Evidence for persistent dysfunction of wild-type aldosterone synthase gene in glucocorticoid-treated familial hyperaldosteronism type I

Background In familial hyperaldosteronism type I (FH-I), glucocorticoid treatment suppresses adrenocorticotrophic hormone-regulated hybrid gene expression and corrects hyperaldosteronism. Objective To determine whether the wild-type aldosterone synthase genes, thereby released from chronic suppression, are capable of functioning normally. Methods We compared mid-morning levels of plasma potassium, plasma aldosterone, plasma renin activity (PRA) and aldosterone : PRA ratios, measured with patients in an upright position, and responsiveness of aldosterone levels to infusion of angiotensin II (AII), for 11 patients with FH-I before and during long-term (0.8-14.3 years) treatment with 0.25-0.75 mg/day dexamethasone or 2.5-10 mg/day prednisolone. Results During glucocorticoid treatment, hypertension was corrected in all. Potassium levels, which had been low (< 3.5 mmol/l) in two patients before treatment, were normal in all during treatment (mean 4.0 +/- 0.1 mmol/l, range 3.5-4.6). Aldosterone levels during treatment [13.2 +/- 2.1 ng/100 ml (mean +/- SEM)] were lower than those before treatment (20.1 +/- 2.5 ng/100 ml, P < 0.05). PRA levels, which had been suppressed before treatment (0.5 +/- 0.2 ng/ml per h), were unsuppressed during treatment (5.1 +/- 1.5 ng/ml per h, P < 0.01) and elevated (> 4 ng/ml per h) in six patients. Aldosterone : PRA ratios, which had been elevated (> 30) before treatment (101.1 +/- 25.9), were much lower during treatment (4.1 +/- 1.0, P < 0.005) and below normal (< 5) in eight patients. Surprisingly, aldosterone level, which had not been responsive (< 50% rise) to infusion of AII for all 11 patients before treatment, remained unresponsive for 10 during treatment. Conclusions Apparently regardless of duration of glucocorticoid treatment in FH-I, aldosterone level remains poorly responsive to AII, with a higher than normal PRA and a low aldosterone : PRA ratio. This is consistent with there being a persistent defect in functioning of wild-type aldosterone synthase gene. (C) Rapid Science Publishers ISSN 0263-6352.

(-)-CGP 12177 causes cardiostimulation and binds to cardiac putative beta(4)-adrenoceptors in both wild-type and beta(3)-adrenoceptor knockout mice

Some blockers of beta(1)- and beta(2)-adrenoceptors cause cardiostimulant effects through an atypical beta-adrenoceptor (putative beta(4)-adrenoceptor) that resembles the beta(3)-adrenoceptor. It is likely but not proven that the putative beta(4)-adrenoceptor is genetically distinct from the beta(3)-adrenoceptor. We therefore investigated whether or not the cardiac atypical beta-adrenoceptor could mediate agonist effects in mice lacking a functional beta(3)-adrenoceptor gene (beta(3)KO). (-)-CGP 12177, a beta(1)- and beta(2)-adrenoceptor blocker that causes agonist effects through both beta(3)-adrenoceptors and cardiac putative beta(4)-adrenoceptors, caused cardiostimulant effects that were not different in atria from wild-type (WT) mice and beta(3)KO mice. The effects of (-)-CGP 12177 were resistant to blockade by (-)-propranolol (200 nM) but were blocked by (-)-bupranolol (1 mu M) with an equilibrium dissociation constant of 15 nM in WT and 17 nM in beta(3)KO. (-)-[H-3]CGP 12177 labeled a similar density of the putative beta(4)-adrenoceptor in ventricular membranes from the hearts of both WT (B-max = 52 fmol/mg protein) and beta(3)KO (B-max = 53 fmol/mg protein) mice. The affinity of (-)-[H-3]CGP 12177 for the cardiac putative beta(4)-adrenoceptor was not different between WT (K-d = 46 nM) and beta(3)KO (K-d = 40 nM). These results provide definitive evidence that the cardiac putative beta(4)-adrenoceptor is distinct from the beta(3)-adrenoceptor.

