First, immunoprecipitation analysis using recombinant Hsp27 and purified tubulin indicated that Hsp27 binds with α/β tubulin heterodimer. Next, the effect of Hsp27 on tubulin polymerization was examined. Hsp27 showed the inhibitory effect on dimethylsulfoxide (DMSO)-induced tubulin polymerization. The inhibitory effect was dose-dependent: about 30% inhibition of polymerization was found after addition of 0.85 mg/mL of Hsp27, 70% inhibition was found by 3.4 mg/mL of Hsp27.
The immunolocalization of periostin, previously termed osteoblast-specific factor 2, was investigated in developing long bones of 17-day-old fetal mice and of 1-, 2-, 3- and 8-week-old mice at the light and electron microscopic levels. Fetal femurs showed innnunoreactions for periostin in the periosteum, perichondrium, articular surface of the epiphyscal cartilage, joint ligaments, and fascias of surrounding muscles. In particular, intense immunoreactivity for periostin was found in the fibrous layer of the periosteum and perichondrium. At postnatal 1-and 2-weeks, in contrast, the immunoreactivity was restricted to the periosteum and thick fascias of surrounding muscles wl1e11 compared with the fetal bones. lmmunoclectron microscopic observation of the periosteum demonstrated immunoreaction products for periostin at the junction of periosteal fibroblasts and collagen bundles, suggesting its competence in the cell-to—matrix interaction. Mice at 3 and 8 weeks, unlike 2-week-old mice, showed a periostin-innnunoreaction dominantly in the osteoblastic layer but not in the fibroblastic layer of the periosteum. Furthermore, the perichondrium and fascias of surrounding muscles were devoid of innnunoreaction. Thus, periostin was confirmed to be widely distributed in bone and concomitant tissues at the fetal stage. As mice grew, however, its immunoreactivity gradually came to be restricted to the osteoblastic layer of the periosteum. Our findings suggest that periostin acts at the site of the cell-to-matrix interaction in periosteum, fascias, and joint ligament during morphogenesis of these tissues.
The effect of undecatungstophosphate (PW11), a type of inorganic polyanion, was studied in vivo. When PW11 was intravenously injected into a cat, strong metabolic acidosis with hypoxia was rapidly induced. Anti-platelet agents partially prevented such a toxic change, but heparin alone did not. PW11 appeared to have a conflicting effect on the haemostasis. Although PW11 had a heparinlike anticoagulant effect, it also induced platelet aggregation (both in vivo and in vitro) and pulmonary embolism. The constituents of PW11 (tungstate and phosphate) had ahnost no effect except for a hypoglycemic reaction induced by tungstate. Therefore, the toxicity of PW11 was considered to be not due to the characteristics of tungsten itself as a heavy metal, but seemed to be due to its unique molecular structure and/or high negative charge of the molecule. An as yet undefined interaction between PW11 and platelets may play an important role in its toxicity.