Regulation of plasminogen activator inhibitor-1 and -2 messenger ribonucleic acid levels in human cumulus and granulosa-luteal cells.

Plasminogen activators and their inhibitors have been implicated in the process of fibrinolysis, tissue remodeling, and ovulation. Epidermal growth factor (EGF), a paracrine hormone found in the human ovary, increases plasminogen activator (PA) activity and the gene expression of PA and plasminogen activator inhibitor (PAI) in human endothelial cells and human cell lines. Gonadotropins also increase PA activity and gene expression in rat preovulatory granulosa cells. We have now analyzed the gene expression of PAI-1 and PAI-2 in uncultured human cumulus cells (CC), uncultured granulosa-luteal cells (GLC), and cultured GLC obtained from preovulatory follicles of patients undergoing assisted reproductive technologies. We also studied the effects of hCG and EGF on PAI-1 and PAI-2 mRNA levels in cultured GLC; GLC were cultured in serum-free medium for various times within 24 h with or without hCG and for 6 h with or without hCG, EGF, or EGF plus hCG. Total RNAs from CC and GLC were extracted, and blot hybridizations with 32P-labeled PAI-1, PAI-2, or 28S ribosomal RNA cDNA probes were performed. Both CC and GLC expressed PAI-1 and PAI-2 genes. In GLC, steady state levels of PAI-1 mRNA levels steadily increased within 24 h of culture, whereas PAI-2 levels peaked at 6 h of culture. PAI-1 mRNA levels were not affected by hCG or EGF at 6 h of culture, but PAI-2 mRNA levels were significantly increased by EGF at 6 h of culture. These studies demonstrate that human GLC PAI-1 and PAI-2 mRNA levels are differentially regulated and suggest that EGF may be involved in modulation of the human ovarian PA system during the periovulatory period.

Use of genetic polymorphisms detected by the random-amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) for differentiation and identification of Aedes aegypti subspecies and populations.

Amplification of random regions of genomic DNA using 10-base primers in the random-amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was used to differentiate and identify mosquito populations based on genetic variation. Genomic DNA was extracted from individual mosquitoes from 11 geographic populations of Aedes aegypti and amplified in PCR reactions using single primers of arbitrary nucleotide sequence. Discriminant analysis of the population frequencies of RAPD fragments produced using three different primers allowed accurate discrimination between the geographic populations in 89% of individuals and between subspecies (Ae. aegypti aegypti versus Ae. aegypti formosus) in 100% of mosquitoes tested. The genetic relatedness of the populations was estimated using three different statistical methods, and unknown populations were correctly classified in a blind test. These results indicate that the RAPD-PCR technique will be useful in studies of arthropod molecular taxonomy and in epidemiologic studies of the relatedness of geographic populations and vector movement.

Partial nucleotide sequence of South American yellow fever virus strain 1899/81: structural proteins and NS1.

We have partially cloned and sequenced the genome of a Peruvian yellow fever virus isolate (1899/81) and compared the nucleotide and deduced amino acid sequences of this strain with the previously published sequence of the West African yellow fever virus strain Asibi. In the 3594 base region sequenced, which contains the structural genes (C, M, E), all but the 72 3'-terminal nucleotides of the NS1 gene and 108 nucleotides of the 5' non-coding region, 515 nucleotide substitutions were detected. Nucleotide divergence was lowest in the 5' non-coding region, 2.8%, compared with an average rate of 14.7% in the coding regions. Over 91% of the 512 nucleotide changes in the coding region were silent; 44 amino acid substitutions resulted. The capsid protein was the least conserved, whereas the M protein was the most highly conserved (6.7% and 1.3% divergence, respectively). The envelope protein had 18 amino acid changes (3.7% divergence), one of which created an additional site for potential glycosylation of the 1899/81 virus. NS1 protein divergence (3.9%) was similar to that seen in the E protein. Of the 44 amino acid substitutions found, 34 (77%) were conservative. The highest number of nonconservative differences occurred in the envelope glycoprotein. These changes may significantly affect the antigenic and biological functions of the viruses.

Different mechanisms contribute to simultaneous inhibition of urokinase and tissue-type plasminogen activators by glucocorticoids in human ovarian carcinoma cells.

Previous studies from our laboratory have demonstrated that OVCA 433 human ovarian carcinoma cells are glucocorticoid responsive by several criteria and contain high affinity, saturable, steroid-specific glucocorticoid receptors. These cells secrete both mammalian plasminogen activators (PAs), urokinase (uPA) and tissue-type PA (tPA). Treatment of OVCA 433 cells with 1 x 10(-7) M dexamethasone (Dex) for 4 days led to 77% and 83% reductions in the extracellular activities of uPA and tPA, respectively, released into serum-free conditioned medium during a 1-h period. Dex treatment led to a 71% decrease in the rate of extracellular uPA antigen accumulation, as determined by enzyme-linked immunosorbent assay, as well as a 73% reduction in steady state uPA mRNA levels. In contrast, Dex treatment led to only a 42% decrease in the rate of extracellular tPA antigen accumulation and a 48% decrease in tPA mRNA levels; such decreases were insufficient to account for the 83% reduction in tPA activity. Thus, while Dex-induced decreases in uPA antigen and mRNA levels accounted for all but 6% of the decrease in uPA activity, a large discrepancy existed between the magnitudes of decreased tPA activity and decreased tPA antigen and mRNA levels. OVCA 433 cells produce both PAI-1 and PAI-2, two specific PA inhibitors. Treatment of cells with 1 x 10(-7) M Dex for 4 days led to a 3.3-fold increase in the rate of extracellular PAI-1 accumulation, with little or no effect on PAI-2 accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)