PCR bias toward the wild-type k-ras and p53 sequences: Implications for PCR detection of mutations and cancer diagnosis

PCR-based cancer diagnosis requires detection of rare mutations in k-ras, p53 or other genes. The assumption has been that mutant and wild-type sequences amplify with near equal efficiency, so that they are eventually present in proportions representative of the starting material. Work factor IX suggests that this assumption is invalid for one case of near-sequence identity To test the generality of this phenomenon and its relevance to cancer diagnosis, primers distant from point mutations in p53 and k-ras were used to amplify, wild-type and mutant sequences from these genes. A substantial bias against PCR amplification of mutants was observed for two regions of the p53 gene and one region of k-ras. For kras and p53, bias was observed when the wild-type and mutant sequences were amplified separately or when mixed in equal proportions before PCR. Bias was present with proofreading and non-proofreading polymerases. Mutant and wild-type segments of the factor V cystic fibrosis transmembrane conductance regulator and prothrombin genes were amplified and did not exhibit PCR bias. Therefore, the assumption of equal PCR efficiency for point mutant and wild-type sequences is invalid in several systems. Quantitative or diagnostic PCR will require validation for each locus, and enrichment strategies may be needed to optimize detection of mutants.

Contribution of beta-adrenoceptor subtypes to relaxation of colon and oesophagus and pacemaker activity of ureter in wild-type and beta(3)-adrenoceptor knockout mice

1 The smooth muscle relaxant responses to the mixed beta(3)-, putative beta(4)-adrenoceptor agonist, (-)-CGP 12177 in rat colon are partially resistant to blockade by the beta(3)-adrenoceptor antagonist SR59230A suggesting involvement of beta(3)- and putative beta(4)-adrenoceptors. We now investigated the function of the putative beta(4)-adrenoceptor and other beta-adrenoceptor subtypes in the colon, oesophagus and ureter of wild-type (WT) and beta(3)-adrenoceptor knockout (beta(3)KO) mice. 2 (-)-Noradrenaline and (-)-adrenaline relaxed KCl (30 mM)-precontracted colon mostly through beta(1)-and beta(3)-adrenoceptors to a similar extent and to a minor extent through beta(2)-adrenoceptors. In colon from beta(3)KO mice, (-)-noradrenaline was as potent as in WT mice but the effects were mediated entirely through beta(1)-adrenoceptors. (-)-CGP 12177 relaxed colon from beta(3)KO mice with 2 fold greater potency than in WT mice. The maintenance of potency for (-)-noradrenaline and increase for (-)-CGP 12177 indicate compensatory increases in beta(1)- and putative beta(4)-adrenoceptor function in beta(3)KO mice. 3 In oesophagi precontracted with 1 mu M carbachol, (-)-noradrenaline caused relaxation mainly through beta(1)-and beta(3)-adrenoceptors. (-)-CGP 12177 (2 mu M) relaxed oesophagi from WT by 61.4+/-5.1% and beta(3)KO by 67.3+/-10.1% of the (-)-isoprenaline-evoked relaxation, consistent with mediation through putative beta(4)-adrenoceptors. 4 In ureter, (-)-CGP 12177 (2 mu M) reduced pacemaker activity by 31.1+/-2.3% in WT and 31.3+/-7.5% in beta(3)KO, consistent with mediation through putative beta(4)-adrenoceptors. 5 Relaxation of mouse colon and oesophagus by catecholamines are mediated through beta(1)- and beta(3)- adrenoceptors in WT. The putative beta(4)-adrenoceptor, which presumably is an atypical state of the beta(1)-adrenoceptor, mediates the effects of(-)-CGP 12177 in colon, oesophagus and ureter.

In familial hyperaldosteronism type I, hybrid gene-induced aldosterone production dominates that induced by wild-type genes

