Tetrahydrobiopterin (BH4) is an unstable natural cofactor for nitric oxide synthase (NOS). Since nitric oxide (NO) is an important mediator of the effects of cytokines and mechanical strain in bone, the expression and cellular localization of mRNA and protein of the BH4-generating enzymes in tissue sections of bone and bone derived cell cultures were investigated. The expression of the mRNA of sepiapterin reductase (SPR), which is the terminal enzyme of the BH4 biosynthetic pathway, was confirmed in osteoblasts on the surface of the trabecular bone and in bone marrow of rat femora. SPR protein was detected by immunohistochemical analysis in similar regions of the femora where SPR mRNA was expressed. Cultured osteoblastic UMR106 cells expressed the mRNAs of all 3 enzymes involved in the BH4 biosynthetic pathway from guanosine 5'-triphosphate (GTP), i.e., GTP cyclohydrolase I, pyruvoyl tetrahydropterin synthase, and SPR, as determined by the reverse transcription-polymerase chain reaction (RT-PCR) technique. Moreover, a significant amount of biopterin, a stable oxide of BH4, was detected in the cell extract by high-performance liquid chromatography (HPLC) analysis. These results indicate that osteoblasts can biosynthesize BH4 by themselves to supply this cofactor to NOS to control NO formation in the bone tissue.
PC12D cells, a subline of PC12 cells, extend neurites within a few hours in response to nerve growth factor (NGF). Multinucleate PC12D cells were produced by cell fusion with polyethylene glycol (PEG). The syncytia extended large neurites following treatment with NGF. In the syncytia, nuclei were distributed in a circular alignment. Many nuclear rings were found in the culture dish 1 day after treatment with PEG, although they were sometimes seen in syncytia within several hours. Colchicine prevented this alignment or disrupted the clusters when added subsequent to their formation. Cytochalasin B did not prevent the formation of these rings, nor affect the alignment of nuclei. Immnofluorescent staining of γ-tubulin, an integral protein of the centrosome, revealed intense dot-like structures in the center of nuclear rings in syncytia. Twice as many dots, or centrioles, as nuclei were present in each syncytium. However, most microtubules were localized around the nuclei and did not appear to extend from the cluster of centrioles. Fluorescent labeling of mitochondria showed them to mainly be localized inside the nuclear rings. Meanwhile, immunofluorescently stained endoplasmic reticulum was mainly localized along with the circular alignment of nuclei. Therefore, not only nuclei but various cell organelles are situated in each characteristic location in the syncytia. These data suggest that microtubules are involved in the migration and alignment of nuclei and presumably of cell organelles in the syncytia.
We investigated the clinical and immunohistochemical availability of DU-PAN-2 (sialyl Lewisc) as a tumor marker for endometrial adenocarcinomas (EMACs) in comparison with that of CA19-9 (sialyl Lewisa). Serum DU-PAN-2 and CA19-9 values of 17 EMACs were measured using an EIA kit and a RIA kit, respectively. The positive/negative cutoff values for both markers were set at the [mean+2×SD] from the serum values of 19 benign uterine cases as follows; DU-PAN-2, 73.9 U/mL; CA19-9, 37.4 U/mL. Immunohistochemical expressions of DU-PAN-2 and CA19-9 in EMACs were analyzed, applying an indirect immunoperoxidase method. The Lewis blood group type was determined using the hemagglutination test. The serum elevations of DU-PAN-2 and CA19-9 higher than the cutoff values before surgery were noted in 29.4% (5/17) and 23.5% (4/17) of EMACs, respectively. Except for one patient who died of systemic tumor dissemination, the elevated serum values of DU-PAN-2 and CA19-9 declined to the levels lower than the cutoff values after surgery. The relationship between immunohistochemical profiles and serum values was statistically proven to be closer in DU-PAN-2 (r=0.609, p=0.0094) than in CA19-9 (r=0.326, p=0.2022). The Lewis negative patients among them were consistently negative for CA19-9 serologically. We believe that DU-PAN-2 could be clinically useful in EMACs as well as immunohistochemically and recommend that this be added into a panel of tumor markers for EMACs.