Angiogenic cytokines, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and angiogenin, are candidates for the induction of pleural effusions because they have been implicated in the induction of neovascularization, vascular permeability, and hemorrhage both in the inflammatory process and in tumor progression. Thus, we hypothesized that these angiogenic factors in effusion might be involved in the clinical manifestation of malignant pleural disease. We measured the levels of VEGF, bFGF, and angiogenin in pleural effusions and sera from 40 patients. Pleural effusions due to malignancy (1,350 pg/ml) contained significantly higher levels of VEGF than effusions due to inflammatory diseases (102 pg/ml; p = 0.034). Furthermore, hemorrhagic effusions showed significantly higher VEGF levels (1,942 pg/ml) than non-hemorrhagic effusions (202 pg/ml; p = 0.016) in malignant patients. In contrast, neither bFGF nor angiogenin were correlated with any clinical manifestation of pleural effusion. Immunohistochemical study revealed that malignant cells in the pleura were stained with anti-VEGF antibody. Our data suggest that VEGF secreted from tumor cells may be involved in the accumulation of pleural effusion in malignancy, and that increased levels of VEGF may induce hemorrhagic effusion.

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Although a number of growth factors and their receptors are involved in the proliferation and differentiation of primordial germ cells (PGCs), the only factor that has been shown to be active in vivo is Steel factor, a ligand for c-Kit. To identify new growth factor receptors that may be required for PGCs function in vivo, we used an reverse transcription-polymerase chain reaction-based strategy to screen for protein kinase genes expressed in PGC-derived embryonic germ cells. We report here that one such gene encoding the receptor tyrosine kinase, Sky, is expressed in both PGCs and their supporting cells in male genital ridges after 11.5 dpc. Interestingly, Sky expression was not detected in female genital ridges, although transcripts were detected in supporting cells in the developing ovary at later stages. Gas 6, a ligand for Sky, was also expressed in interstitial cells which surround Sky positive cells in genital ridges, and, in addition, it supported PGC growth or survival in culture. After birth, Sky expression in testis was restricted to Sertoli cells, and Gas 6 was detected around peritubular cells and Leydig cells. These results suggest that Gas 6-Sky signaling plays a role in PGC growth, sexual differentiation, and Sertoli cell functions in vivo. Sky expression in Sertoli cells diminished by 3 weeks of age, when haploid germ cells first appear. On the other hand, the expression in Sertoli cells was markedly upregulated in the testis of germ cell-deficient W/Wv and jsd/jsd mice. The results suggest that signals from differentiated germ cells suppress Sky gene expression in Sertoli cells. High-resolution chromosomal mapping of Sky is also reported.

To identify key molecules that regulate germ cell proliferation and differentiation, we have attempted to isolate protein kinase genes preferentially expressed in germ line cells. One such cDNA cloned from murine embryonic germ(EG) cells encodes a nonreceptor type serine/threonine kinase and is predominantly expressed in the testis, ovary, and spleen of adult mouse. The nucleotide sequence of the entire coding regions shows that this clone, designated Plk1(polo like kinase 1), is identical with STPK13 previously cloned from murine erythro-leukemia cells. The protein encoded by Plk1 is closely related to the product of Drosophila polo that plays a role in mitosis and meiosis. To define the role of Plk1 in germ cell development, we have examined its expression in murine gonads by in situ hybridization. Here we show that the Plk1 gene is specifically expressed in spermatocytes of diplotene and diakinesis stage, in secondary spermatocytes, and in round spermatids in testes. It is also expressed in growing oocytes and ovulated eggs. The pattern of expression of the Plk1 gene suggests that the gene product is involved in completion of meiotic division, and like the Drosophila polo protein, is a maternal factor active in embryos at the early cleavage stage.

Secretory leukoprotease inhibitor (SLPI) is a serine protease inhibitor involved in antineutrophil elastase protection at inflammatory sites. To elucidate both the function and regulation of SLPI in vivo, we isolated and characterized the mouse Slpi gene. An entire 3-kb mouse Slpi gene fragment was sequenced, including an 0. 8-kb 5'-flanking region, the 2.2-kb Slpi gene, and a 0.1-kb 3'-flanking region. The mouse Slpi gene spans 2,222 base pairs containing four exons and three introns. All splicing borders between exons and introns are conserved as predicted by GT-AG rules. Using primer extension analysis, the transcription start site was located 20 nucleotides upstream from the methionine (ATG) initiation codon. At the defined transcription start site, the sequence TCA+1GAGC is present. These results indicate that both mouse and human genomic structure are highly conserved. Using fluorescence in situ hybridization, we confirmed that, consistent with the genomic similarity, the human SLPI gene is localized on chromosome 20q12-13. 2 and the mouse homologue on chromosome 2H, which are syntenic with each other.