A Raman spectroscopic investigation of the mechanism of the reduction in hair with thioglycerol and the accompanying disulphide conformational changes.

OBJECTIVE: The objective of our research was to investigate the reduction mechanism of thioglycerol (TG) on hair keratin fibres. METHODS: The structure of cross-sections at various depths of virgin white human hair resulting from the permanent waving process with TG was directly analysed at the molecular level using Raman spectroscopy. Also, the penetration of TG for the cross-sectional samples dyed with methylene blue was observed by optical microscopy. RESULTS: The gauche-gauche-gauche (GGG) and gauche-gauche-trans (GGT) conformations of disulphide (-SS-) groups remarkably decreased, while the trans-gauche-trans (TGT) conformation increased by performing the reduction process with TG. In addition, the disconnected -SS- content at various depths of the hair sample reduced with TG adjusted to pH 9.0 with ammonia solution was clearly increased compared with that of the hair sample reduced with TG adjusted to pH 9.0 with monoethanolamine. In the case of adjusting to pH 9.0 with ammonia solution, the tensile strength of the waved hair treated with TG was strong compared with that of the waved hair treated with thioglycolic acid (TGA), but the waving efficiency of the waved hair treated with TG was nevertheless higher than that of the waved hair treated with TGA. CONCLUSION: The author concluded that the waved virgin human hair treated with TG adjusted to pH 9.0 with ammonia solution was less damaged as compared with the waved hair treated with TGA, despite its good waving efficiency, as not only were the GGG and GGT conformations disconnected, but they were also changed to the TGT conformation by performing the reduction process with TG.

Internal structural changes in keratin fibres resulting from combined hair waving and stress relaxation treatments: a Raman spectroscopic investigation.

OBJECTIVE: The objective of our research was to investigate the influence of chemical treatments (reduction, stress relaxation and oxidation) on hair keratin fibres. METHODS: The structure of cross-sections at various depths of virgin white human hair resulting from permanent waving treatments with stress relaxation process was directly analysed at a molecular level using Raman spectroscopy. In particular, the three disulphide (-SS-) conformations in human hair were compared by S-S band analysis. RESULTS: The gauche-gauche-gauche (GGG) and gauche-gauche-trans (GGT) contents of -SS- groups remarkably decreased, while the trans-gauche-trans (TGT) content was not changed by performing the reduction process with thioglycolic acid. In addition, the high-temperature stress relaxation process after reduction accelerated the disconnection of -SS- (GGG and GGT) groups in the human hair, while the low-temperature stress relaxation process after reduction accelerated the reconnection of -SS- (GGG and GGT) groups. Moreover, the S-O band intensity at 1042 cm(-1) , assigned to cysteic acid, existing in the cuticle region and the surface of the cortex region increased, while the GGG content significantly decreased by performing the oxidation process after the reduction and the high-temperature stress relaxation processes. CONCLUSION: The author concluded that the high-temperature relaxation process after reduction accelerated the disconnection of -SS- (GGG and GGT) groups, thereby leading to the remarkable local molecular disorganization (an increase in the cysteic acid content and a decrease in the GGG content) on the cuticle and cortex cells during the oxidation process.

Therapeutic potential of a novel synthetic selectin blocker, OJ-R9188, in allergic dermatitis.

1. We investigated the ability of a newly synthesized sugar derivative, OJ-R9188, [N-(2-tetradecylhexadecanoyl)-O-(L-alpha-fucofuranosyl)-D-seryl]-L-glutamic acid 1-methylamide 5-L-arginine salt, to block binding of selectins to their ligands in vitro and inhibit the infiltration of leukocytes in vivo. 2. OJ-R9188 prevented the binding of human E-, P- and L-selectin-IgG fusion proteins to immobilized sialyl Lewis(x) (sLe(x))-pentasaccharide glycolipid, with IC(50) values of 4.3, 1.3, and 1.2 microM, respectively. 3. In a mouse model of thioglycollate-induced peritonitis, OJ-R9188 at 10 mg kg(-1), i.v. inhibited neutrophil accumulation in the peritoneal cavity. In the IgE-mediated skin reaction, OJ-R9188 at 3 and 10 mg kg(-1), i.v. significantly inhibited extravasation of neutrophils and eosinophils into the inflammatory sites and at 10 mg kg(-1), i.v. also inhibited infiltration caused by picryl chloride-induced delayed-type hypersensitivity in mice. These results suggest that OJ-R9188 may be a useful selectin blocker, with activity against human and mouse E-, P- and L-selectins in vitro and in vivo, and that blocking selectin-sLe(x) binding is a promising strategy for the treatment of allergic skin diseases.

