Neuronal STAT3 activation is essential for CNTF- and inflammatory stimulation-induced CNS axon regeneration.

CNS neurons, such as retinal ganglion cells (RGCs), do not normally regenerate injured axons, but instead undergo apoptotic cell death. Regenerative failure is due to inhibitory factors in the myelin and forming glial scar as well as due to an insufficient intrinsic capability of mature neurons to regrow axons. Nevertheless, RGCs can be transformed into an active regenerative state upon inflammatory stimulation (IS) in the inner eye, for instance by lens injury, enabling these RGCs to survive axotomy and to regenerate axons into the lesioned optic nerve. The beneficial effects of IS are mediated by various factors, including CNTF, LIF and IL-6. Consistently, IS activates various signaling pathways, such as JAK/STAT3 and PI3K/AKT/mTOR, in several retinal cell types. Using a conditional knockdown approach to specifically delete STAT3 in adult RGCs, we investigated the role of STAT3 in IS-induced neuroprotection and axon regeneration. Conditional STAT3 knockdown in RGCs did not affect the survival of RGCs after optic nerve injury compared with controls, but significantly reduced the neuroprotective effects of IS. STAT3 depletion significantly compromised CNTF-stimulated neurite growth in culture and IS-induced transformation of RGCs into an active regenerative state in vivo. As a consequence, IS-mediated axonal regeneration into the injured optic nerve was almost completely abolished in mice with STAT3 depleted in RGCs. In conclusion, STAT3 activation in RGCs is involved in neuroprotection and is a necessary prerequisite for optic nerve regeneration upon IS.

Interleukin-6 contributes to CNS axon regeneration upon inflammatory stimulation.

Mature retinal ganglion cells (RGCs) do not normally regenerate injured axons and undergo apoptosis after axotomy. Inflammatory stimulation (IS) in the eye mediates neuroprotection and induces axon regeneration into the injured optic nerve. Ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) have been identified as key mediators of these effects. Here, we investigated the role of interleukin-6 (IL-6), another member of the glycoprotein 130-activating cytokine family, as additional contributing factor. Expression of IL-6 was markedly induced in the retina upon optic nerve injury and IS, and mature RGCs expressed the IL-6 receptor. Treatment of cultured RGCs with IL-6 or specific IL-6 receptor agonist, significantly increased neurite outgrowth janus kinase/signal transducers and activators of transcription-3 (JAK/STAT3) and phosphatidylinositide 3-kinase/protein kinase B (PI3K/Akt) dependently. Moreover, IL-6 reduced myelin, but not neurocan-mediated growth inhibition mammalian target of rapamycin (mTOR) dependently in cultured RGCs. In vivo, intravitreal application of IL-6 transformed RGCs into a regenerative state, enabling axon regeneration beyond the lesion site of the optic nerve. On the other hand, genetic ablation of IL-6 in mice significantly reduced IS-mediated myelin disinhibition and axon regeneration in the optic nerve. Therefore, IL-6 contributes to the beneficial effects of IS and its disinhibitory effect adds an important feature to the effects of so far identified IS-mediating factors. Consequently, application of IL-6 or activation of its receptor might provide suitable strategies for enhancing optic nerve regeneration.

Wild-type but not mutant huntingtin modulates the transcriptional activity of liver X receptors.

