The elastin gene is disrupted in a family with a balanced translocation t(7;16)(q11.23;q13) associated with a variable expression of the Williams-Beuren syndrome

The Williams-Beuren syndrome (WBS) is a complex developmental disorder with multisystemic manifestations including supravalvular aortic stenosis (SVAS), a so-called elfin face, a hoarse voice, and a specific cognitive phenotype. Most WBS patients have a >1 Mb deletion on one of their chromosomes 7 in q11 but except for elastin, whose haploinsufficiency causes the cardiovascular malformations, it is unknown which genes in the deletion area contribute to the phenotype. We have investigated a family with a cytogenetically balanced translocation t(7;16)(q11.23;q13) in which affected individuals manifested a broad spectrum of clinical phenotypes ranging from a hoarse voice as the only feature to the full WBS phenotype. Molecular cytogenetic and DNA sequence analyses of the translocation breakpoint showed that the cytogenetic rearrangement disrupts the elastin gene locus within intron 5 in the exact same manner in all translocation carriers. The recently described large inversion of the 7q11.23 region was not present in this family. Our data demonstrate that disruption of the elastin gene by a translocation breakpoint may cause classical WBS, atypical WBS, SVAS, or no recognisable phenotype, and provide a clear example for extensive phenotypic variability associated with a position effect in humans.     

Differential effects of calcium channel antagonists (ω-conotoxin GVIA,nifedipine, verapamil) on the electrically-evoked release of [3H]acetylcholine from the myenteric plexus,phrenic nerve and neocortex of rats

Summary Electrically-evoked release of [³H]acetylcholine from autonomic neurons (myenteric plexus), motoneurons (phrenic nerve) and the central nevous system (neocortex) was investigated in the presence and absence of the calcium channel antagonists -conotoxin GVIA, nifedipine and verapamil, whereby the same species (rat) was used in all experiments. Release of [³H]acetylcholine was measured after incubation of the tissue with [³H]choline.-Conotoxin GVIA markedly reduced (70%) the evoked release of [³H]acetylcholine from the myenteric plexus of the small intestine (IC₅₀: 0.7 nmol/l) with a similar potency at 3 and 10 Hz stimulation. An increase in the extracellular calcium concentration attenuated the inhibitory effect of -conotoxin GVIA. Release of [³H]acetylcholine from the rat neocortex was also inhibited (90%) by -conotoxin GVIA, but the potency was 19-fold lower (IC₅₀: 13 nmol/l). However, the release of [³H]acetylcholine from the phrenic nerve was not reduced by -conotoxin GVIA (100 nmol/l) at 1.8 mmol/l calcium (normal concentration), whereas -conotoxin GVIA inhibited evoked [³H]acetylcholine release by 47% at 0.9 mmol/l calcium. Neither nifedipine (0.1 and 1 mol/l) nor verapamil (0.1, 1 and 10 mol/l) modified the evoked release of [3H]acetylcholine from the myenteric plexus and the phrenic nerve.Acetylcholine release from different neurons appears to be regulated by different types of calcium channels. N-type channels play the dominant role in regulating acetylcholine release from both the myenteric plexus and the neocortex, whereas acetylcholine release from motor nerves is regulated by calcium channel(s) not yet characterized. Send offprint requests to I. Wessler at the above address     

Epithelium-derived inhibition of [3H]acetylcholine release from the isolated guinea-pig trachea

Summary To investigate presynaptic, regulatory mechanisms on parasympathetic nerve fibres innervating the airways, the release of newly-synthesized [³H]acetylcholine from the isolated trachea was studied. Reverse phase HPLC followed by liquid scintillation spectrometry was used to separate and quantify the radioactive compounds choline, phosphorylcholine and acetylcholine in the incubation medium and the tissue.During the incubation of the tracheae with [³H]choline a significant synthesis of [³H]acetylcholine (35,000 dpm/preparation) and [³H]phosphorylcholine (500,000 dpm/preparation) occurred. In epithelium-deficient tracheae the formation of [³H]phosphorylcholine was enhanced, whereas the content of [³H]acetylcholine remained unchanged. The spontaneous outflow of tritium consisted mainly of [³H]phosphorylcholine (900 dpm/3 min) and [³H]choline (800 dpm/3 min); [³H]acetylcholine was only a minor fraction (50 dpm/3 min). Electrical stimulation of tracheae with intact epithelium caused only a small release of [³H]acetylcholine (460 dpm in the sample obtained during stimulation), but a considerable outflow of [³H]phosphorylcholine (1,900 dpm) without affecting the outflow of [³H]choline. Electrical stimulation of epithelium-deficient tracheae, however, induced a substantial release of [³H]acetylcholine (2,400 dpm), but only a small outflow of [³H]phosphorylcholine. Chemical stimulation (30 mol/1 veratridine) also caused a large release of [³H]acetylcholine (1,700 dpm) without affecting the outflow of [³H]phosphorylcholine or [³H]choline. Indomethacin (3 mol/1) enhanced the electrically-evoked release of [³H]acetylcholine from tracheae with intact epithelium by 89%.The present experiments demonstrate a strong inhibition by the epithelium of the electrically-evoked release of [³H]acetylcholine from the isolated guinea-pig trachea. Cyclooxygenase products of arachidonic acid do not appear as the main mediators of the epithelium-derived inhibition of acetylcholine release. Send offprint requests to I. Wessler at the above address     

