Establishment of vascular endothelial cell lines in a serum-free culture and the discovery of endothelin and a Vasoactive Intestinal Contractor (VIC)

Immortal vascular endothelial cell lines were established and utilized for the production of an endothelium-derived contraction factor (EDCF) in a serum-free medium. After the discovery of Endothelin (21 amino acid peptide, ET) as an EDCF, a prepro ET cDNA isolated from human tissue was used to examine the expression of ET and its regulation in human endothelial cells. A gene family of ET was shown in mouse by using prepro ET cDNA as a probe. Thus, a novel peptide, Vasoactive Intestinal Contractor (VIC) homologous to ET was deduced from the sequence of one of these genes. VIC was confirmed to induce vasocontraction as well as intestinal contraction. Northern blot analysis indicated that this gene was expressed in the intestine but not in endothelial cells. A cloning and sequencing of prepro VIC cDNA from mouse intestine suggest that a VIC-like peptide, as well as VIC, are co-synthesized by cleavage from prepro VIC with 160 amino acids.     

The gene and the primary structure of acidic ribosomal protein A0 from yeast Saccharomyces cerevisiae which shows partial homology to bacterial ribosomal protein L10

Eukaryotic ribosomes contain an acidic ribosomal protein of about 38 kDa which shows immunological cross-reactivity with the 13 kDa-type acidic ribosomal proteins that are related to L7/L12 of bacterial ribosomes. By using a cDNA clone for 38 kDa-type acidic ribosomal protein A0 from the yeast Saccharomyces cerevisiae, we have cloned a genomic DNA encoding A0 and determined the sequence of 1,614 nucleotides including about 500 nucleotides in the 5'-flanking region. The gene lacks introns and possesses two boxes homologous to upstream activation sequences (UASrpg) in the 5'-flanking region. The amino acid sequence of A0 deduced from the nucleotide sequence shows that A0 shares a highly similar carboxyl-terminal region of about 40 amino acids in length with 13 kDa-type acidic ribosomal proteins, including an identical carboxyl-terminal, DDDMGFGLFD. In the amino-terminal region A0 contains an arginine-rich segment which shows a low but distinct similarity to that of bacterial ribosomal protein L10 through which L10 is thought to bind to 23S rRNA. On the other hand, the carboxyl-terminal half of A0 is enriched with hydrophobic amino acid residues including four pairs of phenylalanine residues which are all conserved in a human homologue.     

Construction of Signature-tagged Mutant Library in Mesorhizobium loti as a Powerful Tool for Functional Genomics

Rhizobia are nitrogen-fixing soil bacteria that establish endosymbiosis with some leguminous plants. The completion of several rhizobial genome sequences provides opportunities for genome-wide functional studies of the physiological roles of many rhizobial genes. In order to carry out genome-wide phenotypic screenings, we have constructed a large mutant library of the nitrogen-fixing symbiotic bacterium, Mesorhizobium loti, by transposon mutagenesis. Transposon insertion mutants were generated using the signature-tagged mutagenesis (STM) technique and a total of 29 330 independent mutants were obtained. Along with the collection of transposon mutants, we have determined the transposon insertion sites for 7892 clones, and confirmed insertions in 3680 non-redundant M. loti genes (50.5% of the total number of M. loti genes). Transposon insertions were randomly distributed throughout the M. loti genome without any bias toward G+C contents of insertion target sites and transposon plasmids used for the mutagenesis. We also show the utility of STM mutants by examining the specificity of signature tags and test screenings for growth- and nodulation-deficient mutants. This defined mutant library allows for genome-wide forward- and reverse-genetic functional studies of M. loti and will serve as an invaluable resource for researchers to further our understanding of rhizobial biology.Key words: Mesorhizobium loti, signature-tagged mutagenesis, mutant library, reverse genetics     

Fluorinated and rearranged gedunin derivatives

When treated with (diethylamino)sulfur trifluoride (DAST), 11α-hydroxygedunin gave 11β-fluorogedunin and 9,11-didehydrogedunin, whereas deacetylgedunin afforded two skeletal rearranged products 6 and 7, in which the Me-30 had shifted from position 8 to position 7. Of those products, 11β-fluorogedunin and 6 were shown to be more cytotoxic than gedunin on P-388 leukemia cells.     

Enhanced Expression of Fibronectin during in Vivo Cellular Aging of Human Vascular Endothelial Cells and Skin Fibroblasts

Vascular endothelial cells are thought to play an important role in human aging as their senescence and/or detachment from vascular wall contribute to arteriosclerosis and high blood pressure in elderly persons. Since fibronectin is necessary for cell attachment and spreading and its increased expression has been reported in aging fibroblasts, we checked its expression in aortic endothelial cells aged in vivo. We found that the steady-state level of fibronectin expression increases with increasing donor age, while the labeling index of cultured cells decreases with age. The increased level of fibronectin expression correlated well with an increase in cell area. To explore whether these changes were a reflection of exhaustion of proliferation potential in vivo, we examined fibronectin expression in human umbilical vein endothelial cells aging in vitro. Very similar results were obtained, supporting the idea that vascular endothelial cells age in vivo by using up division potential. When we examined the expression of fibronectin in human skin fibroblasts aged in vivo and fetal lung fibroblasts aged in vitro, we obtained similar results. In conclusion, the level of expression of fibronectin and cell size increase during in vivo and in vitro aging of both endothelial cells and fibroblasts in a coordinate manner.     

