No abstract

In this study we report the isolation and characterization of three overlapping cDNA clones for the type I/3 isozyme of cGMP-dependent protein kinase (cGK) from human placenta libraries. The composite sequence was 3740 nucleotides long and contained 58 nucleotides from the S-noncoding region, an open reading frame of 2061 bases including the stop codon, and a 3'noncoding region of 1621 nucleotides. The predicted full-length human type IB cGK protein contained 686 amino acids including the imtiator me~o~ne, and had an estimated molecular mass of 77 803 Da. On comparison to the published amino acid sequence of bovine lung Ia, human placenta I/I cGK differed by only two amino acids in the carboxyl-terminal region (amino acids 105686). In contrast, the amino-terminal region of the two proteins was markedly different (only 36% similarity), and human I/I cGK was 16 amino acids longer. In a specific region in the aminoterminus (amino acids 6375) 12 out of 13 amino acids of the human I/f cGK were identical to the partial amino acid sequence recently published for a new I/l isoform of cGK from bovine aorta. Northern blot analysis demonstrated a human I/l cGK mRNA, 7 kb in size, in human uterus and weakly in placenta. An mRNA of 7 kb was also observed in rat cerebellum, cerebrum, lung, kidney, and adrenal, whereas an mRNA doublet of 7.5 and 6.5 kb were observed in rat heart. Comparison of Northern and Western blot analyses demon~rat~ that the mRNA and protein for cerebellar cGK increased during the development of rats from 5 to 30 days old, whereas the 6.5 kb mRNA in rat heart declined.

No abstract

We have previously shown that retinoids inhibit activation of human peripheral blood B-lymphocytes. In the present paper, we wished to explore the involvement of nuclear retinoid-specific receptors in this process by using ligands specific for the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We found that the RAR-specific ligand TTAB reduced anti-IgM-induced B-cell activation in a dose-dependent manner. Thus, at 100 nM of TTAB, DNA synthesis was reduced by approximately 60%. In contrast, the RXR-selective ligand SR11217 had no effect on DNA synthesis. Similar findings were obtained when the expression of the activation antigen CD71 (appears late in G1) was examined. The role of retinoids in apoptosis of resting peripheral blood B-lymphocytes was examined using the same receptor-selective ligands. Again, we found that the RAR-selective ligands were more potent effectors than were the RXR-selective ligands. In spite of the inhibitory effects of retinoids on B-cell proliferation, the same retinoids significantly promoted the survival of the cells. Thus, 10 nM TTAB significantly reduced spontaneous apoptosis of in vitro cultured B-cells at day 3 from 45% to 30%, as determined by vital dye staining and DNA end-labeling. Again, the RXR-specific ligand SR11217 had no effect. Interestingly, we found that CD40 ligand was able to potentiate the retinoid-mediated inhibition of apoptosis. By reverse transcriptase polymerase chain reaction (PCR), we found that peripheral blood B-lymphocytes expressed RARalpha, RARgamma, and RXRalpha, but not RARbeta, RXRbeta, or RXRgamma. Hence, the lack of effect of the RXR-specific ligand SR11217 on growth and apoptosis was not due to absence of RXRs. In conclusion, the ability of retinoids to inhibit growth and prevent apoptosis of normal human B-lymphocytes indicates a dual role of retinoids in this cell compartment, and it appears that both effects of retinoids are mediated via RARs and not RXRs.