Saburo Kakuta

Relationship between parathyroid hormone and adenosine 3′,5′-monophosphate metabolism in the kidney of vitamin D-deficient rats

Abstract The effect of vitamin D administration on cyclic AMP metabolism in the kidney was examined in rats fed a vitamin D-deficient, low Ca diet. The renal cyclic AMP level in vitamin D-deficient rats was higher than that in normal rats fed a laboratory chow, and in significantly decreased after thyroparathyroidectomy. Parathyroid hormone administered in vitro and in vivo did not cause as great a cyclic AMP response in vitamin D-deficient rats as that seen in the normal rats. The response to calcitonin, however, was not blunted in vitamin D-deficient animals. The blunted cyclic AMP accumulation in the kidney seemed to be related to formation, rather than degradation, of the nucleotide. The rats fed the low Ca diet were still hypocalcemic even after supplementation of the diet with a daily dose of either 0.625 μg of vitamin D-3 for 3 weeks or 2.5 μg of vitamin D-3 for the last 3 days. Vitamin D supplementation did not influence either the basal level or parathyroid hormone-stimulated increase of cyclic AMP in the kidney. On the contrary, when animals maintained on the vitamin D-deficient, low Ca diet were switched to the vitamin D-deficient, high Ca diet containing lactose for several days, they recovered normocalcemia and a normal response. These results suggest that the blunted cyclic AMP response to parathyroid hormone in vitamin D deficiency is due to hypocalcemia or associated secondary hyperparathyroidism and not due to deficiency of vitamin D action.

Localization of types I, II and X collagen in the transplanted tumor derived from human osteogenic sarcoma

We have succeeded in transplanting human osteogenic sarcoma of the mandible into nude mice. As the transplanted tumor shows features of calcified chondrosarcoma, this tumor is thought to be an excellent model for study of the process of dystrophic endochondral calcification. Using this model, we studied relations between the expression of types I, II, and X collagen and chondrogenic differentiation of the transplanted tumor. Collagen distribution during the development and growth of the transplanted tumor was investigated by immunofluorescence with specific antibodies against type I, II or X collagen. Type X collagen was intensely stained in the mineralized region. Almost all tumor cells in this region were hypertrophic. Type II collagen was chiefly distributed in the unmineralized region where tumor cells showed chondrocytic or hypertrophic feature. These results indicate that the type of collagen changes from type II to type X in the hypertrophic region and the type X collagen may be synthesized by hypertrophic tumor cells. Type I collagen was localized in the marginal region of the tumor, though it disappeared in the mineralized region.

Expression of collagen species in a cartilaginous tumor derived from a human osteogenic sarcoma

We have succeeded in transplanting human osteogenic sarcoma into nude mice. Morphologically, the transplanted tumor is chondrosarcoma and manifests calcification, but not ossification. This tumor is thought to be an excellent model for studying the process of morbid endochondral calcification. In this study, we have used in situ hybridization to examine expression of collagen type I, II, and III mRNAs in this tumor. In situ hybridization was carried out using biotinylated DNA probes. Hybridized probes were detected using a streptavidin-biotin-alkaline phosphatease reagent. The results showed that collagen type I and II mRNAs were produced by cells of the transplanted tumor. Collagen type I mRNA was chiefly localized in the marginal region of the tumor. Collagen type II mRNA, which was predominantly found in the premineralized region of the transplanted tumor, gradually decreased toward the mineralized region. Collagen type III mRNA was not expressed in the transplanted tumor. These results suggest that the character of progenitor chondrogenic cells might be transferred to the transplanted tumor, and that the tumor cells may change the expression of collagen genes with the differentiation or maturation.