Tissue distribution of placental leucine aminopeptidase/oxytocinase during mouse pregnancy.

Placental leucine aminopeptidase (P-LAP), also called oxytocinase, is an enzyme responsible for hydrolyzing oxytocin. This enzyme is identical to cystine aminopeptidase. We examined the tissue distribution of P-LAP in normal adult mice and also in mothers and fetuses during mouse pregnancy using immunohistochemical (IHC) analysis. P-LAP-immunoreactive protein was expressed in various organs in a cell- and gestational stage-dependent manner. In the kidney, P-LAP was located in distal and collecting tubules but not in proximal tubules. The islet of Langerhans in the maternal pancreas stained positively for P-LAP in the periphery in early gestation. This staining pattern changed so that both the periphery and inner cells were positive during mid-gestation and finally only inner cells were positive in late gestation. Among the hematopoietic cells in the fetal liver, only megakaryocytes showed strong expression of P-LAP. The staining intensity increased with gestation on the apical surface of trophoblasts in the placental labyrinth. These data demonstrate that P-LAP is present in a variety of tissues, and its presence is affected by pregnancy and fetal development. Therefore, P-LAP may play a significant role in various physiological processes in non-pregnant, pregnant, and fetal mice.     

Detection and partial characterization of the chromatin-associated proteases of yeast Saccharomyces cerevisiae

The chromatin fraction was prepared from yeast Saccharomyces cerevisiae free from cytoplasmic contamination except for a trace of mitochondria. When the yeast chromatin was incubated with histones as a substrate it showed three peaks of proteolytic activity as approximately pH 4, pH 7 and pH 11. These activities were separated from each other by differential extractions from chromatin and successive gel filtration through Sephadex G-100. Proteases were partially characterized by affinity labeling with [3H]diisopropylfluorophosphate (iPr2P-F) and by various protease inhibitors. The neutral and the alkaline proteases were serine proteases with a molecular mass of 35 kDa and 25 kDa respectively. The acidic protease showed a molecular size larger than 100 kDa on the gel filtration, and was probably an aspartyl protease because it was most strongly inhibited by pepstatin. A iPr2P-F-binding protein with a molecular mass of 66 kDa, found in chromatin, was likely to be converted to the alkaline protease of 25 kDa when chromatin was incubated at pH 10 or in 6 M urea/0.1 M phosphoric acid at the extraction. The distribution of proteolytic activities and iPr2P-F-binding proteins were compared among chromatins from different strains and from cells in different growth phases and it was found that these three proteases were present in all of them but with different proportions. Considering that rat liver chromatin contains equivalents to these proteases [Tsurugi, K. and Ogata, K. (1982) J. Biochem. (Tokyo) 92, 1369-1381], the results suggested that they play some important roles in the function of eukaryotic chromatin.     

DNA sequence and mutational analysis of rhizobitoxine biosynthesis genes in Bradyrhizobium elkanii.

We cloned and sequenced a cluster of genes involved in the biosynthesis of rhizobitoxine, a nodulation enhancer produced by Bradyrhizobium elkanii. The nucleotide sequence of the cloned 28.4-kb DNA region encompassing rtxA showed that several open reading frames (ORFs) were located downstream of rtxA. A large-deletion mutant of B. elkanii, USDA94 Delta rtx::Omega 1, which lacks rtxA, ORF1 (rtxC), ORF2, and ORF3, did not produce rhizobitoxine, dihydrorhizobitoxine, or serinol. The broad-host-range cosmid pLAFR1, which contains rtxA and these ORFs, complemented rhizobitoxine production in USDA94 Delta rtx::Omega 1. Further complementation experiments involving cosmid derivatives obtained by random mutagenesis with a kanamycin cassette revealed that at least rtxA and rtxC are necessary for rhizobitoxine production. Insertional mutagenesis of the N-terminal and C-terminal regions of rtxA indicated that rtxA is responsible for two crucial steps, serinol formation and dihydrorhizobitoxine biosynthesis. An insertional mutant of rtxC produced serinol and dihydrorhizobitoxine but no rhizobitoxine. Moreover, the rtxC product was highly homologous to the fatty acid desaturase of Pseudomonas syringae and included the copper-binding signature and eight histidine residues conserved in membrane-bound desaturase. This result suggested that rtxC encodes dihydrorhizobitoxine desaturase for the final step of rhizobitoxine production. In light of results from DNA sequence comparison, gene disruption experiments, and dihydrorhizobitoxine production from various substrates, we discuss the biosynthetic pathway of rhizobitoxine and its evolutionary significance in bradyrhizobia.