In vitro, the multienzyme preparation catalyzes the formation of several different D-lysergyl peptide lactams according to the amino acids supplied. Specific antiserum was used to detect LPS 1 in various C. purpurea strains. In C. purpurea wild type, the enzyme was expressed at all stages of cultivation and in different media, suggesting that it is produced constitutively.

We have shown that the mechanism of D-lysergyl peptide synthesis is an ordered process of successive acyl transfers on a multienzyme complex. This knowledge opens the way for enzymatic and genetic investigations into the formation of novel alkaloid cyclopeptides.

A detrimental perceptive consequence of damaged auditory sensory hair cells consists in a pronounced masking effect exerted by low-frequency sounds, thought to occur when auditory threshold elevation substantially exceeds 40 dB. Here, we identified the submembrane scaffold protein Nherf1 as a hair-bundle component of the differentiating outer hair cells (OHCs). Nherf1 −/− mice displayed OHC hair-bundle shape anomalies in the mid and basal cochlea, normally tuned to mid-and high-frequency tones, and mild (22-35 dB) hearing-threshold elevations restricted to midhigh sound frequencies. This mild decrease in hearing sensitivity was, however, discordant with almost nonresponding OHCs at the cochlear base as assessed by distortion-product otoacoustic emissions and cochlear microphonic potentials. Moreover, unlike wild-type mice, responses of Nherf1 −/− mice to high-frequency (20-40 kHz) test tones were not masked by tones of neighboring frequencies. Instead, efficient maskers were characterized by their frequencies up to two octaves below the probe-tone frequency, unusually low intensities up to 25 dB below probe-tone level, and growth-ofmasker slope (2.2 dB/dB) reflecting their compressive amplification. Together, these properties do not fit the current acknowledged features of a hypersensitivity of the basal cochlea to lower frequencies, but rather suggest a previously unidentified mechanism. Low-frequency maskers, we propose, may interact within the unaffected cochlear apical region with midhigh frequency sounds propagated there via a mode possibly using the persistent contact of misshaped OHC hair bundles with the tectorial membrane. Our findings thus reveal a source of misleading interpretations of hearing thresholds and of hypervulnerability to low-frequency sound interference.off-frequency detection | Usher syndrome | hearing impairment | Nherf2 | tail hypersensitivity M ammalian hearing displays remarkable sensitivity, fine temporal acuity, and exquisite frequency selectivity, which contribute to auditory scene analysis and speech intelligibility. The first steps of sound processing, i.e., sound wave detection and neuronal encoding in the cochlea, are performed by two populations of hair cells, the inner hair cells (IHCs) and the outer hair cells (OHCs). These cells are sandwiched between the underlying basilar membrane (BM) and the overlying tectorial membrane (TM) (SI Appendix, Fig. S1A). IHCs are the genuine sensory cells that transduce the sound stimuli into electrical signals in the primary auditory neurons. OHCs are mechanical effectors that amplify the sound-evoked movements of the cochlear partition, sharpen its frequency selectivity, and produce waveform distortions (1, 2). A pure-tone stimulus entering the cochlea elicits a traveling wave that propagates along the BM from the cochlear base toward its apex, increasing in amplitude until it peaks at a characteristic place, where the mechanical properties of the cochlea are best tuned to the stimulus frequency. Beyond this characteristic place, the ampl...

The ionic composition of airway surface liquid (ASL) has been debated, and, in particular for the mouse, a wide range of values has been published. Two techniques were developed to measure the elemental composition of the ASL. X-ray microanalysis of ASL was carried out at low temperature on trachea removed from isofluraneanesthetized animals and shock-frozen. In the second technique, dextran beads were placed on top of the epithelium of the trachea removed from pentobarbital-anesthetized animals, left to equilibrate with the ASL, dried, and subjected to X-ray microanalysis. Both techniques showed that mouse tracheal ASL has significantly lower concentrations of Na and Cl (ϳ60 -80 mM) than serum. Differences between the two techniques were due to different sampling of mucus. CFTR(Ϫ/Ϫ) mice had significantly higher concentrations of Na and Cl in their ASL than age-matched controls. Pilocarpine or isoproterenol stimulation significantly reduced the ion concentrations in tracheal ASL. ASL was also collected with the dextran bead method from the nasal cavity in situ in pentobarbital-anesthetized animals. In control animals, the elemental composition of nasal fluid was similar to that of tracheal ASL. Pilocarpine stimulation caused a significant increase in Na, Cl, and K; stimulation with isoproterenol or phenylephrine caused a significant increase only in K. It is concluded that mouse ASL under unstimulated conditions is hypotonic, which may be related to the relative paucity of submucosal glands in the mouse trachea. cystic fibrosis; ion transport THE AIRWAY EPITHELIUM IS COVERED by a thin layer of liquid, the airway surface liquid (ASL), which is mainly secreted by the submucosal glands. ASL consists of mucus and an underlying periciliary watery layer that enables the cilia to clear the mucus. From studies of patients with cystic fibrosis (CF), as well as studies of patients with exercise-induced asthma, it has become clear that the volume and/or ionic composition of the ASL are important for lung function (1,3,4,23). In its turn, volume and/or ionic composition are affected by ion transport mechanisms in the airway epithelium (11,22). With regard to the airways in CF patients, several different theories have been proposed (reviewed in Ref. 22). Already early on, it was suggested that the ASL in CF patients would have higher concentrations of Na and Cl than normal (13). One of the current theories (17,23) claims that the ASL normally is hypotonic. This would provide an optimal environment for the defensins, proteins that play a role in the defense against bacteria. According to this view, the ASL in CF patients, although still hypotonic, would have a higher salt content than normal and, therefore, a reduced activity of defensins. Another theory (3, 4) claims that the ASL is normally isotonic and that it is isotonic in CF patients but has a reduced volume, which would lead to the formation of viscous mucus that facilitates bacterial colonization.It has been difficult to settle this dispute by determining the exact c...

The first intron of the mitochondrial gene coding for cytochrome oxidase subunit I (COI I1) of Podospora anserina can undergo self‐splicing in vitro at high concentrations of NH4Cl or KCl. Under these conditions cleavage at the 5′ splice junction takes place without branch formation probably via hydrolysis by water or OH‐ and the intron is released in a linear form. In vitro transcripts that contain mutated introns with large deletions in nonconserved domain IV comprising greater than 50% of the intronic sequence display a more efficient splicing reaction and, surprisingly, 5′ cleavage via transesterification and lariat formation is re‐established to a low degree under NH4Cl. In contrast to the self‐splicing group II introns aI5 gamma and bI1 from yeast mitochondria cleavage at the 3′ splice site of the Podospora intron is reduced and cleavage by hydrolysis in trans (i.e. exon reopening) is almost completely suppressed. Both observations could be interpreted as a result of unfavourable spatial conformations of the intron that (i) lead to a steric hindrance of the 5′ exon to attack the 3′ splice site in cis and (ii) block intron‐dependent cleavage reaction of the ligated exons in trans. Alternatively, the possibility that a weak overall interaction of the postulated exon‐ with the corresponding intron‐binding sites (EBS‐IBS pairings) is responsible for the remarkable differences to the self‐splicing reaction of other group II introns is discussed.