Just in time and place: NOS/NO system assembly in neuromuscular junction formation.

Recent advances in the molecular, biochemical, and anatomical aspects of postsynaptic membrane components at the neuromuscular junction (NMJ) are briefly reviewed focussing on assembly, architecture, and function of the multi-subunit dystrophin-protein complex (DPC) and its associated nitric oxide (NO)-signaling complex. Elucidation of unique structural binding motifs of NO-synthases (NOS), and microscopical codistribution of neuronal NOS (nNOS), the major isoform of NOS expressed at the NMJ, with known synaptic proteins, i.e., family members of the DPC, nicotinic acetylcholine receptor (AChR), NMDA-receptor, type-1 sodium and Shaker K(+)-channel proteins, and linker proteins (e.g., PSD-95, 43K-rapsyn), suggests targeting and assembly of the NO-signaling pathway at postsynaptic membrane components. NO mediates agrin-induced AChR-aggregation and downstream signal transduction in C2 skeletal myotubes while administration of L-arginine, the limiting substrate for NO-biosynthesis, enhances aggregation of synapse-specific components such as utrophin. At the NMJ, NO appears to be a mediator of (1) early synaptic protein clustering, (2) synaptic receptor activity and transmitter release, or (3) downstream signaling for transcriptional control. Multidisciplinary data obtained from cellular and molecular studies and from immunolocalization investigations have led us to propose a working model for step-by-step binding of nNOS, e.g., to subunit domains of targeted and/or preexisting membrane components. Formation of NOS-membrane complexes appears to be governed by agrin-signaling as well as by NO-signaling, supporting the idea that parallel signaling pathways may account for the spatiotemporally defined postsynaptic assembly thereby linking the NOS/NO-signaling cascade to early membrane aggregations and at the right places nearby preexisting targets (e.g., juxtaposition of NO source and target) in synapse formation.

Double-blind clinical trial of sertraline treatment for alcohol dependence.

Clinical studies that have evaluated serotonergic medications to reduce alcohol consumption have yielded conflicting results. These studies primarily treated patients with alcohol dependence, excluding those with a current depressive disorder, in an effort to differentiate any medication effects directly on drinking from those on mood. Yet despite the exclusion of current depression, a group of alcohol-dependent patients who are not depressed can be highly heterogeneous. For example, this subgroup can include those with a lifetime depressive disorder. If these patients were more sensitive to serotonergic medications than patients without a lifetime depressive disorder, medication effects in a subgroup of patients who were not depressed could be obscured. Thus, the purpose of this study was to examine the efficacy of sertraline for treating alcohol dependence in patient groups that were differentiated by the presence or absence of lifetime depression. This study examined the effectiveness of sertraline (200 mg/day) or placebo for 14 weeks in 100 alcohol-dependent subjects with (N = 53) or without (N = 47) a lifetime diagnosis of comorbid depression. Sertraline treatment seemed to provide an advantage in reducing drinking in alcohol-dependent patients without lifetime depression, illustrated best with a measure of drinking frequency during treatment. However, sertraline was no better than placebo in patients with a diagnosis of lifetime comorbid depression, and current depression did not change the results. Treatment with selective serotonin reuptake inhibitors may be useful in alcohol-dependent patients who are not depressed. Subtyping those with alcohol dependence on the basis of the absence versus the presence of a lifetime depressive disorder may help to resolve conflicting findings in the literature on the treatment of alcohol dependence with serotonergic medications.

Gender and psychiatric comorbidity: impact on clinical presentation of alcohol dependence.

We examined differences in clinical presentation for outpatient alcohol treatment in: 1) males and females, considering comorbidity; and 2) three comorbid groups, considering gender. Drinking indices and emotional, physical, and sexual abuse reports were compared in 127 male and 69 female alcohol-dependent patients who have a current (36.2%) or lifetime (20.4%) psychiatric disorder or who never had a psychiatric disorder (43.4%). Females reported more emotional and physical abuse than males. Females reported drinking smaller volumes of alcohol but on more days than males. All with current comorbidity, irrespective of gender, reported more days of heavy drinking than other groups. When evaluating drinking status, gender and comorbidity should be considered.

Nitric oxide synthase (NOS-1) coclustered with agrin-induced AChR-specializations on cultured skeletal myotubes.