BACKGROUND: Huntington's disease is caused by expansion of a polyglutamine tract found in the amino-terminal of the ubiquitously expressed protein huntingtin. Well studied in its mutant form, huntingtin has a wide variety of normal functions, loss of which may also contribute to disease progression. Widespread transcriptional dysfunction occurs in brains of Huntington's disease patients and in transgenic mouse and cell models of Huntington's disease. METHODS: To identify new transcriptional pathways altered by the normal and/or abnormal function of huntingtin, we probed several nuclear receptors, normally expressed in the brain, for binding to huntingtin in its mutant and wild-type forms. RESULTS: Wild-type huntingtin could bind to a number of nuclear receptors; LXRalpha, PPARgamma, VDR and TRalpha1. Over-expression of huntingtin activated, while knockout of huntingtin decreased, LXR mediated transcription of a reporter gene. Loss of huntingtin also decreased expression of the LXR target gene, ABCA1. In vivo, huntingtin deficient zebrafish had a severe phenotype and reduced expression of LXR regulated genes. An LXR agonist was able to partially rescue the phenotype and the expression of LXR target genes in huntingtin deficient zebrafish during early development. CONCLUSION: Our data suggest a novel function for wild-type huntingtin as a co-factor of LXR. However, this activity is lost by mutant huntingtin that only interacts weakly with LXR.

Function of Neurolin (DM-GRASP/SC-1) in guidance of motor axons during zebrafish development.

Neurolin (zf DM-GRASP), a transmembrane protein with five extracellular immunoglobulin domains, is expressed by secondary but not primary motoneurons during zebrafish development. The spatiotemporally restricted expression pattern suggests that Neurolin plays a role in motor axon growth and guidance. To test this hypothesis, we injected zebrafish embryos with function-blocking Neurolin antibodies. In injected embryos, secondary motor axons form a broadened bundle along the common path and ectopic branches leave the common path at right angles. Moreover, the formation of the ventral and the rostral projection of secondary motor axons is inhibited during the second day of development. Pathfinding errors, resulting in secondary motor axons growing through ectopic regions of the somites, occur along the common path and in the dorsal and rostral projection. Our data are compatible with the view that Neurolin is involved in the recognition of guidance cues and acts as a receptor on secondary motor axons. Consistent with this idea is the binding pattern of a soluble Neurolin-Fc construct showing that putative ligands are distributed along the common path, the ventral projection, and in the area where the rostral projection develops.

Thauera mechernichensis sp. nov., an aerobic denitrifier from a leachate treatment plant.

A heterotrophic bacterial strain TL1T capable of aerobic denitrification was previously enriched in continuous culture from a landfill leachate treatment plant and isolated as a pure culture. The taxonomic position of this isolate within the beta-subclass of the Proteobacteria was determined by 16S rDNA sequence analysis and by conventional taxonomy including substrate spectrum, quinone type (ubiquinone Q-8) and cellular fatty acid composition. Detection of the specific polyamine 2-hydroxyputrescine supports the membership of strain TL1T in the beta-subclass of the Proteobacteria. The results of 16S rDNA sequencing showed that the strain clustered with, but was separate from, Thauera aromatica and Thauera selenatis. DNA-DNA hybridization experiments indicated that the new isolate represents a new species of the genus, for which the name Thauera mechernichensis is proposed; the type strain is DSM 12266T.

Isolation, primary structure characterization and identification of the glycosylation pattern of recombinant goldfish neurolin, a neuronal cell adhesion protein.

Neurolin is a growth-associated cell surface glycoprotein from goldfish and zebra fish which has been shown to be involved in axonal path-finding in the goldfish retina and suggested to function as a receptor for axon guidance molecules. Being a member of the immunoglobulin superfamily of cell adhesion proteins, neurolin consists of five N-terminal extracellular immunoglobulin (Ig)-like domains, a transmembrane and a short cytoplasmatic domain. Repeated injections of polyclonal Fab fragments against neurolin and of monoclonal antibodies against either Ig domains cause path-finding errors and disturbance of axonal fasciculation. In order to obtain a complete structural characterization and a molecular basis for structure-function determination, recombinant neurolin with the complete extracellular part but lacking the transmembrane and cytoplasmatic domain was expressed in Chinese hamster ovary (CHO) cells (CHO-neurolin). The isolation of CHO-neurolin was carried out by Ni-affinity chromatography and subsequent high-performance liquid chromatography (HPLC). An exact molecular mass determination was obtained by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) and revealed 60.9 kDa, which suggested that approximately 10 kDa are due to glycosylation. The predicted molecular mass is 51.5 kDa, whereas sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) yielded an apparent molecular mass of 72 kDa. Gel shift assays using SDS-PAGE and Western blot analysis with anti-neurolin antibodies provided consistent molecular mass data. The complete primary structure and N-glycosylation patterns were identified using specific lectin assays, MALDI/MS peptide mapping analysis by proteolytic and in-gel digestion, electrospray ionization MS and MALDI/MS in combination with specific glycosidase degradation. HPLC isolation of glycosylated peptide fragments and MS after selective deglycosylation revealed heterogeneous glycosylations at all five N-glycosylation consensus sites. All attached N-glycans are of the complex type and show a mainly biantennary structure; they are fucosylated with alpha(2,3)-terminal neuraminic acid. These data serve as a first detailed model to characterize the molecular recognition structures exhibited by the extracellular domains.

