Background Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO) when the latter is produced at low concentrations. This enzyme exists mainly as a heterodimer consisting of one α and one β subunit and converts GTP to the second intracellular messenger cGMP. In turn, cGMP plays a key role in regulating several physiological processes in the nervous system. The aim of the present study was to explore the effects of a NO donor on sGC activity and its protein and subunit mRNA levels in a neural cell model. Results Continuous exposure of bovine adrenal chromaffin cells in culture to the nitric oxide donor, diethylenetriamine NONOate (DETA/NO), resulted in a lower capacity of the cells to synthesize cGMP in response to a subsequent NO stimulus. This effect was not prevented by an increase of intracellular reduced glutathione level. DETA/NO treatment decreased sGC subunit mRNA and β1 subunit protein levels. Both sGC activity and β1 subunit levels decreased more rapidly in chromaffin cells exposed to NO than in cells exposed to the protein synthesis inhibitor, cycloheximide, suggesting that NO decreases β1 subunit stability. The presence of cGMP-dependent protein kinase (PKG) inhibitors effectively prevented the DETA/NO-induced down regulation of sGC subunit mRNA and partially inhibited the reduction in β1 subunits. Conclusions These results suggest that activation of PKG mediates the drop in sGC subunit mRNA levels, and that NO down-regulates sGC activity by decreasing subunit mRNA levels through a cGMP-dependent mechanism, and by reducing β1 subunit stability.

The discovery of small molecules that act as agonists and antagonists of the Hedgehog-Gli signaling pathway, which plays important roles in the embryo and adult, opens a new avenue for the treatment of diseases caused by aberrant suppression or activation of this complex pathway.

Background NGF exerts a variety of actions including promotion of neuronal differentiation and survival. The PC12 rat pheochromocytoma cell line has proved valuable for studying how NGF works and has revealed that the NGF mechanism includes regulation of gene expression. Accordingly, we used SAGE (Serial Analysis of Gene Expression) to compare levels of specific transcripts in PC12 cells before and after long-term NGF exposure. Of the approximately 22,000 transcripts detected and quantified, 4% are NGF-regulated by 6-fold or more. Here, we used database information to identify transcripts in our SAGE libraries that encode ribosomal proteins and have compared the effect of NGF on their relative levels of expression. Results Among the transcripts detected in our SAGE analysis, 74 were identified as encoding ribosomal proteins. Ribosomal protein transcripts were among the most abundantly expressed and, for naive and NGF-treated PC12 cells, represented 5.2% and 3.5%, respectively, of total transcripts analyzed. Surprisingly, nearly half of ribosomal protein transcripts underwent statistically significant NGF-promoted alterations in relative abundance, with changes of up to 5-fold. Of the changes, approximately 2/3 represented decreases. A time course revealed that the relative abundance of transcripts encoding RPL9 increases within 1 hr of NGF treatment and is maximally elevated by 8 hr. Conclusions These data establish that NGF selectively changes expression of ribosomal protein transcripts. These findings raise potential roles for regulation of ribosomal protein transcripts in NGF-promoted withdrawal from the cell cycle and neuronal differentiation and indicate that regulation of individual ribosomal protein transcripts is cell- and stimulus-specific.

Background The P-glycoprotein (P-gp), an ATP binding cassette transmembrane transporter, is expressed by astrocytes in the adult brain, and is positively modulated during astrogliosis. In a search for factors involved in this modulation, P-gp overexpression was studied in long-term in vitro astroglial cultures. Results Surprisingly, most factors that are known to induce astroglial activation in astroglial cultures failed to increase P-gp expression. The only effective proteins were IFNγ and those belonging to the IL-6 family of cytokines (IL-6, LIF, CT-1 and CNTF). As well as P-gp expression, the IL-6 type cytokines - but not IFNγ - stimulated the expression of endogenous CNTF in astrocytes. In order to see whether an increased intracellular level of CNTF was necessary for induction of P-gp overexpression by IL-6 type cytokines, by the same cytokines analysis was carried out on astrocytes obtained from CNTF knockout mice. In these conditions, IFNγ produced increased P-gp expression, but no overexpression of P-gp was observed with either IL-6, LIF or CT-1, pointing to a role of CNTF in the intracellular signalling pathway leading to P-gp overexpression. In agreement with this suggestion, application of exogenous CNTF -which is internalised with its receptor - produced an overexpression of P-gp in CNTF-deficient astrocytes. Conclusions These results reveal two different pathways regulating P-gp expression and activity in reactive astrocytes, one of which depends upon the intracellular concentration of CNTF. This regulation of P-gp may be one of the long searched for physiological roles of CNTF.

Background Nitrogen fixation gene expression in Sinorhizobium meliloti, the alfalfa symbiont, depends on a cascade of regulation that involves both positive and negative control. On top of the cascade, the two-component regulatory system FixLJ is activated under the microoxic conditions of the nodule. In addition, activity of the FixLJ system is inhibited by a specific anti-kinase protein, FixT. The physiological significance of this negative regulation by FixT was so far unknown. Results We have isolated by random Tn5 mutagenesis a S. meliloti mutant strain that escapes repression by FixT. Complementation test and DNA analysis revealed that inactivation of an asparagine synthetase-like gene was responsible for the phenotype of the mutant. This gene, that was named asnO, encodes a protein homologous to glutamine-dependent asparagine synthetases. The asnO gene did not appear to affect asparagine biosynthesis and may instead serve a regulatory function in S. meliloti. We provide evidence that asnO is active during symbiosis . Conclusions Isolation of the asnO mutant argues for the existence of a physiological regulation associated with fixT and makes it unlikely that fixT serves a mere homeostatic function in S. meliloti. Our data suggest that asnO might control activity of the FixT protein, in a way that remains to be elucidated. A proposed role for asnO might be to couple nitrogen fixation gene expression in S. meliloti to the nitrogen needs of the cells.

