Familial hyperaldosteronism type II: Description of a large kindred and exclusion of the aldosterone synthase (CYP11B2) gene

Familial hyperaldosteronism type II (FH-II) is characterized by autosomal dominant inheritance and hypersecretion of aldosterone due to adrenocortical hyperplasia or an aldosterone-producing adenoma; unlike FH type I (FH-I), hyperaldosteronism in FH-II is not suppressible by dexamethasone. Of a total of 17 FH-II families with 44 affected members, we studied a large kindred with 7 affected members that was informative for linkage analysis. Family members were screened with the aldosterone/PRA ratio test; patients with aldosterone/PRA ratio greater than 25 underwent fludrocortisone/salt suppression testing for confirmation of autonomous aldosterone secretion. Postural testing, adrenal gland imaging, and adrenal venous sampling were also performed. Individuals affected by FH-II demonstrated lack of suppression of plasma A levels after 4 days of dexamethasone treatment (0.5 mg every 6 h). All patients had neg ative genetic testing for the defect associated with FH-I, the CYP11B1/CYP11B2 hybrid gene. Genetic linkage was then examined between FH-II and aldosterone synthase (the CYP11B2 gene) on chromosome 8q. A polyadenylase repeat within the 5'-region of the CYP11B2 gene and 9 other markers covering an approximately 80-centimorgan area on chromosome 8q21-8qtel were genotyped and analyzed for linkage. Two-point logarithm of odds scores were negative and ranged from -12.6 for the CYP11B2 polymorphic marker to -0.98 for the D8S527 marker at a recombination distance (theta) of 0. Multipoint logarithm of odds score analysis confirmed the exclusion of the chromosome 8q21-8qtel area as a region harboring the candidate gene for FH-II in this family. We conclude that FH-II shares autosomal dominant inheritance and hyperaldosteronism with FH-I, but, as demonstrated by the large kindred investigated in this report, it is clinically and genetically distinct. Linkage analysis demonstrated that the CYP11B2 gene is not responsible for FH-II in this family; furthermore, chromosome 8q21-8qtel most likely does not harbor the genetic defect in this kindred.

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.

Retinal and lenticular ultrastructure in the aestivating salamanderfish, Lepidogalaxias salamandroides (Galaxiidae, Teleostei) with special reference to a new type of photoreceptor mosaic

The salamanderfish, Lepidogalaxias salamandroides (Galaxiidae, Teleostei) is endemic to southwestern Australia and inhabits shallow, freshwater pools which evaporate during the hot summer months. Burrowing into the substrate in response to falling water levels allows these fish to aestivate for extended periods of time while encapsulated in a mucous cocoon even when the pools contain no water. Only a few minutes after a major rainfall, these fish emerge into relatively clear water which subsequently becomes laden with tannin, turning the water black and reducing the pH to approximately 4.3. As part of a large study of the visual adaptations of this unique species, the retinal and lenticular morphology of the aestivating salamanderfish is examined at the level of the light and electron microscopes. The inner retina is highly vascularised by a complex system of vitreal blood vessels, while the outer retina receives a blood supply by diffusion from a choriocapillaris. This increased retinal blood supply may be an adaptation for reducing the oxygen tension during critical periods of aestivation. Large numbers of Muller cells traverse the thickness of the retina from the inner to the outer limiting membranes. The ganglion cells are arranged in two ill-defined layers, separated from a thick inner nuclear layer containing two layers of horizontal cells by a soma-free inner plexiform layer. The photoreceptors can be divided into three types typical of many early actinopterygian representatives; equal double cones, small single cones and large rods (2:1:1). These photoreceptors are arranged into a unique regular square mosaic comprising a large rod bordered by four equal double cones with a small single cone located at the corner of each repeating unit. The double cones may optimise perception of mobile prey which it tracks by flexion of its head and neck and the large rods may increase sensitivity in the dark tannin-rich waters in which it lives. Each single cone also possesses a dense collection of polysomes and glycogen (a paraboloid) beneath its ellipsoid, the first such finding in teleosts. The retinal pigment epithelium possesses melanosomes, pha,oocytes and a large number of mitochondria. The anatomy of the retina and the photoreceptor mosaic is discussed in relation to the primitive phylogeny of this species and its unique life history.

NMR structure of C-terminally tagged gramicidin channels

A biotin group was covalently attached to the C terminus of gramicidin A (gA) through a linker arm comprising a glycine residue with either one (gAXB) or two caproyl groups (gAXXB). High-resolution two-dimensional NMR spectroscopy was used to determine the structure of these modified gA analogues and [Lys(16)]gramicidin A (gA-Lys) in sodium dodecyl-d(25) sulphate micelles. Gated gA ion channels based on linking a receptor group to these gA analogues have been used recently as a component in a sensing device. The conformations of the gA backbones and amino acid side chains of lysinated gA and biotinylated gA in detergent micelles were found to be almost identical to that of native gA, i.e. that of an N-terminal to N-terminal (head to head) dimer formed by two right-handed, single-stranded beta(6.3) helices. The biotin tail of the gAXB and gAXXB and the lysine extremity of gA-Lys appeared to lie outside the micelle. Thus it appears that the covalent attachment of functional groups to the C terminus of gA does not disrupt the peptide's helical configuration. Further, single channel measurements of all three gA analogues showed that functioning ion channels were preserved within a membrane environment. (C) 1999 Elsevier Science B.V. All rights reserved.

alpha-Conotoxin ImI inhibits the alpha-bungarotoxin-resistant nicotinic response in bovine adrenal chromaffin cells

The activity of alpha-conotoxin (alpha-CTX) lml, from the vermivorous marine snail Conus imperialis, has been studied on mammalian nicotinic receptors on bovine chromaffin cells and at the rat neuromuscular junction. Synthetic alpha-CTX lml was a potent inhibitor of the neuronal[ nicotinic response in bovine adrenal chromaffin cells (IC50 = 2.5 mu M, log IC50 = 0.4 +/- 0.07), showing competitive inhibition of nicotine-evoked catecholamine secretion. (alpha-CTX lml also inhibited nicotine-evoked Ca-45(2+) uptake but not Ca-45(2+) uptake stimulated by 56 mM Kr. In contrast, alpha-CTX lml had no effect at the neuromuscular junction over the concentration range 1-20 mu M. Bovine chromaffin cells are known to contain the alpha 3 beta 4, alpha 7, and (possibly) alpha 3 beta 4 alpha 5 subtypes. However, the secretory response of bovine chromaffin cells is not inhibited by alpha-bungarotoxin, indicating that alpha 7 nicotinic receptors are not involved. We propose that alpha-CTX lml interacts selectively with the functional (alpha 3 beta 4 or alpha 3 beta 4 alpha 5) nicotinic acetylcholine receptor to inhibit the neuronal-type nicotinic response in bovine chromaffin cells.

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.