Previously we reported that neuronal nitric oxide synthase type-1 (NOS-1) is expressed in skeletal myotubes in vitro. In the present paper we sought to determine whether agrin-induced membrane specializations known to include the nicotinic acetylcholine receptor (AChR) on cultured myotubes may also contain NOS-1 and related molecules. After treatment with various agrin constructs containing the full C-terminally AChR-clustering domain (fragments N2, N4), but not with fragment C2 (truncated), NOS-1 expressed in the cytosol of mouse C2C12 skeletal myotubes coclustered with AChR, 43K rapsyn, MuSK, and the dystrophin/utrophin glycoprotein-complex (DUGC). Agrin-induced specializations also included coaggregates of N-methyl-d-aspartic acid (NMDA)-receptor, alpha-sodium (NaCh), or Shaker-type K+ channel (KCh)/PSD-95 complexes, and NOS-1. We conclude that agrin is crucial for recruitment of preassembled multimolecular membrane clusters, including AChR, NMDAR, and ion channels linked to NOS-1. Coassembly of NOS-1 to postsynaptic molecules may reflect site-specific NO-signaling pathways in neuromuscular junction formation and functions.

Sertraline treatment for alcohol dependence: interactive effects of medication and alcoholic subtype.

BACKGROUND: Characteristic behaviors of some alcohol-dependent individuals, e.g., binge drinking, comorbid psychopathology, and some types of alcohol-related problems, have been linked to abnormalities in serotonergic neurotransmission. However, studies that have evaluated serotonergic pharmacotherapy for reducing drinking have yielded conflicting results. One explanation for these findings is a general failure to distinguish alcohol subgroups that may be differentiated on the basis of serotonergic abnormalities. However, in 1996, Kranzler and colleagues reported that Type B alcoholics, who are characterized by high levels of premorbid vulnerability, alcohol dependence severity, and comorbid psychopathology, showed less favorable drinking outcomes in response to treatment with fluoxetine, a serotonin reuptake inhibitor, than with placebo. This medication effect was not seen in Type A alcoholics, i.e., those with lower risk/severity of alcoholism and psychopathology. The aim of the present study was to explore the validity of differential responding by alcohol-dependent subtypes using the serotonin reuptake inhibitor, sertraline. METHODS: A k-means clustering procedure was applied to a sample of alcohol-dependent subjects enrolled in a 14-week, placebo-controlled trial of 200 mg/day of sertraline, classifying them into lower-risk/severity (Type A: n = 55) and higher-risk/severity (Type B: n = 45) subgroups. RESULTS: A significant interaction between alcoholic subtype and medication condition was found, confirming the findings of Kranzler and colleagues that alcoholic subtypes responded differentially to serotonergic medication. Somewhat at variance with their results, however, the present study showed that the lower risk/severity (Type A) subjects had more favorable outcomes when treated with sertraline compared to placebo. CONCLUSIONS: Alcoholic subtypes differentially responded to sertraline when used as a treatment to reduce alcohol drinking, with one subtype having more favorable outcomes. Subtyping alcoholics may help to resolve conflicting findings in the literature on serotonergic treatment of alcohol dependence.

[Effect of the long-acting beta-2 agonist inhalant formoterol on the quality of sleep of patients with bronchial asthma]. / Einfluss des inhalativen, langwirksamen Beta-2-Agonisten Formoterol auf die Schlafqualität von Patienten mit Asthma bronchiale.

BACKGROUND: Patients with asthma bronchiale often complain of exacerbations during the night. Some anti-asthmatic drugs are known to impair the quality of sleep. HYPOTHESIS: The study was undertaken to prove the effect of the long-acting, inhaled beta-2-agonist formoterol on the quality of sleep in patients with mild or moderate asthma bronchiale. METHODS: 20 patients with asthma bronchiale (15 female, 21-75 years, O 33.3 years) without sleep disorder were evaluated by polysomnography during 3 nights. After one adaptation night, the patients were randomly assigned 24 micrograms of formoterol during one and placebo during the other night. The frequency of respiratory disturbances, arousals, sleep latency, sleep stages, sleep efficiency, motoric activity, and subjective impression of sleep quality were compared in both groups. RESULTS: Respiratory disturbances were less, and the subjective impression of sleep quality was better with formoterol in 50% of the patients. The objective quality of sleep was slightly better with formoterol, but not significant (Tab. 1). CONCLUSIONS: In patients with mild or moderate asthma bronchiale, formoterol does not impair, probably slightly improves the quality of sleep.

Cotinine replacement levels for a 21 mg/day transdermal nicotine patch in an outpatient treatment setting.