Proliferation and Differentiation of Bone Marrow Cells on Titanium Plates Treated With a Wire-type Electrical Discharge Machine

For successful dental implants, it is necessary to obtain satisfactory osteointegration at the site of both the cortical and trabecular bones in the jaw. Bone marrow stromal cells differentiate into osteoblast-lineage cells and have an important role in bone remodeling. In this experiment, the responsiveness of bone marrow cells to a titanium plate with a rough surface was compared with that of a titanium plate with a smooth surface. The rough surface was created by treating with a wire-type electrical discharge machine, and the smooth plate was produced by polishing with 1.500-grade emery paper. The results indicated that, though bone marrow cells proliferated on both plates, the proliferation pattern and cell growing time on the plates were different. While the cells on the smooth plate proliferated along the grooves produced by polishing, the cells on the rough plate proliferated randomly and more rapidly. As bone marrow cells consisted of heterogeneous cell populations involving hematopoietic cells, we collected bone marrow mesenchymal stromal cells that proliferated on plastic dishes and studied the proliferation and differentiation of these cells. Stromal cells on the rough plate more actively proliferated than those on the smooth plate. In long-term culture, the cells on the rough plate showed higher alkaline phosphatase activity and produced cell nodules. The cells on the smooth plate were stripped off the plate without nodule formation. These results indicated that bone marrow stromal cells on the rough plate could more rapidly proliferate and differentiate into osteoblast-lineage cells compared with those on the smooth plate.

Isolation of matrix vesicles by isoelectric focusing in Pevikon-Sephadex

We have investigated the use of an isoelectric focusing (IEF) technique for isolating and characterizing matrix vesicles. Focusing was performed on crude preparations of matrix vesicles isolated from collagenase digests of chick epiphyseal cartilage and purified by discontinuous sucrose gradient centrifugation. Crude and partially purified vesicle preparations were subjected to flat bed IEF in a slurry of Pevikon-Sephadex. Partially purified matrix vesicles focused as a narrow band (pI congruent to to 6.5). Alkaline phosphatase, solubilized from matrix vesicles, focused with a pl of 4.0-4.5. The IEF profile of matrix vesicles also differed from that of chondrocyte membranes. Thus, the membrane pls were congruent to to 5.4 and 6.6-7.8, respectively. The latter peak probably corresponded to the pl of the matrix vesicle preparation. This observation lends support to the view that vesicles originate from distinct regions of the chondrocyte membrane.

Clinical trial of recombinant granulocyte colony-stimulating factor for chemotherapy-induced neutropenia in patients with oral cancer

PURPOSE This study was undertaken to evaluate the efficacy and toxicity of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in reducing neutropenia in patients with oral cancer undergoing intensive chemotherapy. MATERIALS AND METHODS Patients with chemotherapy-induced neutropenia (< 1 x 10(9)/L) were divided into two groups: control group (n = 13) and rhG-CSF administration group (n = 16). rhG-CSF was administered subcutaneously at a dose of 75 micrograms/day on consecutive days. Peripheral blood cell counts and oral complications were investigated in each group. RESULTS The duration of neutropenia and absolute neutrophil nadir counts were significantly improved by administration of G-CSF. No consistent effect on thrombocytopenia was noted. Administration of rhG-CSF also reduced the duration and degree of oral complications associated with chemotherapy-induced neutropenia. Intolerable side effects associated with administration of rhG-CSF were not observed. CONCLUSION It was concluded that rhG-CSF is effective in shortening the duration of neutropenia after chemotherapy at a dose of 75 micrograms/day.

Age-related increases in LPS-stimulated nitric oxide production from cultured rat bone marrow cells

OBJECTIVE To understand bone metabolism during senescence, we examined age-related change in nitric oxide (NO) production from bone marrow cells stimulated by lipopolysaccharide (LPS). METHODS We evaluated the age-related change in the NO production and expression of iNOS protein and mRNA of LPS-stimulated bone marrow cells collected from the tibiae of young and retired female and young and retired male rats. In addition, we used flow cytometry to assess changes in the distribution of CD14, a cell surface receptor of LPS. RESULTS The results revealed that NO production from bone marrow cells stimulated with LPS changed with aging. The NO levels in old rats were significantly higher (P<0.05) than those in young rats. Polymerase chain reaction (PCR) analysis indicated that the LPS-induced expression of iNOS mRNA was augmented in retired rats. Although the distribution pattern of the bone marrow cells was similar between young and retired rats, the percentage of CD14-positive cells in specific populations differed between the age groups. Specifically, in the granule-containing bone marrow cells, the percentage of CD14-positive cells was increased in retired rats. CONCLUSION Our results indicate that LPS-stimulated NO production from rat bone marrow cells increased with age and that the difference in responsiveness might be due to changes in the percentage of CD14-positive cells in the bone marrow.