A multipotent clonal human periodontal ligament cell line with neural crest cell phenotypes promotes neurocytic differentiation, migration, and survival

Repair of injured peripheral nerve is thought to play important roles in tissue homeostasis and regeneration. Recent experiments have demonstrated enhanced functional recovery of damaged neurons by some types of somatic stem cells. It remains unclear, however, if periodontal ligament (PDL) stem cells possess such functions. We recently developed a multipotent clonal human PDL cell line, termed cell line 1-17. Here, we investigated the effects of this cell line on neurocytic differentiation, migration, and survival. This cell line expressed the neural crest cell marker genes Slug, SOX10, Nestin, p75NTR, and CD49d and mesenchymal stem cell-related markers CD13, CD29, CD44, CD71, CD90, CD105, and CD166. Rat adrenal pheochromocytoma cells (PC12 cells) underwent neurocytic differentiation when co-cultured with cell line 1-17 or in conditioned medium from cell line 1-17 (1-17CM). ELISA analysis revealed that 1-17CM contained approximately 50 pg/ml nerve growth factor (NGF). Cell line 1-17-induced migration of PC12 cells, which was inhibited by a neutralizing antibody against NGF. Furthermore, 1-17CM exerted antiapoptotic effects on differentiated PC12 cells as evidenced by inhibition of neurite retraction, reduction in annexin V and caspase-3/7 staining, and induction of Bcl-2 and Bcl-xL mRNA expression. Thus, cell line 1-17 promoted neurocytic differentiation, migration, and survival through secretion of NGF and possibly synergistic factors. PDL stem cells may play a role in peripheral nerve reinnervation during PDL regeneration.     

Cytologic features of functioning stromal cells in a yolk sac tumor of the ovary. A case report

BACKGROUND: Functioning stromal cells are sometimes seen in primary and metastatic ovarian neoplasms. However, the cytologic features of functioning stromal cells have been described only rarely. CASE: A 19-year-old woman had an alpha-fetoprotein-producing ovarian yolk sac tumor with functioning stroma. Her preoperative serum testosterone level was elevated. Imprint cytology showed that the functioning stromal cells had centrally located nuclei with low nuclear/cytoplasmic ratios. Occasionally these cells had vacuolated cytoplasm, suggesting the presence of lipids. In sharp contrast, the yolk sac tumor cells had more pleomorphic and hyperchromatic nuclei. We were able to distinguish between neoplastic and functioning stromal cells on the basis of these findings. In addition, immunostaining for inhibin on imprint cytologic slides was of great help in identifying functioning stromal cells. CONCLUSION: Because functioning stromal cells may unexpectedly induce hormonal effects in a variety of ovarian tumors, it is important to identify such cells in cytologic specimens.     

An assay method for the prediction of tumor promoting potential of chemicals by the use of Bhas 42 cells

It has become an important task to develop a simple in vitro method for the detection of non-genotoxic carcinogens, among which tumor promoters are included. Bhas 42 cells are v-Ha-ras-transfected BALB/c 3T3 cells and are regarded as initiated cells in the 2-stage transformation paradigm. We designed a method for detecting tumor promoters by the use of Bhas 42 cells at advanced passage generation. In this method, the cells are cultured in six-well plates for 17 days during which test chemicals are added in the medium for 11 days from days 3 to 14. The end-point of the assay is the induction of transformed foci. When the tumor promoter TPA was used, a significant number of transformed foci were induced concentration-dependently, whereas only a few foci were observed in control cultures. When various chemicals were examined by the method, a reasonable correlation was observed with the reported tumor-promoting ability in animal experiments. We propose that the Bhas 42 cell transformation method is practical and useful for the detection of tumor promoters.     

Loss-of-function of an N-acetylglucosaminyltransferase,POMGnT1, in muscle-eye-brain disease

Muscle-eye-brain disease (MEB), an autosomal recessive disorder, is characterized by congenital muscular dystrophy, brain malformation, and ocular abnormalities. Previously, we found that MEB is caused by mutations in the gene encoding the protein O-linked mannose beta1,2-N-acetylglucosaminyltransferase 1 (POMGnT1), which is responsible for the formation of the GlcNAcbeta1-2Man linkage of O-mannosyl glycan. Although 13 mutations have been identified in patients with MEB, only the protein with the most frequently observed splicing site mutation has been studied. This protein was found to have no activity. Here, we expressed the remaining mutant POMGnT1s and found that none of them had any activity. These results clearly demonstrate that MEB is inherited as a loss-of-function of POMGnT1.     

Gravimorphism in rice and barley: Promotion of leaf elongation by vertical inversion in agravitropically growing plants

We have compared shoot responses of agravitropic rice and barley plants to vertical inversion with those of normal ones. When rice plants were vertically inverted, the main stems of a japonica type of rice, cv. Kamenoo, showed negative gravitropism at nodes 2–15 of both elongated and non-elongated intermodes. However, shoots of lazy line of rice, lazy-Kamenoo, bent gravitropically at nodes 11–15 only elongated internodes but not at nodes 2–10 of non-elongated ones. Thus, shoots of Kamenoo responded gravitropically at all stages of growth, whereas shoots of lazy-Kamenoo did not show gravitropic response before heading. In Kamenoo plants, lengths of both leaf-sheath and leaf-blade were shortened by vertical inversion, but those of the vertically inverted plants of lazy-Kamenoo were significantly longer than the plants in an upright position. When agravitropic and normal plants of barley were vertically inverted, the same results as in rice were obtained; elongation of both leaf-sheath and leaf-blade was inhibited in normal barley plants, Chikurin-Ibaragi No. 1, but significantly stimulated in agravitropic plants ofserpentina barley. These results suggest that vertical inversion of rice and barley plants enhances the elongation growth of leaves in the absence of tropistic response.     