This study examined plasma cotinine replacement levels of 56 outpatient smokers administered a 21 mg/day transdermal nicotine patch (Nicoderm CQ ). The percentage of cotinine replacement ranged from 35 to 232% (mean 107%; median 90.5%). Four subject variables were found to be significantly correlated with percentage of cotinine replacement-baseline cotinine level, prior quit attempts, gender, and the Fagerström Tolerance Questionnaire score. A two-variable model consisting of baseline cotinine level and gender provided the most powerful predictor combination. The percentage of cotinine replacement was not predictive of post-treatment smoking. The relatively high levels of cotinine replacement obtained using the Nicoderm CQ 21 mg/day patch suggest cautious use of higher dose treatment with this particular patch.

Nitric oxide synthase (NOS) in mouse skeletal muscle development and differentiated myoblasts.

The neuronal isoform of nitric oxide synthase (nNOS, termed also NOS-I) is expressed in normal adult skeletal muscle, suggesting important functions for NO in muscle biology. However, the expression and subcellular localization of NOS in muscle development and myoblast differentiation are largely unknown. In the present study, NOS was immunolocalized with isoform-specific antibodies in developing muscle and in differentiated myoblast cultures (mouse C2C12) together with histochemical NADPH-dependent diaphorase activity that is blocked by specific NOS inhibitors and therefore designated as NOS-associated diaphorase activity (NOSaD). Western blot analysis revealed immunoreactive bands for NOS-I-III in lysates from perinatal and adult muscle tissue and C2C12-myotubes that comigrated with prototypical proteins. In embryonic skeletal muscle, but not in adult myofibers, diffuse cytosolic staining and lack of sarcolemmal NOSaD activity and NOS-I immunoreaction were evident. In both myoblasts and fusioned myotubes, NOSaD and NOS isoforms I-III colocalize in the cytosol. Additionally, members of the sarcolemmal dystrophin-glycoprotein complex (i.e., dystrophin, adhalin, beta1-dystroglycan) immunolocalize in the cytosol of differentiating myoblasts, whereas anti-dystrophin and anti-beta1-dystroglycan clearly delineate the sarcolemma in myotubes. Thus, expression of NOS isoforms I-III and NOSaD is cytosolic in fusion-competent myoblasts during myotube formation in vitro. Interaction of NOSaD/NOS-I with the sarcolemmal dystrophin-complex known from mature myofibers is apparently lacking in prenatal muscle development and differentiating myoblasts. Localization of NOS isoforms thus characterized in myogenic cultures may help further to investigate regulated NO formation in muscle cells in vitro.

In situ identification of neuronal nitric oxide synthase (NOS-I) mRNA in mouse and rat skeletal muscle.

Skeletal muscle provides a major source of the signaling molecule nitric oxide (NO) however in situ identification of NO-synthase (NOS) mRNA has not been verified. We have used NOS-I (neuronal NOS) probes prepared from plasmid DNA by reverse transcription-polymerase chain reaction (RT-PCR) to detect mRNA transcripts in skeletal muscle cells and myofibers of rat and mouse. Mouse C2C12 myoblasts and myotubes reveal strong cytosolic in situ hybridization (ISH) signals in vitro. In adult animals, ISH signals are detectable in striated myofibers at subsarcolemmal and perinuclear regions whilst the myofibrillar compartment is devoid of signals. Expression of NOS-I mRNA in fusion-competent myoblasts suggests that the NOS/NO system is of relevance to myogenic differentiation. Compartmentalization of NOS-I mRNA may reflect spatiofunctional actions between NOS message and protein and the putative subcellular NO targets.

Clinical correlations with carbohydrate-deficient transferrin levels in women with alcoholism.

Carbohydrate-deficient transferrin (CDT) has received increasing attention as a potential biological marker for heavy drinking or as an objective marker of relapse in patients who are treated for alcohol dependence. Previous studies have demonstrated the utility of CDT among men, but there are fewer and inconsistent reports on the utility of CDT among women. This study reports in a sample of 40 alcohol-dependent women, the association between CDT levels, and several different types of measures of drinking intensity including frequency of heavy drinking. Although the majority of drinking indices correlated with CDT levels in men, among women, CDT levels were significantly correlated with the percentage of days of heavy drinking when heavy drinking day was defined as drinking 6 or more drinks per drinking day. The results also support an association between current menstrual function, CDT levels, and drinking indices. These findings suggest that the pattern of drinking (combining high frequency and high intensity) may be an important determinant of CDT levels in women with alcohol dependence, compared with men.

NOS type-1 mRNA expression and protein localization in spinal autonomic neurons.