Oxygen, pH value, and carbon source induced changes of the mode of oscillation in synchronous continuous culture of Saccharomyces cerevisiae.

Oscillations of measured process parameters occur in continuous cultures of Saccharomyces cerevisiae owing to a partial synchronization of budding. Intentional changes of the oxygen concentration, pH value, and carbon source cause effects on the period length similar to those known from variations of the dilution rate. The generation times of parent and daughter cells frequently differ in synchronous culture. To analyze the oscillation the term mode IJ of oscillation is used, which is defined as the ratio IJ of the generation times of parent and daughter cells. When the dissolved oxygen concentration was reduced to zero, the mode of oscillation changed within two periods from mode 12 to mode 11, caused by a decrease of the generation time of daughter cells and an increase of that of the parent cells. When the pH value was slowly reduced from 5.0 to 3.9, a change from mode 112 to mode 13 was observed. Mode 13, representing one parent and three daughter cell populations (the start of budding of each of the three being delayed by one period), denotes an elongated generation time of the daughter cells compared to mode 112, marked by one parent and two different daughter cell classes. When the carbon source galactose was replaced by glucose a mode change from mode 12 to mode 11 was observed. This alteration of the mode was found to be dependent on the status of the cell cycle at the time when the carbon source is changed. The population distribution in batch cultures with glucose or galactose as a substrate was analysed by dyeing the DNA and counting the bud scars. Galactose provoked higher growth rates for the older cells. According to the model for stationary synchronous growth parameters like DO, pH value or the type of carbon source can be varied within a certain range without effecting the period length. If the variation imposes a certain stress, the culture switches to a new mode. These kinds of parameters therefore provide selective measures to influence the period lengths and the modes of oscillation.

Studies with lysine N6-hydroxylase. Effect of a mutation in the assumed FAD binding site on coenzyme affinities and on lysine hydroxylating activity.

The proposed FAD binding site of L-lysine N6-hydroxylase (EC 1.14.13.99) exhibits an unusual proline in a position where a highly conserved glycine is found in other FAD dependent hydroxylases. We have studied the role of this proline by mutating it to glycine in [P14G]aerA, which was expressed in Escherichia coli M15-2 and purified to homogeneity. The mutation has marked effects on the affinities of the cofactors FAD and NADPH as well as the substrate, lysine. Compared to the wild-type enzyme, the activity vs. pH profile of the mutant protein indicates a shift of the apparent pK'(a)s (7.8 and 8.7 for wild-type and 6.8 and 7.7 for the P14G-mutant enzyme) and of the activity maximum (pH 8 for wild-type and pH 7 for the P14G-mutant enzyme). While the activity of the mutant enzyme is much lower under conditions found to be optimal for the wild-type enzyme, adjustment of substrate and cofactor concentrations and pH leads to comparable activities for the mutant enzyme. These results suggest that the proline fulfils an important structural role in the proposed FAD binding site.

Neurolin Ig domain 2 participates in retinal axon guidance and Ig domains 1 and 3 in fasciculation.