Nitric oxide synthase activity is inducible in rat,but not rabbit alveolar macrophages,with a concomitant reduction in arginase activity

Alveolar macrophages were obtained by broncho-alveolar lavage of isolated rat and rabbit lungs and cultured (2.5 × 10⁶ cells/dish) for 18 h in the absence or presence of bacterial lipopolysaccharides (LPS) alone or in combination with cytokines. Thereafter, accumulation of ³H-citrulline (NO synthase activity) and ³H-ornithine (arginase activity) were determined.During incubation of rat alveolar macrophages with ³H-arginine clear amounts of ³H-citrulline and ³H-ornithine (3.8 and 4.6% of the added ³H-arginine, respectively) were formed and most of these metabolites appeared in the incubation medium (ratios extra-/intracellular of 17 and 70 for ³H-citrulline and ³H-ornithine, respectively). When rat alveolar macrophages had been cultured with LPS the formation of ³H-citrulline was increased about 30-fold and this was accompanied by a reduction in ³H-ornithine formation of about 60%. The effects of LPS were largely attenuated by dexamethasone (10 mol/1). Inhibition of NO synthase by N^G-monomethyl-l,-arginine (l-NMMA, 100 mol/1) in LPS treated alveolar macrophages reduced the formation ³H-citrulline by more than 90% and restored the ³H-ornithine formation. After culturing in the presence of LPS the ratios extra/intracellular of ³H-citrulline and ³H-ornithine were markedly enhanced and this effect was not dexamethasone sensitive. During incubation of rabbit alveolar macrophages a marked formation of ³H-ornithine (about 5.3% of the added ³H-arginine), but no significant formation of ³H-citrulline could be detected. Pretreatment with LPS tended to enhance the formation of ³H-ornithine (by 50%) without effects on ³H-citrulline. Rabbit-interferon and/or tumor necrosis factor- present together with LPS during the culture period did not result in a significant ³H-citrulline formation. Under all conditions tested, culture media of rabbit alveolar macrophages did not contain significant amounts of nitrite (less than 0.5 nmol) whereas in culture media of untreated rat alveolar macrophages 22 nmol nitrite (per 18 h) were detected, and LPS induced a 3-fold nitrite accumulation, an effect prevented by dexamethasone.In conclusion, in rabbit alveolar macrophages NO synthase activity was not detectable and could also not be induced by LPS and different cytokines, whereas in rat alveolar macrophages NO synthase was readily inducible. Alveolar macrophages of both species showed marked arginase activity. After induction of marked NO synthase activity, ornithine formation was largely reduced possibly by concomitant inhibition of arginase and/or withdrawn of arginine from arginase.     

Effects of nicotine receptor agonists on acetylcholine release from the isolated motor nerve,small intestine and trachea of rats and guinea-pigs

Summary The effects of nicotine receptor agonists on the release of [³H]acetylcholine from the phrenic nerve, the small intestine and the trachea were investigated to characterize neuronal nicotine receptors within the peripheral nervous system. Contraction of the indirectly-stimulated hemidiaphragm was recorded to investigate desensitization of the postsynaptic muscular nicotine receptors. Nicotine, cytisine, 1,1-dimethyl-4-phenylpiperazinium and 2-(4-aminophenyl)-ethyl-trimethyl-ammoniumiodide caused a concentration-dependent (0.1–30 M) increase in evoked [³H]acetylcholine release from the phrenic nerve, whereby bell-shaped concentration-response curves were obtained. The rank order of decreasing potency was: nicotine > cytisine > 1,1-dimethyl-4-phenylpiperazinium > 2-(4-aminophenyl)-ethyl-trimethyl-ammoniumiodide. The presynaptic effects of nicotine depended strongly on the exposure time: facilitation occurred after a short 20 s exposure and inhibition after a 3 min exposure, whereas nicotine no longer affected evoked [³H]acetylcholine release after a 15 min exposure. Pre-exposure (40 min) of the phrenic nerve to 0.3 M nicotine prevented any subsequent modulatory effect of a high nicotine concentration. In contrast, the contraction of the indirectly-stimulated hemidiaphragm remained unaffected in the presence of 0.3–30 M nicotine, but a concentration of 1 mM nicotine abolished skeletal muscle contraction. Nicotine (10 M) produced a substantial release of [³H]acetylcholine in the small intestine but not in the isolated trachea. The present experiments show presynaptic nicotine receptors at the phrenic nerve, which, under appropriate conditions, can mediate facilitation of evoked transmitter release. These neuronal receptors appear more sensitive to desensitizing conditions than the postsynaptic muscular nicotine receptors. Nicotine also mediates a transient release of acetylcholine in the myenteric plexus but not in the trachea, and as a consequence, applied nicotine preferentially activates smooth muscle activity in the intestine.Abbreviations DMPP 1,1-dimethyl-4-phenylpiperazinium - PAPETA 2-(4-aminophenyl)-ethyl-trimethylammoniumiodiderine of [³H]acetylcholine release     

