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K Kusuzaki

Publications and source records attributed to K Kusuzaki.

At least 55 records · Page 3Linked to original sources
PubMed1991

Cloning and expression of the rfe-rff gene cluster of Escherichia coli.

We have cloned a 13 kb Escherichia coli DNA fragment which complemented the rfe mutation to recover the biosynthesis of E. coli O9 polysaccharide. Using Tn5 insertion inactivation, the rfe gene was localized at the 1.5 kb HindIII-EcoRI region flanking the rho gene. We constructed an rfe-deficient E. coli K-12 mutant by site-directed inactivation using a DNA fragment of the cloned 1.5 kb rfe gene. This also confirmed the presence of the rfe gene in the 1.5 kb region. By simultaneous introduction of both the rfe plasmid and the plasmid of our previously cloned E. coli O9 rfb into this rfe mutant, we succeeded in achieving in vivo reconstitution of O9 polysaccharide biosynthesis. From sequence analysis of the rfe gene, a putative promoter followed by an open reading frame (ORF) was identified downstream of the rho gene. This ORF coincided with the position of the rfe gene determined by Tn5 analysis and site-directed mutagenesis. Furthermore, we identified the rff genes in the 10.5 kb DNA flanking the rfe gene. We recognized at least two functional domains on this cloned rff region. Region I complemented a newly found K-12 rff mutant, A238, to synthesize the enterobacterial common antigen (ECA). Deletion of region II resulted in the synthesis of ECAs with shorter sugar chains. When the 10.5 kb rff genes of the plasmid were inactivated by either deletion or Tn5 insertion, the plasmid lost its ability to give rise to transformants of the rfe mutants.

Amino Acid Sequence↗
PubMed1991

[Staining of bone canaliculi using decalcified bone tissues].

Recently we were able to stain bone canaliculi using the decalcified bone tissues, according to a modified method of the Bodian staining method, which is commonly used for staining the nerve fibers with silver impregnation of protargol. The bone tissues from normal rabbits, rats, and humans were decalcified with buffered EDTA solution after 10% formalin fixation. The tissues were embedded in paraffin and were sliced at 4 microns then the sections were attached to the gelatin-coated glass slides. These sections were treated first with potassium dichromate followed by 2% protargol solution containing copper. After gold chrolide treatment, they were immersed in oxalic acid. Both the lacunae and canaliculi of osteocytes were stained black in sharp contrast to bone matrix of red purple. Using the present staining method, the bone canaliculi were clearly detectable in fine details without producing apparent artificial damage of soft tissues. The canaliculi showed also markedly complex fine structures. By combining the present staining method with eosin or toluidine blue staining, we were able to recognize even immature osteocytes in enchondral ossification at growth cartilage with fine bone canaliculi. From these results, we conclude that the present method is very useful in histopathological studies of the lacuna-canalicular system of osteocytes in the decalcified bone tissues.

Animals↗
PubMed1991

[The distribution of S-100 protein positive chondrocytes in the human articular cartilages under aging or diseased conditions].

There have been few reports on the localization of S-100 protein positive chondrocytes in the human articular cartilages. We studied 59 articular cartilages of the aged subjects, 65 osteoarthritic (OA) and 39 rheumatoid arthritic (RA) articular cartilages, to detect the histological localization of S-100 protein using immunoperoxidase method (ABC). The results obtained from normal cartilages demonstrated strongly positive cells representing hypertrophic chondrocytes in the perivascular areas of the neonatal articular cartilage and in the deep zone of the infant articular cartilage. The moderately positive cells were found in the intermediate zone of infant and adult articular cartilages. In mild OA, there were many positive chondrocytes in the intermediate zone with erosion of the surface layer, while in moderate or severe OA many strongly positive cells were found in clusters. The hypertrophic cells in the metaplastic cartilage arising from bone marrow in subjects with severe OA, or from pannus after RA were also positive. It is therefore, suggested that S-100 protein may be correlated with the metabolic activity of the cartilage matrix such as collagen and proteoglycan, as reported in the literature. S-100 protein further, appears to be useful for evaluating histologically the activity of cartilage repair in the pathologic human articular cartilages.

Adolescent↗
PubMed1991

In vitro assay of nuclear uptake of doxorubicin hydrochloride in osteosarcoma cells of dogs.

