Distribution of new human beta-defensin genes clustered on chromosome 20 in functionally different segments of epididymis.

Human beta-defensins are a family of cationic peptides that share a pattern of six conserved cysteine residues. We describe the cloning and characterization of the cDNAs of five novel beta-defensin genes (DEFB25-DEFB29) clustered on chromosome 20p13, which were identified using a bioinformatics approach. Expression analysis revealed the occurrence of the transcripts in only a few organs, with the highest abundance in the male genital tract. Examination of beta-defensin expression in human epididymis by real-time quantitative RT-PCR revealed a distribution along the functionally different segments of the epididymal duct. In situ hybridization for one of the cDNAs shows mRNA restriction to the epithelial cell layer of the epididymis, known to secrete factors responsible for sperm maturation. We suggest that the novel peptides carry out physiological functions in the male genital tract that may not be directly related to bacterial growth inhibition in host defense.

Expression of endocrine gland-derived vascular endothelial growth factor in ovarian carcinoma.

The first tissue-specific angiogenic molecule, endocrine gland-derived vascular endothelial growth factor (EG-VEGF), was identified recently in human ovary, raising hopes of developing tumor type-specific angiogenesis inhibitors. In the present study, we analyzed the expression of EG-VEGF mRNA in normal human tissues and ovarian neoplasms by quantitative real-time reverse transcription-PCR. EG-VEGF mRNA was expressed in all ovarian neoplasms examined. No significant difference was identified among benign, low malignant potential neoplasms or stage I ovarian cancer, all of which exhibited 2-fold lower mRNA levels compared with normal premenopausal ovaries. EG-VEGF mRNA levels further decreased in late stage compared with early stage carcinomas (P < 0.05) and were consistently lower in laser capture microdissected tumor islets compared with surrounding stroma. EG-VEGF was undetectable by reverse transcription-PCR in 17 established epithelial ovarian cancer cell lines or in cultured human ovarian surface epithelial cells, whereas it was detected in peripheral blood as well as tumor-infiltrating T lymphocytes. Finally, in contrast to VEGF, EG-VEGF mRNA levels did not correlate with clinical outcome in advanced ovarian carcinoma. These results suggest that EG-VEGF is most likely derived from nonepithelial components of ovarian carcinomas and may play a marginal role in promoting angiogenesis in advanced ovarian carcinoma. We postulate that EG-VEGF-targeted antiangiogenic therapy may prove useful in early stage but not in advanced stage ovarian carcinoma.

Isolation and biochemical characterization of LEAP-2, a novel blood peptide expressed in the liver.

The human genome contains numerous genes whose protein products are unknown in terms of structure, interaction partner, expression, and function. To unravel the function of these orphan genes, it is of particular value to isolate native forms of protein and peptide products derived from these genes. From human blood ultrafiltrate, we characterized a novel gene-encoded, cysteine-rich, and cationic peptide that we termed liver-expressed antimicrobial peptide 2 (LEAP-2). We identified several circulating forms of LEAP-2 differing in their amino-terminal length, all containing a core structure with two disulfide bonds formed by cysteine residues in relative 1-3 and 2-4 positions. Molecular cloning of the cDNA showed that LEAP-2 is synthesized as a 77-residue precursor, which is predominantly expressed in the liver and highly conserved among mammals. This makes it a unique peptide that does not exhibit similarity with any known human peptide regarding its primary structure, disulfide motif, and expression. Analysis of the LEAP-2 gene resulted in the identification of an alternative promoter and at least four different splicing variants, with the two dominating transcripts being tissue-specifically expressed. The largest native LEAP-2 form of 40 amino acid residues is generated from the precursor at a putative cleavage site for a furin-like endoprotease. In contrast to smaller LEAP-2 variants, this peptide exhibited dose-dependent antimicrobial activity against selected microbial model organisms. LEAP-2 shares some characteristic properties with classic peptide hormones and it is expected that the isolation of this novel peptide will help to unravel its physiological role.

Different effects of glucose starvation on expression and stability of VEGF mRNA isoforms in murine ovarian cancer cells.

Vascular endothelial growth factor (VEGF) has been implicated as a potent regulator of angiogenesis in tumors, and its protein exists as at least five isoforms with distinct biologic activities and clinical significance. Tumors under metabolic stress conditions dramatically increase VEGF expression due to both increased transcription and decreased mRNA degradation. However, it is not known how stress conditions regulate expression of each VEGF isoform. Here, we report a novel Taqman real-time RT-PCR strategy for quantification of all murine VEGF isoforms and find that (1) glucose starvation dramatically up-regulates the mRNA level of all VEGF isoforms, with the three abundant isoforms, VEGF120, VEGF164, and VEGF188, increasing at a similar rate, while the rare isoform VEGF144 is more markedly up-regulated; (2) glucose starvation induces a significant increase of the relative abundance of VEGF144 mRNA, but not the more prevalent isoforms VEGF120, VEGF164, and VEGF188, compared to total VEGF; and (3) the stability of each isoform mRNA differs under the control conditions as well as glucose starvation. The latter significantly stabilizes mRNA of all VEGF isoforms at a different rate, with VEGF144 most significantly stabilized. Our results indicate that under metabolic stress conditions VEGF144 is the most dramatically up-regulated VEGF isoform, probably through mechanism(s) different from the three abundant VEGF isoforms.