Vasanti Natarajan

Molecular cloning and predicted full‐length amino acid sequence of the type Iβ isozyme of cGMP‐dependent protein kinase from human placenta

In this study we report the isolation and characterization of three overlapping cDNA clones for the type Iβ isozyme of cGMP‐dependent protein kinase (cGK) from human placenta libraries. The composite sequence was 3740 nucleotides long and contained 58 nucleotides from the 5′‐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 Iβ cGK protein contained 686 amino acids including the initiator methionine, and had an estimated molecular mass of 77 803 Da. On comparison to the published amino acid sequence of bovine lung Iα, human placenta Iβ cGK differed by only two amino acids in the carboxyl‐terminal region (amino acids 105–686). In contrast, the amino‐terminal region of the two proteins was markedly different (only 36% similarity), and human Iβ cGK was 16 amino acids longer. In a specific region in the amino‐terminus (amino acids 63–75), 12 out of 13 amino acids of the human Iβ cGK were identical to the partial amino acid sequence recently published for a new Iβ isoform of cGK from bovine aorta. Northern blot analysis demonstrated a human Iβ 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 demonstrated 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.

RAR-, not RXR, ligands inhibit cell activation and prevent apoptosis in B-Lymphocytes

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 RARα, RARγ, and RXRα, but not RARβ, RXRβ, or RXRγ. 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. J. Cell. Physiol. 175:68–77, 1998.

The Human Gene for the Regulatory Subunit RIα of Cyclic Adenosine 3′,5′-Monophosphate-Dependent Protein Kinase: Two Distinct Promoters Provide Differential Regulation of Alternately Spliced Messenger Ribonucleic Acids1

The present study reports the exon-intron organization of the human RIα gene of cAMP-dependent protein kinase and approximately 2 kilobases (kb) of the 5′-flanking region obtained by isolation and sequencing of several phage clones from human genomic libraries. The RIα gene is composed of nine coding exons of varying lengths, separated by introns, giving the gene a total length of at least 21 kb. Our recent cloning of a processed RIα pseudogene with a 5′-noncoding region different from the previously reported RIα complementary DNA indicated that the RIα gene may have multiple leader exons giving rise to alternately spliced messenger RNAs (mRNAs). Reverse transcription of human testis RNA followed by PCR identified two different RIα mRNA species (RIα1a and RIα1b) containing distinct 5′-sequences due to alternately splicing the gene. The previously known RIα1b mRNA revealed low constitutive expression in a human B lymphoid cell line (Reh) and was stimulated only 4- to 6-fold by treatment with cAMP. In contr...

Expression of retinoic acid receptor and retinoid X receptor subtypes in rat liver cells: implications for retinoid signalling in parenchymal, endothelial, Kupffer and stellate cells.

In the present study, a systematic examination of the relative expression pattern of the nuclear retinoid receptors (RAR and RXR) in various liver cells was performed. Our data demonstrate that RXRalpha is the dominant receptor in all liver cells, and that RARbeta is also expressed at a high level in most cells. More specifically, RARbeta and RARalpha were the most predominant of the RAR subtypes in parenchymal cells, while all three RAR subtypes were equally expressed in endothelial and Kupffer cells. The total level of expression of all the RXR subtypes were larger than the total level of expression of all the RAR subtypes in all liver cells. This is in agreement with the observation that RXR is a heterodimer partner not only for RAR, but also for other members in the steroid/thyroid receptor superfamily of ligand-dependent transcription factors.

Secretion of N-(4-hydroxyphenyl) retinamide-retinol-binding protein from liver parenchymal cells: Evidence for reduced affinity of the complex for transthyretin

The synthetic retinoid 4‐HPR has been shown to markedly lower the plasma concentration of both retinol and RBP in rats and humans. We have studied the effect of 4‐HPR on the secretion of retinol‐RBP from liver cells in vivo and in vitro. In rats maintained with a normal diet, a vitamin A‐deficient diet or a normal diet supplemented with 4‐HPR, chylomicrons [3H]retinyl esters were rapidly cleared from the plasma. The secretion of chylomicron‐derived [3H]retinol from tissues to the circulation, however, was different. In control rats, the lymph‐derived [3H]retinol peaked after about 2 hr, whereas 4‐HPR treatment effectively reduced this peak of [3H]retinol. Our results suggest that 4‐HPR inhibits secretion of retinol‐RBP from the liver. Therefore, we decided to study the effect of 4‐HPR on the secretion of RBP using the human hepatoma cell line HepG2. Retinol and 4‐HPR were found to induce the secretion of RBP. The medium from cells treated with 4‐HPR was immunoprecipitated with antibodies against human RBP. HPLC analysis of the precipitated RBP revealed the presence of 4‐HPR. When the medium from cells incubated with either 4‐HPR or retinol was applied to a TTR affinity column, we found that RBP from cells incubated with 4‐HPR had a considerably reduced affinity for TTR. We conclude that 4‐HPR binds RBP and thereby induces secretion of RBP in HepG2 cells, and that the secreted 4‐HPR‐RBP complex has a reduced affinity for TTR. This observation may explain the 4‐HPR‐induced reduction of plasma retinol and RBP observed in in vivo studies. Int. J. Cancer 71:654‐659, 1997.

Localization of the human gene for the type I cyclic GMP-dependent protein kinase to chromosome 10.

We have recently characterized cDNAs and genomic DNA fragments for human type I cGMP-dependent protein kinase (cGK). By probing human x hamster hybrid cell lines with a 1.2-kb intron fragment from the human type I cGK gene, we identified a 5.9-kb BglII restriction fragment and localized it to human chromosome 10. In situ hybridization analyses using 3H-labeled cDNA and genomic DNA probes for the human type I cGK to human metaphase chromosomes supported the somatic cell hybrid data and indicated that the gene (PRKG1B; protein kinase, cGMP-dependent) maps to 10p11.2----q11.2.

The Human Type I cGMP-Dependent Protein Kinase Gene

The type I cGMP-dependent protein kinase has been shown to play a crucial role in relaxation of vascular smooth muscle and is believed to mediate its effects via regulation of the calcium levels in the cell. Two isoforms of type I cGMP-dependent protein kinase, designated type Iα and type Iβ have been shown to exist. With exception of the amino terminal part of the molecules the two isoforms are identical. To elucidate which genetic mechanism is responsible for the existence of two isoforms we have undertaken the task of characterizing the human gene(s) for these proteins. Using cDNA probes encoding the type Iβ cGMP-dependent protein kinase (Sandberg et al 1989), 8 exons covering approximately half the coding region have been isolated so far. Several exon/intron splice junctions have been conserved between Drosophila and human cGMP-dependent protein kinase.