A rapid, simple chemosensitivity assay, assessing tumor cell nuclear uptake of doxorubicin hydrochloride, was evaluated in 16 dogs with appendicular osteosarcoma. Doxorubicin was administered to dogs in 5 biweekly treatments, and surgical resection was performed after the second or third treatment. The chemosensitivity assay was performed on biopsy specimens from all dogs before chemotherapy. It was repeated on tissue from resected tumors, and tumors were evaluated histologically to determine the degree of necrosis resulting from chemotherapy. Disease-free and total survival time correlated significantly (P less than 0.05 in both cases) with the degree of postchemotherapy necrosis of the primary tumors. Significant correlation was not apparent between the percentage of tumor cells with nuclear uptake of doxorubicin (in either biopsy or resection samples) and disease-free or total survival time. The percentage of cells with nuclear uptake of doxorubicin in surgically resected tumors correlated significantly (P less than 0.05) with percentage of necrosis.

Animals↗
PubMed1989

Immunohistochemical localization of interleukin 1 in human growth cartilage.

Although it has been reported that interleukin 1 (IL-1) stimulate chondrocytes to produce collagenase and proteoglycanase in vitro, IL-1 producing cells and the function of IL-1 have not been demonstrated in osteocartilaginous tissue in vivo. Immunohistochemical studies of human cartilaginous epiphysis and growth cartilage demonstrated that IL-1 was detected in: (1) chondrocytes surrounding cartilage canal, (2) hypertrophic chondrocytes in cartilaginous epiphysis, (3) chondrocytes at the hypertrophic and calcified zones in the growth cartilage of actively growing bone. In contrast, few hypertrophic chondrocytes showed positive reactions to IL-1 in growth plates nearing physiologic closure. Furthermore, IL-1 was detected in chondrocytes cultured from human growth cartilage. These results show that IL-1 is produced by matured chondrocytes of human growth cartilage in vivo. Chondrocyte-derived IL-1 might play a key role in the hypertrophy of chondrocytes, in the vascularization of cartilage and in the formation of bone.

Adolescent↗
PubMed1986

[The relationship between cell kinetics and histological features of giant cell tumor of the bone].

This study was undertaken to clarify the relationship between the proliferative activity and histological findings of the giant cell tumor (GCT) of bone by means of an epi-illumination cytofluorometer (NIKON SPM-RF1-D). Fresh tissues of GCT were surgically obtained from two cases. In both cases, small pieces of tumor tissues were obtained from several different regions based on the macroscopic characteristics of the cut surface, and processed for single cell preparation using enzymatic method. These isolated cells were smeared and stained with acridine orange, and then analyzed cytofluorometrically to determine simultaneously DNA and RNA contents of the individual cells. The results showed that the proliferative activity of tumor cells was much higher in the regions composed of both many histiocytic stromal cells having polygonal or ovoid shape and many multinucleated giant cells, than either in the regions composed of fibrocytic stromal cells accompanying abundant collagen fibers or in the regions composed of foamy cells.

Adult↗
PubMed1985

[DNA-RNA cytofluorometric analysis of a case of osteosarcoma in relation to histological characteristics].

We applied DNA-RNA cytofluorometry with AO stain to cell kinetic analysis of osteosarcoma in a 12-year-old girl in relation to its histological characteristics. Histological findings obtained for 9 macroscopically different lesions were grouped into 4 main structural characteristics, but their cytofluorometric results were classified into 2 main patterns of DNA-RNA distribution. One showed remarkable polyploidization with many DNA synthetic cells in the invasive lesions, which were composed of pleomorphic cells forming osteoid or occasionally cartilaginous matrix. The other showed marked accumulation of tetraploid cells almost without DNA synthetic cells, being composed of relatively uniform fibroblastic or stellate-like cells in the cartilaginous matrix. These results indicated a close relationship between cell proliferative activity and the tissue environment.

Bone Neoplasms↗
PubMed1985

[DNA cytofluorometric analysis of nerve sheath tumors].