The optic disk-directed growth of retinal ganglion cell axons is markedly disturbed in the presence of polyclonal antineurolin antibodies, which mildly affect fasciculation (Ott, H., M. Bastmeyer, and C.A.O. Stuermer, 1998. J. Neurosci. 18:3363-3372). New monoclonal antibodies (mAbs) against goldfish neurolin, an immunoglobulin (Ig) superfamily cell adhesion/recognition molecule with five Ig domains, were generated to assign function (guidance versus fasciculation) to specific Ig domains. By their ability or failure to recognize Chinese hamster ovary cells expressing recombinant neurolin with deletions of defined Ig domains, mAbs were identified as being directed against Ig domains 1, 2, or 3, respectively. Repeated intraocular injections of a mAb against Ig domain 2 disturb the disk-directed growth: axons grow in aberrant routes and fail to reach the optic disk, but remain fasciculated. mAbs against Ig domains 1 and 3 disturb the formation of tight fascicles. mAb against Ig domain 2 significantly increases the incidence of growth cone departure from the disk-oriented fascicle track, while mAbs against Ig domains 1 and 3 do not. This was demonstrated by time-lapse videorecording of labeled growth cones. Thus, Ig domain 2 of neurolin is apparently essential for growth cone guidance towards the disk, presumably by being part of a receptor (or complex) for an axon guidance component.

Stereoselective synthesis and receptor binding of conformationally restricted and flexible 2,4-disubstituted 1,3-dioxanes derived from benzomorphans.

The key steps in the stereoselective synthesis of the tricyclic aminomethyl derivatives 19 and 20 and the aminoethyl substituted 1,3-dioxanes 24 and 25 are nucleophilic addition of aryllithium intermediates to the nitroalkene 13, intramolecular transacetalization of the addition products 15 and 16 (only for the tricyclic derivatives 19 and 20) and subsequent reduction of the nitro group. The affinities of the secondary and tertiary amines 19c,d, 20c,d, 24c,d, and 25c,d for the ion channel binding site of the NMDA receptor, for mu-, kappa-, and sigma-receptors have been investigated. In the group of tricyclic compounds only 19d shows remarkable sigma-receptor affinity (Ki = 21.6/1.10 microM). In the 1,3-dioxane series the moderate mu- (Ki = 27.8 microM) and kappa-receptor affinity (Ki = 36 microM) as well as the high sigma-receptor affinity (Ki = 3.3 microM) of the (S,S,S)-configurated methylamine 24c should be emphasized. The pentan-1-ol 26, the side product isolated during the synthesis of 24c, is of particular interest because of its considerable affinity to mu- (Ki = 16.0 microM), kappa- (Ki = 2.8 microM), and sigma-receptors (Ki = 14.5/1.26 microM). The biphasic competition curves obtained during sigma-receptor binding studies of 19d and 26 (two Ki values) may be explained by different interaction with sigma-receptor subtypes.

Cross-modality priming between odors and odor-congruent words.

It is well known that the Stroop effect is subject to influence by same-modality primes, but the possibility of cross-modal priming effects is unclear. Smell is a fundamental sensory system that is assumed to have potent cross-modality priming effects. We might expect the presence of a specific odor to interfere with performance on Stroop cards containing odor-congruent words. Forty participants, half of whom were primed with an unpleasant odor and half with a pleasant odor, were examined with modified Stroop cards containing pleasant and unpleasant descriptive words. A significant Stroop card by odor group interaction was found, indicating that the presence of an odor interferes with the performance on an odor-congruent Stroop card. These findings demonstrate cross-modality priming between olfaction and vision.

Effect of the dilution rate on the mode of oscillation in continuous cultures of Saccharomyces cerevisiae.