Recent, extensive, and preferential insertion of members of the miniature inverted-repeat transposable element family Heartbreaker into genic regions of maize

A 314-bp DNA element called Heartbreaker-hm1 (Hbr-hm1) was previously identified in the 3' untranslated region of a mutant allele of the maize disease resistance gene HM1. This element has structural features of miniature inverted-repeat transposable elements (MITEs) and is a member of a large family of approximately 4,000 copies in the maize genome. Unlike previously described MITEs, most members of the Hbr family display over 90% sequence identity. This, coupled with the insertion of an Hbr element into an allele of the HM1 gene, suggested that this family might have spread recently throughout the genome. Consistent with this view is the finding that Hbr insertion sites are remarkably polymorphic. Ten of ten loci containing Hbr elements were found to be polymorphic for the presence or absence of Hbr among a collection of maize inbred lines and teosinte strains. Despite the fact that over 80% of the maize genome contain moderate to highly repetitive DNA, we find that randomly chosen Hbr elements are predominantly in single or low copy regions. Furthermore, when used to query both the public and private databases of plant genes, over 50% of the sequences flanking these Hbr elements resulted in significant "hits." Taken together, these data indicate that the presence or absence of Hbr elements is a significant contributory factor to the high level of polymorphism associated with maize genic regions.     

Dietary Interventions for Heart Failure in Older Adults: Re-Emergence of the Hedonic Shift

Dietary non-adherence to sodium restriction is an important contribution to heart failure (HF) symptom burden, particularly in older adults. While knowledge, skills, and attitudes toward sodium restriction are important, sodium intake is closely linked to the ability to taste salt. The ‘hedonic shift’ occurs when sodium restriction induces changes in an individual’s salt taste that lower subsequent salt affinity. Older adults often have compromised salt taste and higher dietary salt affinity due to age-related changes. Older HF patients may have additional loss of salt taste and elevated salt appetite due to comorbid conditions, medication use, and micronutrient or electrolyte abnormalities, creating a significant barrier to dietary adherence. Induction of the hedonic shift has the potential to improve long-term dietary sodium restriction and significantly impact HF outcomes in older adults.     

The variation of acetylcholine release from myenteric neurones with stimulation frequency and train length

The effects of scopolamine on the release of 3H-acetylcholine (ACh) from the guinea-pig myenteric plexus were studied at different stimulation frequencies (0.03-10 Hz) and train lengths (1-180 pulses). Release of 3H-ACh was measured in the absence of cholinesterase inhibitors as the outflow of tritium from myenteric plexus-longitudinal muscle preparations preloaded with 3H-choline. In control experiments the volley output of 3H-ACh declined with increasing train length and increasing stimulation frequency. Stimulation by one pulse produced the highest volley output. Scopolamine facilitated the evoked output of 3H-ACh via blockade of presynaptic muscarine receptors. A significant increase was already observed when the preparation was stimulated with 3 pulses at 10 Hz which indicates that the inhibitory muscarinic mechanism becomes operational within 200 ms. The facilitatory effects of scopolamine depended on both train length and frequency of stimulation. Maximal increases in 3H-ACh output were seen with brief trains (3 and 6 pulses) at a high frequency (10 Hz), or with longer trains (20 and 180 pulses) at lower frequencies (0.3 and 1 Hz). Scopolamine compensated for the frequency-dependent decline of 3H-ACh volley output only during brief periods of stimulation (3 and 6 pulses). It is therefore concluded that the decline in volley output during the first few pulses of a train is due to the negative feedback mechanism which is activated by the transmitter released by the preceding impulses. With longer trains of stimulation the negative feedback mechanism plays only a minor role in regulating the output per pulse.