DNA strand breakage by hydroxyphenyl radicals generated from mutagenic diazoquinone compounds.

The mutagenic diazoquinone compounds p-diazoquinone (p-DQ), o-diazoquinone (o-DQ) and 3-diazo-N-nitrosobamethan (D-BM) cleaved the phosphodiester bond of lambda DNA, phi X174 RFI DNA and M13mp8ss DNA. p-DQ also cleaved the phosphodiester bond of bis(p-nitrophenyl)phosphate. The breakage of the phosphodiester bond was inhibited by the antioxidant butyl hydroxyanisole (BHA), ethanol, the spin trapping agent DMPO, cysteine and 2-mercaptoethanol. While incubation of p-DQ and o-DQ alone gave p-hydroquinone and catechol, respectively, incubation of these compounds in the presence of BHA and ethanol gave phenol in large yields. Incubation of p-DQ and o-DQ with the spin trapping agents DMPO and PBN gave spin adducts assignable as p- and o-hydroxyphenyl adducts, respectively. The breakage of the phosphodiester bond of DNA by the diazoquinone compounds is suggested to be due to the hydroxyphenyl radicals generated during incubation.     

Cytogenetic studies in a Y-to-X translocation observed in three members of one family,with evidence of infertility in male carriers

Summary A family is reported in which the mother and two sons are carriers of a Y-to-X translocation, der(X)t(X;Y) (p22;q11). All of the three carriers have short statute and disproportion of extremities, but otherwise normal phenotype. One of the sons, the propositus, has been affected with schizophrenia. Evidence was obtained that male carriers are probable sterile; both sons aged 26 and 30 years had azoospermia and the biopsied specimens of the testis had histologic pictures showing spermatogenetic arrest. The mother was H-Y weakly positive, and the normal X chromosome was inactivated in the majority of the cells analyzed. Dermatoglyphics of the three carriers were unusual and dissimilar to the features of Turner's syndrome. The clinical and cytogenetic findings in the present study are compared with those of the previously reported familial cases, and the genetic background causing phenotypic abnormalities in the male and female carriers is discussed.     

Tumor necrosis factor and vascular endothelial growth factor induce endothelial integrin repertories, regulating endovascular differentiation and apoptosis in a human extravillous trophoblast cell line

Angiogenesis is crucial in human development. Extravillous trophoblast (EVT) cells mimic endothelial cells in angiogenesis during endovascular differentiation, inducing a remodeling of spiral arteries that increases blood flow toward the intravillous space. We have previously shown that tumor necrosis factor (TNF) alpha regulates expression of ITGA6 and ITGA1, which are involved in cell survival, in the human EVT cell line TCL1. To further investigate endovascular differentiation, we examined the effects of vascular endothelial growth factor (VEGF), TNF, and extracellular matrix (ECM) on TCL1 cells. Seeded on Matrigel, TCL1 cells show tube-like formation that specifically recalls morphological changes in endothelial cells. Anti-ITGAV/ITGB3 antibodies significantly reduced the size of the capillary network (P < 0.05) on Matrigel and also suppressed TNF-induced apoptosis (P < 0.05) in TCL1 cells. VEGF induced expression of ITGAV/ITGB3 subunits and protein aggregation, as in the case of TNF, which in turn, induces synthesis of VEGF in TCL1 cells. Soluble FLT1 suppressed these activities in TCL1 cells, indicating that signals involving VEGF axis are essential for endovascular differentiation. These results suggest that TNF, VEGF, and ECM collaboratively regulate EVT behavior, including cell survival and endovascular differentiation, through integrin signaling during establishment and maintenance of successful human pregnancies.     

Tight junctions in Schwann cells of peripheral myelinated axons: a lesson from claudin-19-deficient mice

Tight junction (TJ)-like structures have been reported in Schwann cells, but their molecular composition and physiological function remain elusive. We found that claudin-19, a novel member of the claudin family (TJ adhesion molecules in epithelia), constituted these structures. Claudin-19-deficient mice were generated, and they exhibited behavioral abnormalities that could be attributed to peripheral nervous system deficits. Electrophysiological analyses showed that the claudin-19 deficiency affected the nerve conduction of peripheral myelinated fibers. Interestingly, the overall morphology of Schwann cells lacking claudin-19 expression appeared to be normal not only in the internodal region but also at the node of Ranvier, except that TJs completely disappeared, at least from the outer/inner mesaxons. These findings have indicated that, similar to epithelial cells, Schwann cells also bear claudin-based TJs, and they have also suggested that these TJs are not involved in the polarized morphogenesis but are involved in the electrophysiological "sealing" function of Schwann cells.