Relationship between histologic grade and cytofluorometric cellular DNA and RNA content in primary bone tumors.

The diagnosis and grading of bone tumors remains a challenging problem. We studied the relationship between histologic grade and cytofluorometric cellular DNA and RNA content in 108 primary bone tumors. The data included DNA ploidy, mean DNA content (MDC), S-phase fraction (SPF), mean RNA content (MRC) and RNA/DNA ratio (RDR; MRC/MDC) which represents the RNA content normalized for the DNA content. Benign tumors had a diploid stem line with low MDC (mean; 1.04), low SPF (0.9), high MRC (2.41) and high RDR (2.31). Giant cell tumors of bone, which are locally aggressive benign tumors, showed diploidy with relatively higher MDC (1.07, p < 0.01) and SPF (2.6, p < 0.01) and lower MRC (1.81, p < 0.01) and RDR (1.69, p < 0.01). Similar results were obtained in low-grade sarcomas. In high-grade sarcomas, the data depended on the histologic findings. Pleomorphic sarcomas such as osteosarcomas revealed aneuploidy with remarkably higher MDC (1.70 in osteosarcomas, p < 0.01) and SPF (6.5, p < 0.01), but lower RDR (1.70, p < 0.01). In contrast, small cell sarcomas, such as Ewing's sarcomas, showed diploidy with low MDC (1.11 in Ewing's sarcomas, N.S.) and SPF (2.5, p < 0.01) and extremely low RDR (1.34, p < 0.01). The RDR value was higher in well-differentiated tumors than in primitive tumors, rendering it useful in grading bone tumors with a diploid stem line. By combining the RDR value with the MDC value, 96% of diploid sarcomas could be distinguished from benign tumors. These results indicate that cellular DNA and RNA content analysis may be of value in assessing the malignant potential of diploid as well as aneuploid bone sarcomas.

Oral administration of 1 alpha hydroxyvitamin D3 inhibits tumor growth and metastasis of a murine osteosarcoma model.

We studied the effect of oral administration of 1 alpha hydroxyvitamin D3 (1-D3) on the growth and metastatic ability of Dunn murine osteosarcoma model. A solution of 1-D3 or vehicle alone was administered daily for 2 weeks to tumor-bearing mice using an esophageal tube and tumor size was serially monitored. In 1-D3-treated mice, the growth of Dunn osteosarcoma was significantly suppressed in a dose-dependent manner. Histologically, tumor cells in the control mice proliferated in marginal regions of the tumor with wide central necrosis, whereas in the 1-D3-treated mice, tumor cells were distributed as scattered islands among extensive necrotic tissue. The mean tumor necrosis area was 55.7% in the control tumors and 94.6% in 1-D3-treated tumors (p < 0.001). There were no substantial differences in the cytofluorometric cell cycle distribution or the histological mitotic index between control and 1-D3-treated tumors. When 1-D3 was administered to mice from 2 days before to 2 weeks after transplantation of the tumor, there were significantly fewer metastatic foci in the lungs in 1-D3-treated mice than in control mice. We also tested the effect of coadministration of 1-D3 and doxorubicin on the growth of Dunn osteosarcoma and found that these two drugs act additively to suppress tumor growth. These results indicated that 1-D3 given orally inhibits tumor growth and metastases in a Dunn osteosarcoma model. Although the mechanism remains unknown, oral administration of 1-D3 might be promising as a new method of treating human osteosarcoma.

DNA cytofluorometric analysis of benign and malignant nerve sheath tumors.

