The periodontal ligament (PDL) is a highly specialized tissue connecting the cementum with the tooth socket bone and affects the life span of the tooth. However, little is known about the precise characteristics and regenerative mechanism of PDL cells because of the absence of specific markers and cell lines. Therefore, we aimed to establish three immortalized human PDL fibroblast cell lines by using simian virus40 T-antigen (SV40T-Ag) and human telomerase reverse transcriptase (hTERT) transfection, expecting these cells to have the characteristics of primary cells. The transfected cells were named STPLF. The expression of SV40T-Ag and hTERT in all STPLF lines was verified by using the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method, stretch PCR analysis, or Western blotting analysis. All STPLF showed stable proliferation at more than 120 population doublings (PD), whereas primary human PDL fibroblasts (HPLF) stopped at 10-20 PD. Characterization by RT-PCR analysis revealed that all STPLF genes mimicked the expression of their respective original HPLF genes. STPLF expressed runt-related transcription factor-2, osterix, alkaline phosphatase, osteopontin, osteocalcin, periostin, receptor activator of NF-kappa B ligand, osteoprotegerin, epidermal growth factor receptor, alpha-smooth muscle actin, and type XII collagen. STPLF stimulated with 50 micro g/ml ascorbic acid and 2 mM beta-glycerophosphate for 4 weeks produced more calcified deposits than did HPLF cultured with the same reagents. These results suggest that each STPLF line retained the characteristics of the respective original HPLF, that STPLF gained increased calcification activity, and that STPLF are helpful tools for studying the biology and regenerative mechanisms of human PDL.
Microspectrophotometric evaluation of differentially stained sister chromatids made it possible to analyse precisely the factors involved in the Giemsa methods. The concentration of Hoechst 33258, pH of the mounting medium temperature during UV-exposure and the quality (wavelength)of UV-light influenced the differential staining. Exposure of blacklight of 10(-5) M Hoechst 33528-stained brdU-labeled chromosome specimens mounted in McIlvaine buffer (pH 8.0) at 50 degrees C reproducibly allowed differential staining of sister chromatids within 15 min. On the other hand, Korenberg-Freedlender's method using no Hoechst 33258 was also UV-light-dependent. Thus, photolysis of BrdU-substituted DNA was considered the basic mechanism of the Giemsa methods where the photosensitive Hoechst 33258 played a role as a sensitizer.
Aims/hypothesis Maternal diabetes during pregnancy increases the risk of congenital malformations such as neural tube defects (NTDs). Although the mechanism of this effect is uncertain, it is known that levels of nitric oxide synthase (NOS) and nitric oxide are elevated in embryos of a mouse model of diabetes. We postulated that overproduction of nitric oxide causes diabetes-induced congenital malformations and that inhibition of inducible NOS (iNOS) might prevent diabetic embryopathy. Methods Mice were rendered hyperglycaemic by intraperitoneal injection of streptozotocin. The incidence of congenital malformations including NTDs was evaluated on gestational day 18.5. We assessed the involvement of iNOS in diabetesinduced malformation by administering ONO-1714, a specific inhibitor of iNOS, to pregnant mice with streptozotocininduced diabetic mice and by screening mice with iNOS deficiency due to genetic knockout (iNos −/− ). Results ONO-1714 markedly reduced the incidence of congenital anomalies, including NTDs, in fetuses of a mouse model of diabetes. It also prevented apoptosis in the head region of fetuses, indicating that iNOS is involved in diabetesrelated congenital malformations. Indeed, no NTDs were observed in fetuses of diabetic iNos −/− mice and the incidence of other malformations was also markedly reduced. Conclusions/interpretation We conclude that increased iNOS activity during organogenesis plays a crucial role in the pathogenesis of diabetes-induced malformations and suggest that inhibitors of iNOS might help prevent malformations, especially NTDs, in diabetic pregnancy.
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