The growth properties of the asymmetric budding yeast Saccharomyces cerevisiae were analysed during spontaneous oscillations in continuous cultures at varying dilution rates D. The length of the oscillation period changed between 1.4 and 14 h in response to the decrease of dilution rate from 0.15 to 0.05 h-1. The distribution of parent and daughter cells in the population was determined microscopically after staining the bud scars and DNA. Most of the data obtained fits a theoretical population balance model assuming two-classes of subpopulations and integer ratios between the generation times of both classes. Some data has to be described by an extended population model assuming there is one parent and two daughter cell classes. How changes of dilution rate may cause an accidental switch of the mode of oscillation is demonstrated. Glucose consumption and metabolite production were measured off-line by enzymatic methods and gas exchange was monitored on-line. All these data of one period point to internal and external signals responsible for the synchronisation of the cell cycle.

Stereoselective syntheses and CNS activity of novel tricyclic amines.

The enantiomerically pure amines (+)-5, (-)-9, (+)-11 and (+)-12 are stereoselectively prepared by reductive amination of the ketone (-)-4 [-->(+)-5], LiAlH4 reduction of the oxime (-)-7 followed by reductive methylation [-->(-)-9], SN2-substitution of the benzenesulfonate 10 [-->(+)-11] and reductive methylation of (+)-11 [-->(+)-12]. In the same way the racemic amines (+/-)-5, (+/-)-9, (+/-)-11 and (+/-)-12 are accessible starting from the racemic ketone (+/-)-4. Kinetic resolution of the racemic ketone (+/-)-4 with baker's yeast leads to the dextrorotatory ketone (+)-4 (86% ec), which is transformed via the methanesulfonate 16 into the tricyclic amines (-)-11 and (+)-17. Weak sedative effects are observed after application of the amines (+)-5, (+/-)-5, 0-9, (+/-)-9(+)-12, and (+/-)-12 to mice. Strong sedation is caused by (+/-)-11 with the dextrorotatory enantiomer (+/-)-11 being more effective than the levorotatory enantiomer (-)-11.

Lactobacillus panis sp. nov., from sourdough with a long fermentation period.

Two Lactobacillus strains isolated from rye sourdough most closely resemble Lactobacillus oris on the basis of their physiological, morphological, and chemotaxonomic characteristics. A 16S ribosomal DNA sequence analysis showed that these two strains clustered with, but were separate from, L. oris, Lactobacillus vaginalis, and Lactobacillus pontis. The results of DNA-DNA hybridization experiments indicated that the new two isolates represent a new Lactobacillus species, for which we propose the name Lactobacillus panis; strain DSM 6035T is the type strain of this species.

Degradation of prochloraz and 2,4,6-trichlorophenol by environmental bacterial strains.

Eight bacterial isolates from enrichment with 2,4,6-trichlorophenol (TCP) as sole carbon source were tested for their potential to degrade prochloraz. None of them could grow on prochloraz. Strain C964, identified as Aureobacterium sp., effectively reduced the fungitoxic activity of prochloraz in a bioassay and degradation was confirmed by HPLC. Two other isolates, strain C611 and C961, using TCP as a carbon source, belong to the beta subclass of the proteobacteria and presumely degrade TCP via 2,4-dichlorohydroquinone and hydroxyhydroquinone as indicated by oxygen-consumption tests.

The 92-kDa chitinase from Streptomyces olivaceoviridis contains a lysine-C endoproteinase at its N-terminus.

Serine proteinases of 42, 22 and 14 kDa were purified from the culture fluid of Streptomyces olivaceoviridis by FPLC. The first 14 amino acids at their N-termini were identical and coincide with the N-terminal amino acid sequence of 92-kDa chitinase, which was found to hydrolyse casein. The four proteins hydrolyse synthetic substrates at the carboxyl group of lysine and (more slowly) arginine. The 14-kDa endoproteinase releases only two fragments of 42 and 43 kDa from beta-galactosidase. When the pure 92-kDa chitinase was incubated at 37 degrees C in Tris.HCl buffer, it was cleaved into a 70-kDa chitinase and a 22-kDa proteinase which in its part is rapidly degraded to a 14-kDa proteinase.