The present study was undertaken to examine the ploidy patterns of benign and malignant nerve sheath tumors using PI-DNA cytofluorometry and to clarify the relationship between the ploidy patterns and pathological findings for these lesions. The benign nerve sheath tumors (schwannomas and neurofibromas) were found to be dipolid or to be euploid-polyploidization, with a few DNA synthetic cells. The number of polyploid cells increased with increase in the frequency of cells with large and atypical nuclei in histology, regardless of the natures of cellular arrangements. Malignant schwannomas were composed of many polyploid and aneuploid cells with DNA synthetic cells, indicating their active cell proliferation. Thus, benign nerve sheath tumors exhibited low-proliferative ploidy patterns that were clearly different from those of malignant nerve sheath tumors. As Takeshita has already stated, it is difficult to ascertain why some of the neurogenic tumors show euploid-polyploidization (19). Further investigations will be needed in this direction. Finally, it is clear from the results of this study that the cytofluorometric analysis mentioned above is useful for assessing malignancy and that it may be useful for predicting prognosis of nerve sheath tumors.

Role of selectins on IgE-mediated skin reaction.

Selectins play an important role on leukocytes infiltration into inflammatory tissues. To understand the role of selectins, we investigated the effects of selectin-IgG chimeras and anti selectin antibodies on the murine IgE-mediated skin inflammation model. Biphasic skin reactions were induced by intradermal challenge with ovalbumin (OA) to ears of actively sensitized mice. This reaction was characterized by immediate and late phase responses observed as which were induced via a rapid increase in capillary permeability and leukocyte infiltration, respectively. The expression of E-selectin mRNA was significantly increased to reach its highest level at 2 h after OA challenge. E-, P-, and L-selectin-IgG chimeras inhibited the late phase responses, i.e. ear swelling, neutrophil infiltration and eosinophil infiltration at 24 h after OA challenge in a dose-dependent manner at dose range of 0.1 - 10 mg kg(-1), i.v. Antiselectin antibodies did not inhibit the increase of ear swelling. But anti E- and P-selectin antibodies significantly inhibited neutrophil infiltration and eosinophil infiltration. These results indicate that selectins play an important role on the late phase response of the murine IgE-mediated skin inflammation model by mediating inflammatory cell adhesion to endothelium.

DNA ploidy alterations detected during dedifferentiation of periosteal chondrosarcoma.

DNA ploidy of a case with dedifferentiated periosteal chondrosarcoma was analyzed by DNA cytofluorometry. The diagnosis of primary periosteal chondrosarcoma was made on the basis of the radiographic and histological findings. At 4 years after marginal resection, the tumor recurred locally and metastasized to various organs. The patient died of disease 2 years later. Histologically, there were two components, chondrosarcoma and malignant fibrous histiocytoma, in the recurrent and metastatic tumors. DNA ploidy analysis of multiple samples revealed that the primary lesion was composed of many diploid cells with some tetraploid and octaploid cells, whereas in the dedifferentiated area, there were many aneuploid cells which were not recognized in any area of the primary tumors. This case illustrated that DNA ploidy alteration of euploidy to aneuploidy is closely correlated with the process of dedifferentiation in chondrosarcoma.

[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.

Concentration of interleukin-1 beta in serum and synovial fluid in patients with rheumatoid arthritis and those with osteoarthritis.

The concentration of interleukin-1 beta (IL-1 beta) in serum and synovial fluid of the knee of patients with rheumatoid arthritis (RA) and those with osteoarthritis (OA) was examined using a recently developed radioimmunoassay method. The serum concentration of IL-1 beta was below the detection limit of 0.25 ng/ml in all RA and OA patients. The IL-1 beta concentration in the synovial fluid was higher in RA patients than in OA patients. In RA patients, a significant correlation was demonstrated between the synovial fluid IL-1 beta concentration and the erythrocyte sedimentation rate, C-reactive protein level, synovial fluid white blood cell count and Lansbury index. The synovial fluid IL-1 beta concentration decreased with the reduction of the rheumatoid activity by the medication, but tended to increase or remain high when inflammatory activity of RA could not be adequately controlled. The IL-1 beta concentration, easily determined, shows promise as a new quantitative indicator of rheumatoid activity.

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.

[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.

[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.

[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.

[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.