The present study was undertaken to clarify the ploidy patterns of peripheral nerve sheath tumors by determining nuclear DNA content of the individual tumor cells using PI-DNA cytofluorometry (NIKON SPM-RF1-D), and to investigate the relation between the ploidy patterns and histopathological findings. Most of the solitary neurilemmomas and neurofibromas studied were found to be associated with euploid-polyploidization, almost without DNA synthetic cells. It was also shown that the number of polyploid cells increased in accordance with an increase in the frequency of cells having large, atypical nuclei in histological picture, regardless of the natures of cellular arrangements. Malignant schwannomas, however, were composed of many polyploid and aneuploid cells with an increase of DNA synthetic cells, indicating their active cell proliferation. Thus, the ploidy patterns of nerve sheath tumors were remarkably different between benign and malignant ones. Furthermore, a case of multiple schwannoma suspected to be a variant of von Recklinghausen's disease, showed euploid-polyploidization with many DNA synthetic cells, indicating a ploidy pattern intermediate between benign and malignant tumors. This tumor thus appears to be a premalignant tumor or in the early stages of malignancy.

Adolescent↗
PubMed1984

[DNA cytofluorometry of soft tissue tumors].

We carried out DNA cytofluorometry with propidium iodide stain on the 17 cases of soft tissue tumors including giant cell tumor of the tendon sheath, pigmented villonodular synovitis, 2 hemangiomas, 3 lipomas, 5 schwannomas, 3 neurofibromas, liposarcoma and synovial sarcoma. The benign tumors were characterized by regular polyploidization with very few S-phase cells, indicating slow tumor growth. Most of the malignant soft tissue tumors were associated with remarkable polyploidization with an increase in S-phase cells. However, some malignant tumors did not show polyploidization. We concluded, therefore, that an increase in S-phase cells is an important, cytofluorometric criterion for malignancy of soft tissue tumors.

Adult↗
PubMed1984

[Multiparametric cytofluorometry of the process of cellular proliferation, differentiation and maturation of the chondrocytes in the growing rat].

The present study was undertaken to analyze quantitatively the process of cellular proliferation, differentiation and maturation of the chondrocytes in both the epiphyseal cartilage plates and the articular cartilages of the growing rats, using the multiparametric cytofluorometry (NIKON SPM-RFl-D). The analyses involve the simultaneous determinations by cytofluorometry of nuclear DNA vs. cellular RNA contents, nuclear DNA vs. cellular protein contents, and nuclear DNA vs. silver grain amount of the 3H-leucine, 3H-glycine, 3H-thymidine and the 35SO4 autoradiography. In addition, for the detailed analysis of cell kinetics, DNA-RNA cytofluorometry was carried out in combination with 3H-thymidine autoradiography. According to the results obtained, the chondrocytes were quantitatively divided into subpopulations of G0-G1, S and G2 in the cell cycle by their nuclear DNA and cellular RNA contents, and the maturative chondrocytes had higher cellular RNA content than the proliferative ones. It was suggested that there were, among the chondrocytes in the epiphyseal cartilage plate, two other subpopulations of binuclear cells and mononuclear tetraploid cells, both having higher cellular RNA content than the mononuclear G2 cells. It is reasonable to assume that these two subpopulations are the polyploid cell groups accompanying highly active cell proliferation.

Animals↗
PubMed1984

[DNA-RNA cytofluorometry for a primary cancer of the stomach and choledochus].

We standardized DNA-RNA cytofluorometry with AO stain using a NIKON SPM-RFI-D instrument for cell smears. We applied this technique in a 72-year-old woman with primary double cancer of the stomach and choledochus. We first suspected malignancy of the choledochus and examined the desquamated cells in the bile cytofluorometrically to obtain by X-Y plotting the irregularly increased polyploid cells with a high percentage of cells in S phase. These polyploid cells and S phase cells were found to have an increased RNA content. Before operation, the presence of another malignancy of the stomach was also ascertained. The surgical specimens were diagnosed histopathologically as well differentiated, tubular adenocarcinoma of the stomach and moderately differentiated, tubular adenocarcinoma of the choledochus. By cytofluorometric analysis similar results were obtained, i.e., polyploidization with increase of S phase cells. Based on these results, we concluded that our method is useful for the diagnosis of alimentary tract malignancy, and that the criteria of malignancy appear to be unusual polyploidization with increased fractions of S phase cells.

Adenocarcinoma↗
PubMed1983

[Quantitative analysis of nuclear DNA of rat chondrocytes during the course of growth and aging--Feulgen-DNA cytofluorometry method].