Autonomic neurons of the rat spinal cord show strong NADPH diaphorase activity and immunoreactivity for nitric oxide synthase (NOS). Here we show mRNA expression of NOS type-1 (neuronal or brain NOS) transcripts in cell bodies of sympathetic preganglionic neurons (SPNs) of the intermediolateral (IML) cell column by non-radioactive in situ hybridization using NOS-I riboprobes. Hybridization signals occurred only in neuronal cell bodies and not outside, in what appeared to be fibers and/or terminals. In preganglionic fibers of SPNs, however, dense axoplasmic immunogold labeling was detected with a monoclonal anti-NOS-I antibody. Expression of NOS-I mRNA in SPN cell bodies and axoplasmic immunolocalization of NOS-I protein suggest that protein translocation is involved in NO-mediated preganglionic control of peripheral targets.

Partial loss of NADPH-diaphorase/nitric oxide synthase-complex in amyotrophic lateral sclerosis and human type-II myofiber atrophy.

To substantiate the role of nitric oxide synthase type-I (NOS-I) in neurogenic muscular disorders we investigated human biopsy samples of type-II fiber atrophy and amyotrophic lateral sclerosis (ALS) by NOS-I immunoreactivity (-IR), NOS-associated NADPH-dependent diaphorase activity (NOSaD) and Western blot analysis. In type-II atrophy, loss of NOSaD and reduced NOS-I-IR was apparent in atrophic myofibers. In atrophic fiber groups lacking NOSaD, both NOS-I and dystrophin-IR was decreased while sarcolemmal beta-dystroglycan- and adhalin-IR (markers of the sarcolemmal dystrophin-glycoprotein complex) was normal. In ALS, groups of scattered angulated atrophic fibers revealed partial loss of NOS-I-IR/NOSaD. Atrophied fibers of either type-I or type-II thus revealed differential sarcolemmal NOS/NOSaD pattern thereby reflecting myopathological alterations of the NO-system in human type-II atrophy and ALS.

Fibroblast growth factor-2 (FGF-2) and FGF-receptor (FGFR-1) immunoreactivity in embryonic spinal autonomic neurons.

The development of the nervous system appears to be under the control of multiple growth factors, neurotrophins and cytokines, which may be expressed either continuously or transiently throughout defined stages of cellular generation, proliferation or differentiation. Fibroblast growth factor (FGF) cytokines and their receptors are abundantly expressed in the embryonic nervous system but their localization at autonomic levels in the fetal spinal cord has not yet been detailed. Immunoreactivity to FGF-2, probably the best characterized member of the FGF family (FGF-1 to FGF-10) and of one of its high affinity receptors, FGFR-1, was found in autonomic neurons at embryonic day E14, the peak day of generation and proliferation in the common ventral motoneuron pool. It was also continuously present throughout the investigated subsequent stages (E15 to postnatal day P30). Immunogold electron microscopy revealed the cytoplasmic localization of FGF-2 and FGFR-1 in intermediolateral neurons, the major group of sympathetic preganglionic neurons in the spinal cord. In these neurons, immunocytochemistry from E14 onwards showed the co-distribution of both markers at the period of axonal outgrowth to peripheral targets, e.g. the adrenal medulla. Our findings suggest autocrine and/or paracrine actions of FGF-2 for sympathetic preganglionic development but do not support its role as a target-derived neurotrophic factor for autonomic neuron development.

On-line column-switching high-performance liquid chromatography analysis of cardiovascular drugs in serum with automated sample clean-up and zone-cutting technique to perform chiral separation.

A selective and highly reproducible, multi-column HPLC method is described for the analysis of the following cardiovascular drugs: lidocaine, pindolol, metoprolol, oxprenolol, diltiazem and verapamil, in serum. Column-switching devices are employed in combination with advanced separation media technologies for the automated analysis of samples containing complex matrices. The method consists of on-line sample clean-up using a restricted access sorbent, HPLC analysis of the drugs on a microsphere non-porous silica RP-18 column, and front-cutting to perform the chiral separation of pindolol enantiomers on a second HPLC system. Simultaneous control of the two HPLC systems and data analysis is achieved from a single centralized software. The R.S.D. values of the peak areas for spiked serum are less than 1% for metoprolol and oxprenolol, 2-5% for lidocaine, diltiazem and verapamil, and 1.2 and 2.4% for the two pindolol enantiomers. Recoveries, limits of detection and linearities are provided.

Polyphenols, astringency and proline-rich proteins.

