Endogenous production of nitric oxide by vascular endothelial growth factor down-regulates proliferation of choriocarcinoma cells.

The trophoblast-like choriocarcinoma cell line BeWo expresses a receptor for vascular endothelial growth factor (VEGF) and proliferates in response to VEGF. Nitric oxide (NO) seems to play a key role in the VEGF-induced proliferation of endothelial cells but the NO mechanistic regulation of VEGF-stimulated trophoblast proliferation is presently unclear. We assessed the effect of exogenous VEGF on BeWo cell proliferation by [3H]thymidine incorporation. The VEGF-induced proliferation of BeWo cells was significantly increased by the NO synthase (NOS) inhibitor, N(omega)-nitro-l-arginine methyl ester (L-NAME), but was inhibited by the NO donor, sodium nitroprusside. Treatment of the cells with 10 ng/ml of VEGF increased not only eNOS expression but also NO production. The extracellular signal-regulated kinase (Erk) of the mitogen-activated protein kinase (MAPK) family was activated by VEGF as demonstrated by the phosphorylation of Erk in Western blots. The effects of VEGF on NO production and the expression of endothelial NOS (eNOS) were attenuated by treating BeWo cells with the selective inhibitor of MAPK kinase, PD98059. VEGF-stimulated proliferation of BeWo cells was inhibited by the tyrosine kinase inhibitor genistein but increased by PD98059. Other kinase inhibitors, including LY294002 (phosphoinositide 3-kinase inhibitor) and SB203580 (P38 MAPK inhibitor), had no effect on the proliferation of the cells and NO production. These results indicate that endogenous NO production down-regulates the VEGF-stimulated proliferation of BeWo cells and that the activation of Erk plays an important role in this mechanism.

Detection of fetal erythroid cells from maternal blood using fluorescence in situ hybridization and liquid culture.

Fetal nucleated erythrocytes circulating in maternal blood are a potential source of fetal DNA for noninvasive prenatal genetic diagnosis. However, the estimated ratio of fetal to maternal cells is extremely small. In order to enrich these cells, we performed direct culture using a two-phase liquid system. Mononuclear cells were obtained from maternal blood samples at 8-10(+3) weeks of gestation and cultured in the first phase. After 4-5 days, the nonadherent cells were harvested and recultured with erythropoietin in the second phase for another 3-5 days. We examined cellular morphology, and counted the number of benzidine- positive cells and the percentage of glycophorin A/CD71 positive erythroid cells. We also did Kleihauer-Betke stain for Hb F, polymerase chain reaction (PCR) for SRY/DYZ1, chromosome analysis, and fluorescence in situ hybridization (FISH). The number of total erythroid cells reached about 0.1 x 10(6)-1.0 x 10(6)/mL with a purity of 84.0-97.3%. Hb F stain showed total erythroid cells of approximately 0.4 x 10(4)-9.8 x 10(4)/mL. Male DNA was detected in one case by PCR. In this case, the XY karyotype was confirmed by FISH and amniocentesis. This approach provides enriched source of fetal cells for further prenatal genetic analysis without complicated separation or sorting procedures.

Partial trisomy of chromosome 10(q22-q24) due to maternal insertional translocation (15;10).

Interchromosomal insertional translocations are rare chromosome rearrangements with an incidence of about 1:80,000 live births. We report on the clinical and cytogenetic findings of a newborn baby with partial trisomy 10q22-10q24 due to a maternal insertional translocation 15;10. Partial trisomy of the long arm of chromosome 10 is a distinctive chromosome aberration characterized by prenatal-onset growth retardation and craniofacial, skeletal, and other somatic anomalies. Most cases are unbalanced products from reciprocal chromosome translocations, and insertional translocations are rarely involved. The proband was initially referred because of severe intrauterine growth retardation, and fluorescence in situ hybridization (FISH) using painting probes confirmed the maternal balanced (15;10) insertion.

Enrichment and detection of fetal erythroid cells from maternal peripheral blood using liquid culture.

Isolating fetal cells from maternal blood for prenatal genetic analysis is the least invasive method currently being investigated. In order to enrich these fetal erythroid cells we employed a two-phase liquid culture system that supports the growth and differentiation of erythroid progenitor cells. Mononuclear cells were separated from ten maternal blood samples at 8-14(+3) weeks of gestation and cultured in the first phase. After 4-5 days, the non-adherent cells were harvested and recultured with erythropoietin for another 3-4 days. The mean number of total erythroid cells reached approximately 0.77x10(6)/ml with an average purity of 90.5%. Hb F stain disclosed fetal erythroid cells averaging approximately 5.7%, therefore the mean number of fetal erythroid cells isolated was 0.49x10(5)/ml. Female karyotypes were only observed while counting 5-66 metaphases. However, male DNA (SRY or DYZ1) was detected by PCR in 7/10 cases; in four of these cases and in one SRY-negative pregnancy the 46,XY karyotype was found by amniocentesis performed during the second trimester. This liquid culture system provided an enriched source of fetal cells without the need for more complicated separation or sorting procedures.

Cytotoxic compounds from the roots of Juglans mandshurica.

Three new compounds, a diarylheptanone glucoside (1), 4,5, 8-trihydroxy-alpha-tetralone 5-O-beta-d-[6'-O-(3", 5"-dimethoxy-4"-hydroxybenzoyl)]glucopyranoside (2), and 1,4, 8-trihydroxy-3-naphthalenecarboxylic acid 1-O-beta-d-glucopyranoside methyl ester (3), were isolated from the roots of Juglans mandshurica, and their structures were elucidated on the basis of spectroscopic studies including 2D-NMR.