The mes1+ gene of the fission yeast Schizosaccharomyces pombe is essential for the second meiotic division. We have cloned a 1.1-kb HindIII fragment containing mes1+ by complementation from an S. pombe genomic library. Sequencing of the genomic and cDNA fragments indicates the existence of one small intron of 75 nucleotides, although both the 5'(G/GTTAGT) and 3'(CAG/T) intron-exon junctions deviate from the consensus sequences proposed for S. pombe. The putative translation product of the mature mes1+ mRNA is a 11-kDa protein of 101 amino acids which has no significant homology to any previously-reported proteins. Disruption of mes1 has no effect on cell growth but causes an arrest of meiosis before the second meiotic division. Northern-blot analysis revealed that mes1+ was preferentially transcribed under conditions of nitrogen starvation. When a h90 homothallic strain was shifted to a nitrogen-deficient medium, a pre-mRNA accumulated and then was gradually processed to generate a mature mRNA. This splicing did not occur in either a heterothallic haploid strain or in a homothallic mei2 mutant strain which was defective in the initiation of meiosis. Expression of the first exon alone was not able to suppress the mes1 null allele. These results indicate that mes1+ is required for the completion of meiosis, that splicing is required for the function of the mes1+ gene, and that this splicing requires the function of the mei2+ product.
This study examined whether tetrahydrobiopterin (BH4) stimulates angiogenesis by measuring the formation of tube-like structures in vascular endothelial cells. Bovine aortic endothelial cells that were cultured between two layers of collagen type I gel formed tube-like networks. Addition of BH4 or sepiapterin, a precursor of BH4 synthesis, stimulated the formation of tube-like structures. The sepiapterin-stimulated tube formation was completely inhibited by co-treatment with N-acetylserotonin, an inhibitor of sepiapterin reductase. These findings show that BH4 stimulates in vitro angiogenesis in vascular endothelial cells.
Using mouse osteoclast-like cells (OCs), we have shown that treatment with glucocorticoids (GCs) resulted in an increase in calcitonin (CT) binding by enhancing CT receptor (CTR) gene transcription. Additionally, treatment with GCs demonstrated increased sensitivity to CT. There is, however, scant information on the effects of GC or CTR regulation by GCs in human osteoclasts. In this study we examined CTR regulation by GCs and the effects of GCs and CT together in human OCs. OCs were prepared by treatment of peripheral blood mononuclear cells in vitro with soluble receptor activator of nuclear factor-kappaB ligand and macrophage colony-stimulating factor. Treatment of mature OCs with dexamethasone (Dex) resulted in a dose- and time-dependent increase in [(125)I]salmon CT (sCT) binding capacity. Treatment with Dex enhanced CTR messenger RNA (mRNA) expression, suggesting that CTR up-regulation is at least partly due to an increase in de novo CTR synthesis. Triamcinolone and prednisolone reproduced the Dex effect on [(125)I]sCT-specific binding and CTR mRNA expression, but 17beta-estradiol, progesterone, dehydroepiandrosterone, and aldosterone did not. A Scatchard plot analysis showed that Dex enhanced CTR number with a minimal change in the affinity to sCT. Autoradiographic studies using [(125)I]sCT showed that Dex enhanced the CTR density on individual multinuclear OCs. Up-regulation of [(125)I]sCT-specific binding and CTR mRNA expression was seen even in the presence of sCT, but the enhancement diminished subsequently at later times (36-48 h after sCT removal), which was consistent with our previous observation in mouse OCs. This suggests that GCs and CTs act on CTR expression differently, consistent with our previous work using mouse OCs, in which we found that GCs increased transcription of CTR gene expression, whereas CT reduced CTR mRNA stability. The results obtained in this study show that GC increased CTR expression and sensitivity to CT in cells of the human osteoclast lineage and provide the basis for understanding the beneficial effects of combination treatment with GCs and CTs in malignancy-associated hypercalcemia.
Summary We examined the effects of insulin on tetrahydrobiopterin (BH4) synthesis in mouse brain microvascular endothelial cells (MBMECs). Treatment of MBMECs with insulin increased the intracellular BH4 content in a time- and concentration-dependent manner. The insulin-induced increase in BH4 content was inhibited by treatment with 2,4-diamino-6-hydroxypyrimidine, a selective inhibitor of GTP cyclohydrolase I, and Nacetylserotonin, a selective inhibitor of sepiapterin reductase. These findings indicate that insulin stimulates BH4 synthesis in MBMECs through a de novo synthetic pathway of BH4.
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