Recent, NMR and precipitation, studies of molecular recognition of proline-rich proteins and peptides by plant polyphenols are described and rationalized. The action of polysaccharides and caseins in the moderation of the astringent response, which is engendered by polyphenols present in foodstuffs and beverages, is described. The possible influence of plant cell wall glycoproteins on the process of lignification is discussed in the light of the observed affinity of phenolic substrates for prolyl residues in protein structures.

Binding to DNA of selected lexitropsins and effects on prokaryotic topoisomerase activity.

The binding behaviour toward DNA of some minor groove binders related to distamycin was studied by means of circular dichroism. In addition their influence on the activity of topoisomerases isolated from Streptomyces noursei has been investigated. The monocationic imidazole containing ligands (lexitropsins) show a decreased affinity to AT pairs but an increased affinity to GC pairs which contrasts the AT-preferred binding of Dst-2 and Dst-3. For the monocationic triimidazole containing lexitropsin the affinity for GC over AT pairs was most pronounced. It was also found that the imidazole containing lexitropsins are inhibitors of topoisomerases. These minor groove binders interfere more strongly with the DNA gyrase activity than with the prokaryotic topoisomerase I. Our results indicate that Dst-3 most effectively inhibits gyrase and topoisomerase I activity. However, the inhibitory effect is neither related to the base pair specificity nor to the binding strength of different ligands. The mechanism of interference of minor groove binders with topoisomerase activity is more complex. It is considered that different factors, such as the nature of the ligand together with their DNA binding parameters and the target sequences of the enzymes play a role in the inhibitory effects of minor groove binders.

DNA binding of the nonintercalative ligands SN-6132, SN-6131 and SN-6113: minor variations of the ligand structure may cause changes in the base pair preference.

The DNA binding selectivity of three ligands of a series of antitumor agents of bisquaternary ammonium heterocycles has been investigated by means of CD spectroscopy and melting measurements. From the spectroscopic results and binding data it is concluded that the agents SN-6132, SN-6131 and SN-6113 have relatively high affinity to AT base pair sequences whereas the binding to GC pairs is very low. The binding selectivity to AT base pair sequences decreases in the order netropsin > SN-6132 > SN-6113 > SN-6131. Poly(dA).poly(dT) has the highest binding preference for SN-6132 relative to that of SN-6131. The different binding behavior of the ligands is related to their distinct changes in the chemical structure and to the DNA minor groove properties which determines the adaptability of the ligands in the groove.

Variation of DNA sequence specificity of DNA-oligopeptide binding ligands related to netropsin: imidazole-containing lexitropsins.

CD binding studies of nonintercalative oligopeptides related to netropsin, named lexitropsins, have been carried out with synthetic duplex DNAs and natural DNA. While netropsin possesses a high dA.dT sequence specificity, these ligands show a progressive lowering of the ability to bind to dA.dT basepairs in DNA and a dramatic reduction of the sequence specificity seen at high salt concentration due to a replacement of pyrrole moieties by imidazoles. This variation in DNA sequence specificity of lexitropsins is mirrored in corresponding large differences in the template inactivation of poly(dA-dT).poly(dA-dT) in the RNA polymerase reaction by these drugs. The presence of imidazole permits binding of the oligopeptide to dG.dC pairs, which is most effective for the triimidazole peptide. Results at increasing salt concentration reveal, however, that a tight binding to pure dG.dC sequences does not occur. A proper sequence containing dG.dC and dA.dT pairs is supposed to be required for a higher specificity. The CD data accord well with previously reported melting studies and are in favor of recent theoretical results suggesting that the diminished AT preference may be due to an increase in the complexation energy with the dG.dC pairs.

Interaction of the nonintercalative antitumour drugs SN-6999 and SN-18071 with DNA: influence of ligand structure on the binding specificity.

Binding to DNA's of the non-intercalative ligands SN-6999 and SN-18071 has been studied by means of circular dichroism, UV absorption, thermal melting and for SN-6999 by viscosity measurements. Both antitumour drugs show a preference for dA.dT rich DNA's, but the base pair selectivity of SN-18071 is lower as indicated by some affinity to dG.dC containing duplex DNA. The dA.dT base pair specificity of SN-6999 is comparable to that of netropsin. It forms very stable complexes with dA.dT containing duplex DNA and competes with netropsin binding on DNA. The ligands SN-18071 and pentamidine are totally released from their complexes with poly(dA-dT).poly(dA-dT) by competitive netropsin binding. The results demonstrate that hydrogen bonding capacity of the ligand in addition to other factors strongly contribute to the base sequence specificity in the recognition process of the ligand with DNA. A binding model of SN-6999 with five dA.dT pairs in the minor groove of B-DNA is suggested.