We compared the aldosterone-producing potency of the angiotensin II-sensitive wild-type aldosterone synthase genes and the ACTH-sensitive hybrid 11 beta-hydroxylase/aldosterone synthase gene by examining aldosterone, PRA, and cortisol day-curves (2-hourly levels over 24 h) in patients with familial hyperaldosteronism type I, before and during long-term (0.8-13.5 yr) glucocorticoid treatment. In 8 untreated patients, PRA levels were usually suppressed, and aldosterone correlated strongly with cortisol (r = 0.69-0.99). Fourteen studies were performed on 10 patients receiving glucocorticoid treatment that corrected hypertension, hypokalemia, and PRA suppression in all. ACTH was markedly and continuously suppressed in 6 studies, 3 of which demonstrated strong correlations between aldosterone and PRA (r = 0.77-0.92), ACTH was only partially suppressed in the remaining 8 studies; aldosterone correlated strongly: 1) with cortisol alone in 5 (r = 0.71-0.98); 2) with cortisol (r = 0.90) and PRA (r = 0.74) in one; 3) with PRA only in one (r = 0.80); and 4) with neither PRA nor cortisol in one. Unless ACTH is markedly and continuously suppressed, aldosterone is more responsive to ACTH than to renin/angiotensin II, despite the latter being unsuppressed. This is consistent with the hybrid gene being more powerfully expressed than the wild-type aldosterone synthase genes in familial hyperaldosteronism type I.

Leaf area development of ABA-deficient and wild-type peas at two levels of nitrogen supply

The ABA-deficient wilty pea (Pisum sativum L.) and its wild-type (WT) were grown at two levels of nitrogen supply (0.5 and 5.0 mM) for 5-6 weeks from sowing, to determine whether leaf ABA status altered the leaf growth response to N deprivation. Plants were grown at high relative humidity to prevent wilting of the wilty peas. Irrespective of N supply, expanding wilty leaflets had ca 50% less ABA than WT leaflets but similar ethylene evolution rates. Fully expanded wilty leaflets had lower relative water contents (RWC) and were 10-60% smaller in area (according to the node of measurement) than WT leaflets. However, there were no genotypic differences in plant relative leaf expansion rate (RLER). Growth of both genotypes at 0.5 mM N increased the RWC of fully expanded leaflets, but did not alter ethylene evolution or ABA concentration of expanding leaflets. Plants grown at 0.5 mM N showed a 20-30% reduction in RLER, which was similar in magnitude in both wilty and WT peas. Thus, leaf ABA status did not alter the leaf growth response to N deprivation.

Heterologous RNA encapsidated in pariacoto virus-like particles forms a dodecahedral cage similar to genomic RNA in wild-type virions

The genome of some icosahedral RNA viruses plays an essential role in capsid assembly and structure. In T=3 particles of the nodavirus Pariacoto virus (PaV), a remarkable 35% of the single-stranded RNA genome is icosahedrally ordered. This ordered RNA can be visualized at high resolution by X-ray crystallography as a dodecahedral cage consisting of 30 24-nucleotide A-form RNA duplex segments that each underlie a twofold icosahedral axis of the virus particle and interact extensively with the basic N-terminal region of 60 subunits of the capsid protein. To examine whether the PaV genome is a specific determinant of the RNA structure, we produced virus-like particles (VLPs) by expressing the wild-type capsid protein open reading frame from a recombinant baculovirus. VLPs produced by this system encapsidated similar total amounts of RNA as authentic virus particles, but only about 6% of this RNA was PaV specific, the rest being of cellular or baculovirus origin. Examination of the VLPs by electron cryomicroscopy and image reconstruction at 15.4-Angstrom resolution showed that the encapsidated RNA formed a dodecahedral cage similar to that of wild-type particles. These results demonstrate that the specific nucleotide sequence of the PaV genome is not required to form the dodecahedral cage of ordered RNA.

tRNASer(CGA) differentially regulates expression of wild-type and codon-modified papillomavirus L1 genes

Exogenous transfer RNAs (tRNAs) favor translation of bovine papillomavirus 1 wild-type (wt) L1 mRNA in in vitro translation systems (Zhou et al. 1999, J. Virol., 73, 4972-4982). We, therefore, investigated whether papillomavirus (PV) wt L1 protein expression could be enhanced in eukaryotic cells following exogenous tRNA supplementation. Both Chinese hamster ovary (CHO) and Cos1 cells, transfected with PV1 wt L1 genes, effectively transcribed the genes but did not translate them. However, L1 protein translation was demonstrated following co-transfection with the L1 gene and a gene expressing tRNA(Ser)(CGA). Cell lines, stably transfected with a bovine papillomavirus 1 (BPV1) wt L1 expression construct, produced L1 protein after the transfection of the tRNA(Ser)(CGA) gene, but not following the transfection with basal vectors, suggesting that tRNA(Ser)(CGA) gene enhanced wt L1 translation as a result of endogenous tRNA alterations and phosphorylation of translation initiation factors elF4E and elF2alpha in the tRNA(Ser)(CGA) transfected L1 cell lines. The tRNA(Ser)(CGA) gene expression significantly reduced translation of L1 proteins expressed from codon-modified (HB) PV L1 genes utilizing mammalian preferred codons, but had variable effects on translation of green fluorescent proteins (GFPs) expressed from six serine GFP variants. The changes of tRNA pools appear to match the codon composition of PV wt and HB L1 genes and serine GFP variants to regulate translation of their mRNAs. These findings demonstrate for the first time in eukaryotic cells that translation of the target genes can be differentially influenced by the provision of a single tRNA expression construct.