There have been no reported studies on the Feulgen-DNA cytofluorometry of the cartilage cells. We have attempted to devise a method of cell separation from the epiphyseal and articular cartilages of the rats, and to analyze by cytofluorometry the changes in the ploidy patterns of these chondrocytes during growth and ageing of the animals. Chondrocytes were isolated from the proximal cartilage of tibia by dual enzymatic digestions of the cartilage matrix with papain and collagenase, followed by mechanical cell separation with scissors and a micro-homogenizer, and were smeared onto the object glass with PBS. These procedures were found to be suitable for the Feulgen-DNA cytofluorometry of the chondrocytes from our repeated studies. We also carried out Feulgen-DNA cytofluorometry combined with 3H-thymidine autoradiography to determine cellular DNA content of the DNA synthetic chondrocytes in the epiphyseal cartilage. It has been clarified that during the growth course of the rats, the chondrocytes of the epiphyseal cartilage consist of many mononuclear diploid cells, a few mononuclear tetraploid cells and of some fraction of the cells having intermediate DNA values between the diploid and tetraploid levels. Those cells with intermediate DNA values, after autoradiographic studies, were found to correspond to DNA synthetic cells, indicating cell proliferative activity. It has been shown that during ageing of the rats, most of the chondrocytes from the articular cartilage are mononuclear diploid cells. The distribution of each cellular DNA content at the diploid level as determined by Feulgen-DNA cytofluorometry was shown to become gradually broader.

Aging↗
PubMedDate not supplied

Multiple bone lesions after allogeneic bone marrow transplantation in a patient with relapsed adult acute lymphoblastic leukemia: minimal residual disease analysis may predict extramedullary relapse.

We describe a patient with acute lymphoblastic leukemia (ALL, L2) who relapsed with multiple bone lesions after allogeneic bone marrow transplantation (allo-BMT). Allo-BMT was performed from an HLA-identical sibling during the first hematological complete remission (CR). Minimal residual disease (MRD) assessed by polymerase chain reaction (PCR) with primers for T cell receptor delta (TCRdelta) gene became positive in the bone marrow sample on day 46 after allo-BMT. On day 113, the patient complained of a painful tumor at the right clavicle. The examination of biopsy specimen revealed infiltration of leukemic cells. After partial response was achieved by local radiotherapy, disseminated bone lesions were demonstrated by 99mTC scintigraphy scan, followed by bone marrow relapse on day 137. The patient died of cardiac tamponade on day 236 after Allo-BMT. MRD assessed by PCR assay for TCRdelta gene in the bone marrow is useful for the prediction of extramedullary as well as medullary relapse after BMT.

Adult↗
PubMedDate not supplied

Effectiveness of activated vitamin D3 on improving prognosis of osteosarcoma patients.

We examined the survival prognosis of patients with osteosarcoma after orally administering the active form of vitamin D3, 1alpha (OH) D3 (alphaD3). The 29 patients, ranging in age from 9 to 58 years (mean, 19 years), with osteosarcoma were treated in our department between 1983 and 1995. The surgical stage was IIB in all patients. Among the 29 patients, alphaD3 was administered to 18 patients, and the remaining 11 patients served as controls. These patients underwent chemotherapy mainly with adriamycin and cisplatin, and wide tumor resection. In the alphaD3-treated group, the 5- or 10-year survival rate was 61.1%, while in the untreated group, the 5-year survival rate was 63.6%, and 10-year survival rate was 33.9%. Prognosis of the patients treated with alphaD3 tended to be better than that of untreated patients, but there was no significant difference in the survival rate between the groups (p=0.3823). The survival rate in the group treated with a total dose of alphaD3 more than 1,500 microg was also higher than that in the untreated group, but the difference was not significant (p=0.0740). Therefore, we concluded that alphaD3 at the dose used in this study was not effective in improving the prognosis of osteosarcoma patients.

Adolescent↗
PubMedDate not supplied

Extra-articular wide tumor resection and limb reconstruction in malignant bone tumors invading the knee joint.

We demonstrate our procedure of extra-articular wide tumor resection and its clinical outcome. The knee joint including the femorotibial, patellofemoral and the proximal tibiofibular joints was totally resected without any view of articular cartilage and synovium. The resected limb was reconstructed with endoprosthesis of the knee joint. The average score of the limb function evaluated by ISOLS criteria was 69.3% in all patients. The limb function of these patients was almost the same as that of the patients who received the intra-articular procedure. The procedure of extra-articular resection was theoretically necessary and clinically practical.

Arthroplasty, Replacement, Knee↗