Background NFATp is one member of a family of transcriptional activators whose nuclear accumulation and hence transcriptional activity is regulated in mammalian cells. Human NFATp exists as a phosphoprotein in the cytoplasm of naive T cells. Upon antigen stimulation, NFATp is dephosphorylated, accumulates in nuclei, and functions to regulate transcription of genes including those encoding cytokines. While the properties of the DNA binding domain of NFATp have been investigated in detail, biochemical studies of the transcriptional activation and regulated association with nuclei have remained unexplored because of a lack of full length, purified recombinant NFATp. Results We developed methods for expressing and purifying full length recombinant human NFATp that has all of the properties known to be associated with native NFATp. The recombinant NFATp binds DNA on its own and cooperatively with AP-1 proteins, activates transcription in vitro, is phosphorylated, can be dephosphorylated by calcineurin, and exhibits regulated association with nuclei in vitro. Importantly, activation by recombinant NFATp in a reconstituted transcription system required regions of the protein outside of the central DNA binding domain. Conclusions We conclude that NFATp is a bona fide transcriptional activator. Moreover, the reagents and methods that we developed will facilitate future studies on the mechanisms of transcriptional activation and nuclear accumulation by NFATp, a member of an important family of transcriptional regulatory proteins.

Background: The germ cell nuclear factor (GCNF, also known as retinoid acid receptor-related testis-associated receptor, neuronal cell nuclear receptor or NR6A1) is an orphan receptor in the nuclear receptor superfamily found in mammals, amphibians and fish. The mouse Gcnf gene is expressed in the placenta and the developing nervous system and germ cells, and responds to retinoic acid. Results: We have defined the intron-exon structure of the mouse Gcnf gene and found that it contains 11 exons. Exons 1-4 encode the 75 amino acid amino-terminal domain and exon 4 also encodes the core DNA-binding domain. The carboxy-terminal extension is encoded by exon 5, exons 6 and 7 encode the hinge region, and exons 7-11 encode the putative ligand-binding domain. Unusually, the two zinc-finger motifs in the DNA-binding domain are encoded by separate exons. Conclusions: The protein-coding region of GCNF is contained in 11 exons. The genomic structure of this nuclear receptor gene will be useful for further studies.

Background Luteinizing hormone secreted by the anterior pituitary gland regulates gonadal function. Luteinizing hormone secretion is regulated both by alterations in gonadotrope responsiveness to hypothalamic gonadotropin releasing hormone and by alterations in gonadotropin releasing hormone secretion. The mechanisms that determine gonadotrope responsiveness are unknown but may involve regulators of G protein signaling (RGSs). These proteins act by antagonizing or abbreviating interaction of Gα proteins with effectors such as phospholipase Cβ. Previously, we reported that gonadotropin releasing hormone-stimulated second messenger inositol trisphosphate production was inhibited when RGS3 and gonadotropin releasing hormone receptor cDNAs were co-transfected into the COS cell line. Here, we present evidence for RGS3 inhibition of gonadotropin releasing hormone-induced luteinizing hormone secretion from cultured rat pituitary cells. Results A truncated version of RGS3 (RGS3T = RGS3 314–519) inhibited gonadotropin releasing hormone-stimulated inositol trisphosphate production more potently than did RSG3 in gonadotropin releasing hormone receptor-bearing COS cells. An RSG3/glutathione-S-transferase fusion protein bound more 35S-Gqα than any other member of the G protein family tested. Adenoviral-mediated RGS3 gene transfer in pituitary gonadotropes inhibited gonadotropin releasing hormone-stimulated luteinizing hormone secretion in a dose-related fashion. Adeno-RGS3 also inhibited gonadotropin releasing hormone stimulated 3H-inositol phosphate accumulation, consistent with a molecular site of action at the Gqα protein. Conclusions RGS3 inhibits gonadotropin releasing hormone-stimulated second messenger production (inositol trisphosphate) as well as luteinizing hormone secretion from rat pituitary gonadotropes apparently by binding and suppressing the transduction properties of Gqα protein function. A version of RGS3 that is amino-terminally truncated is even more potent than intact RGS3 at inhibiting gonadotropin releasing hormone-stimulated inositol trisphosphate production.

Background: Germ-cell nuclear factor (GCNF, NR6AI) is an orphan nuclear receptor. Its expression pattern suggests it functions during embryogenesis, in the placenta and in germ-cell development. Mouse GCNF cDNA codes for a protein of 495 amino acids, whereas the four reported human cDNA variants code for proteins of 454 to 480 amino acids. Apart from this size difference, there is sequence conservation of up to 98.7%. To elucidate the genomic structure that gives rise to the different human GCNF mRNAs, the sequence information of the human GCNF locus is compared to the previously reported structure of the mouse locus. Results: The genomic structures of the mouse and human GCNF genes are highly conserved. The comparison reveals that the shorter human protein results from skipping the 45 base-pair third exon. Three different human isoforms - GCNF-1, GCNF-2a and GCNF-2b - are generated by differential usage of alternative splice acceptor sites of the fourth and the seventh exon. Conclusion: By homology with the mouse gene, 11 GCNF coding exons can be defined on human chromosome 9. All human GCNF cDNAs identified so far are, however, derived from mRNAs generated by splicing the fourth to the second exon. Although the genomic sequence is highly conserved, the analysis suggests that alternative splicing generates a higher complexity of human GCNF isoforms compared with the situation in the mouse.