A cell type-specific constitutive point mutant of the common beta-subunit of the human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 receptors requires the GM-CSF receptor alpha-subunit for activation

The high affinity receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a cytokine-specific alpha-subunit (hGMR alpha) and a common signal-transducing beta-subunit (hpc) that is shared with the interleukin-3 and -5 receptors, We have previously identified a constitutively active extracellular point mutant of hpc, I374N, that can confer factor independence on murine FDC-P1 cells but not BAF-B03 or CTLL-2 cells (Jenkins, B. J., D'Andrea, R. J., and Gonda, T. J. (1995) EMBO J. 14, 4276-4287), This restricted activity suggested the involvement of cell type-specific signaling molecules in the activation of this mutant. We report here that one such molecule is the mouse GMR alpha (mGMR alpha) subunit, since introduction of mGMR alpha, but not hGMR alpha, into BAF-B03 or CTLL-2 cells expressing the I374N mutant conferred factor independence, Experiments utilizing mouse/human chimeric GMR alpha subunits indicated that the species specificity lies in the extracellular domain of GMRa. Importantly, the requirement for mGMR alpha correlated with the ability of I374N (but not wild-type hpc) to constitutively associate with mGMRa. Expression of I374N in human factor-dependent UT7 cells also led to factor-independent proliferation, with concomitant up-regulation of hGMR alpha surface expression. Taken together, these findings suggest a critical role for association with GMR alpha in the constitutive activity of I374N.

Murine ventricular L-type Ca2+ current is enhanced by zinterol via beta(1)-adrenoceptors, and is reduced in TG4 mice overexpressing the human beta(2)-adrenoceptor

1 The functional coupling of B-2-adrenoceptors (beta (2)-ARs) to murine L-type Ca2+ current (I-Ca(L)) was investigated with two different approaches. The beta (2)-AR signalling cascade was activated either with the beta (2)-AR selective agonist zinterol (myocytes from wild-type mice), or by spontaneously active, unoccupied beta (2)-ARs (myocytes from TG4 mice with 435 fold overexpression of human beta (2)-ARs). Ca2+ and Ba2+ currents were recorded in the whole-cell and cell-attached configuration of the patch- clamp technique, respectively. 2 Zinterol (10 muM) significantly increased I-Ca(L) amplitude of wild-type myocytes by 19+/-5%, and this effect was markedly enhanced after inactivation of Gi-proteins with pertussis-toxin (PTX; 76+/-13% increase). However, the effect of zinterol was entirely mediated by the beta (1)-AR subtype, since it was blocked by the beta (1)-AR selective antagonist CGP 20712A (300 nM). The beta (2)-AR selective antagonist ICI 118,551 (50 nM) did not affect the response of I-Ca(L) to zinterol. 3 In myocytes with beta (2)-AR overexpression I-Ca(L) was not stimulated by the activated signalling cascade. On the contrary, I-Ca(L) was lower in TG4 myocytes and a significant reduction of single-channel activity was identified as a reason for the lower whole-cell I-Ca(L). The beta (2)-AR inverse agonist ICI 118,551 did not further decrease I-Ca(L). PTX-treatment increased current amplitude to values found in control myocytes. 4 In conclusion, there is no evidence for beta (2)-AR mediated increases of I-Ca(L) in wild-type mouse ventricular myocytes. Inactivation of Gi-proteins does not unmask beta (2)-AR responses to zinterol, but augments beta (1)-AR mediated increases of I-Ca(L). In the mouse model of beta (2)-AR overexpression I-Ca(L) is reduced due to tonic activation of Gi-proteins.