[Sense of smell in deaf and blind patients]. / Die Riechleistungen von Gehörlosen und Blinden.

The olfactory performances of 10 visually-impaired and 9 hearing-impaired subjects were investigated by a psychophysical sniffing test (the Munich Olfaction Test). These subjects were compared with 21 control volunteers who completed the test with covered eyes, 20 control volunteers whose ears were plugged during the test and with 22 unimpaired controls. The test examined discrimination performances, detection and perception thresholds, identification abilities, olfactory memory, and hedonistic evaluations of all subjects. The present results showed that attention focusing on olfactory tasks by lowering of auditory or visual inputs did not enhance the olfactory performances of the control subjects. Also, the visually-impaired subjects did not perform better then the control subjects. The diminished olfactory abilities of the hearing-impaired were discussed against the background of delayed language acquisition.

Emotional qualities of odors and their influence on the startle reflex in humans.

Recent human and animal research suggests that the startle reflex might serve as a psychophysiological indicator of the emotional valence of foreground stimulation. The present experiment was designed to evaluate the emotional effects of positive and negative odorant stimuli. We examined the effects of continuous hydrogen sulphide (H2S) and vanillin stimulation on the magnitude of the acoustic startle reflex (measured at the M. orbicularis oculi) and on ratings of subjective valence in 16 healthy subjects. In accordance with the view that odors have emotional qualities, we found that H2S, a presumed negative foreground stimulus, significantly enhanced the startle-reflex amplitude relative to neutral air stimulation, whereas vanillin, a positive foreground stimulus, tended to reduce the reflex amplitude compared with neutral air stimulation. Both odorant stimuli were rated as equally intense by the subjects, and heart rate and electrodermal activity were not affected differentially by the two odorants.

FAD and substrate analogs as probes for lysine N6-hydroxylase from Escherichia coli EN 222.

Lysine N6-hydroxylase catalyzes the hydroxylation of the N-terminal amino function of L-lysine at the expense of NADPH and molecular oxygen. The enzyme also requires FAD for its catalytic activity. Unlike other flavoprotein monooxygenases, binding of FAD is rather weak with a Kd of 30 microM at 4 degrees C. The spectral properties of FAD bound to lysine N6-hydroxylase are very similar to free oxidized FAD. In the absence of substrate, the enzyme has an NADPH oxidase activity which results in the generation of hydrogen peroxide. With increasing concentration of L-lysine, the NADPH oxidase activity is enhanced up to 10-fold and the generation of hydrogen peroxide decreases. At the same time, the substrate is hydroxylated. Km values for L-lysine and FAD were determined as 105 microM and 0.7 microM, respectively. Utilizing FAD analogs, we could demonstrate that L-lysine exerts its effector role mostly on the reductive half reaction of the overall catalytic cycle. Prolonged incubation of the enzyme with either 8-chloro- or 8-fluoro-FAD gave rise to a covalently attached flavin which is formed as a result of the nucleophilic attack of a thiolate on the 8-position of the flavin. Several lines of evidence indicate that the reaction takes place in the FAD binding site of the protein. The substrate specificity was investigated using amino acids with various lengths of side chain. L-Lysine and derivatives with similar side chain length are hydroxylated by lysine N6-hydroxylase. Ornithine, the lower homolog of lysine, was not hydroxylated and did not affect the NADPH oxidase activity of the enzyme. On the other hand, homolysine accelerated the rate of NADPH oxidation but was not hydroxylated. Additional requirements for efficient hydroxylation were also investigated using a variety of substrate analogs. From these studies a schematic structure of the active site of the enzyme was deduced. Sequence comparison of the FAD binding site of various flavoproteins revealed possible factors for weak binding of the cofactor in the case of lysine N6-hydroxylase.