Gag-Pol supplied in trans is efficiently packaged and supports viral function in human immunodeficiency virus type 1

The intracellular trafficking and subsequent incorporation of Gag-Pol into human immunodeficiency virus type 1 (HIV-1) remains poorly defined. Gag-Pol is encoded by the same mRNA as Gag and is generated by ribosomal frameshifting. The multimerization of Gag and Gag-Pol is an essential step in the formation of infectious viral particles. In this study, we examined whether the interaction between Gag and Gag-Pol is initiated during protein translation in order to facilitate the trafficking and subsequent packaging of Gag-Pol into the virion. A conditional cotransfection system was developed in which virion formation required the coexpression of two HIV-1-based plasmids, one that produces both Gag and Gag-Pol and one that only produces Gag-Pol. The Gag-Pol proteins were either immunotagged with a His epitope or functionally tagged with a mutation (K65R) in reverse transcriptase that is associated with drug resistance. Gag-Pol packaging was assessed to determine whether the Gag-Pol incorporated into the virion was preferentially packaged from the plasmid that expressed both Gag and Gag-Pol or whether it could be packaged from either plasmid. Our data show that translation of Gag and Gag-Pol from the same mRNA is not critical for virion packaging of the Gag-Pol polyprotein or for viral function.

Codon modified human papillomavirus type 16 E7 DNA vaccine enhances cytotoxic T-lymphocyte induction and anti-tumour activity

Polynucleotide immunisation with the E7 gene of human papillomavirus (HPV) type 16 induces only moderate levels of immune response, which may in part be due to limitation in E7 gene expression influenced by biased HPV codon usage. Here we compare for expression and immunogenicity polynucleotide expression plasmids encoding wild-type (pWE7) or synthetic codon optimised (pHE7) HPV16 E7 DNA. Cos-1 cells transfected with pHE7 expressed higher levels of E7 protein than similar cells transfected with pW7. C57BL/6 mice and F1 (C57X FVB) E7 transgenic mice immunised intradermally with E7 plasmids produced high levels of anti-E7 antibody. pHE7 induced a significantly stronger E7-specific cytotoxic T-lymphocyte response than pWE7 and 100% tumour protection in C57BL/6 mice, but neither vaccine induced CTL in partially E7 tolerant K14E7 transgenic mice. The data indicate that immunogenicity of an E7 polynucleotide vaccine can be enhanced by codon modification. However, this may be insufficient for priming E7 responses in animals with split tolerance to E7 as a consequence of expression of E7 in somatic cells. (C) 2002 Elsevier Science (USA).

Prolonged retention after aggregation into secretory granules of human R183H-growth hormone (GH), a mutant that causes autosomal dominant GH deficiency type II

Human R183H-GH causes autosomal dominant GH deficiency type II. Because we show here that the mutant hormone is fully bioactive, we have sought to locate an impairment in its progress through the secretory pathway as assessed by pulse chase experiments. Newly synthesized wild-type and R183H-GH were stable when expressed transiently in AtT20 cells, and both formed equivalent amounts of Lubrol-insoluble aggregates within 40 min after synthesis. There was no evidence for intermolecular disulfide bond formation in aggregates of wild-type hormone or the R183H mutant. Both wildtype and R183H-GH were packaged into secretory granules, assessed by the ability of 1 mm BaCl2 to stimulate release and by immunocytochemistry. The mutant differed from wildtype hormone in its retention in the cells after packaging into secretory granules; 50% more R183H-GH than wild-type aggregates were retained in AtT20 cells 120 min after synthesis, and stimulated release of R183H-GH or a mixture of R183H-GH and wild-type that had been retained in the cell was reduced. The longer retention of R183H-GH aggregates indicates that a single point mutation in a protein contained in secretory granules affects the rate of secretory granule release.

tonB3 is required for normal twitching motility and extracellular assembly of type IV pili

Three mutants with Tn5-B21 insertion in tonB3 (PA0406) of Pseudomonas aeruginosa exhibited defective twitching motility and reduced assembly of extracellular pili. These defects could be complemented with wild-type tonB3.