Paleomagnetism and gondwanaland.

Lower Paleozoic data now available for all the southern continents enable a unique reconstruction of Gondwanaland to be determined from paleomagnetic measurements alone. This reconstruction is corroborated by the computerized fit of the continental shelves and the matching of geological age provinces.

Interaction of nucleic acids with a non-intercalative anti-leukemic compound containing bisquarternary heterocycles.

The binding of an antitumour drug with bisquarternary ammonium heterocyclic structure, NSC-101327, to nucleic acids has been examined by using ultraviolet absorption and CD measurements. Like the minor groove-binding oligopeptides, netropsin and distamycin A, the optically inactive chromophoric system of NSC-101327 shows induced Cotton effects in the CD spectra of complexes with various DNAs, RNA and single-stranded polynucleotides. This property directly reflects interaction of NSC-101327 with different types of nucleic acids at moderate ionic strength, which contrasts with previous findings of a higher selective binding of netropsin to B-DNA. However, an efficient interactin of NSC-101327 with dA X dT basepair sequences is demonstrated by a large melting temperature increase of dA X dT-rich DNAs. NSC-101327 also reacts with dG X dC base pairs of B-DNA and forms a complex with Z-DNA of poly( br8dG -dC) X poly( br8DG -dC). The affinity of NSC-101327 to poly(dG-dC) X poly(dG-dC) is, however, lower, and the CD spectral binding effect depends on the ionic strength. The CD results of the complex with poly(dA-dT) X poly(dA-dT) suggests at least two binding modes, in accordance with previous conclusions. This is indicated by a clear-cut initial increase of the CD signal and a subsequent large decrease to negative CD signals. Competition experiments with netropsin suggest that binding of NSC-101327 occurs preferentially in the minor groove without intercalation. NSC-101327 also tends to interact with lower binding affinity to dG-dC pairs in B-DNA, with rA X rU pairs of RNA and with single-stranded polynucleotides. Thus our results suggest that NSC-101327 represents a DNA groove-binding ligand of lower basepair specificity and lower conformational selectivity compared to the B-specific netropsin probe.

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.

Magnetic circular dichroism study of the binding of netropsin and distamycin A with DNA.

The magnetic circular dichroism (MCD) of netropsin and distamycin-A is reported. New data for the interaction with dA ; dT base pairs in DNA were obtained from the MCD of their complexes with DNA duplex polymers. The MCD results allow an interpretation of the induced Cotton effects in the natural CD spectra of netropsin and distamycin-A complexes with DNA. While large distortions of the bases in DNA by the oligopeptide interaction is excluded, some subtle conformational variations of the DNA might explain the inhibition of the enzyme function of netropsin and distamycin-A on DNA.

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.

Chain length-dependent association of distamycin-type oligopeptides with A X T and G X C pairs in polydeoxynucleotide duplexes.

Different binding affinities of various distamycin analogs including the deformylated derivative with poly(dA-dC) X poly(dG-dT) were investigated using CD measurements. The inhibitory effect of distamycins on the DNAase I cleavage activity of DNA duplexes strongly supports the binding data. The base specificity of the ligand interaction with duplex DNA depends on the chain length of distamycin analogs. Netropsin, distamycin-2 and the deformylated distamycin-3 show no binding to dG X dC containing sequences at moderate ionic strength and are classified as highly dA X dT specific. In contrast distamycin having three, four or five methylpyrrolecarboxamide groups also forms more or less stable complexes with dG X dC-containing duplexes. These ligands possess a lower basepair specificity. The correlation between binding behavior and oligopeptide structure shows that presence of the number of hydrogen acceptor and donor sites determines the basepair and sequence specificity. The additional interaction with dG X dC pairs becomes essential when the number of hydrogen acceptor sites exceeds n = 3.

Studies on the conformation of protonated DNA.

Experiments were made to demonstrate the predominant protonation effects and structural changes of the ordered double helical DNA structure and denatured state of DNA. Spectrophotometric titrations performed at different wavelengths indicate that cytosine can be protonated in the DNA double helical molecule to a high extent without breakdown of the secondary structure. With DNA heat-denatured under severe conditions the protonation of cytosine can be measured at 280, 295, and 300 mµ: the apparent pK value obtained was ∼4.6. The protonated double helical conformation of the DNA molecule differs from the unprotonated state, which follows from the decrease of the thermal stability and from changes in the ORD curves. The ORD of a GC-rich DNA indicates a novel Cotton effect with positive rotations at ∼260 mµ in 0.02M KCl below pH 4.0 to pH 3.3. The occurrence of the new peak parallels the extent of protonated cytosine measured by the spectrophotometric titrations. It is concluded that the protonated cytosine in the double helical structure is responsible for the difference between the protonated DNA conformation and the native state at neutral pH.

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.

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.

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.

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.

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.

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.

Interaction of phenosafranine with nucleic acids and model polyphosphates. III. Heterogeneity in phenosafranine interactions with DNA base pairs.

Fluorescence and circular dichroism spectral measurements, thermal denaturation studies and binding competition experiments with netropsin and actinomycin D were carried out in systems containing phenosafranine bound to DNA's differing in base composition. The investigated properties exhibit a heterogeneity related to the content of A.T and G.C pairs in DNA and to the nature of phenosafranine binding modes. At low level of saturation of binding sites (r less than 0.1) phenosafranine does not show strong preference for any of the DNA base pairs in the overall binding. However, the strong monomer non-cooperative binding outside the helix (mode I1) occurs predominantly, even though not exclusively in G.C rich regions. The strong binding modes involving intercalated dye molecules (mode I2 and eventually mode II1) prevail in A.T rich regions. These binding modes become the principal types of strong phenosafranine interaction with DNA when the level of saturation of binding sites increases, i.e. at r greater than 0.1.20

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.

Two binding modes of netropsin are involved in the complex formation with poly(dA-dT).poly(dA-dT) and other alternating DNA duplex polymers.

Using CD measurements we show that the interaction of netropsin to poly(dA-dT).poly(dA-dT) involves two binding modes at low ionic strength. The first and second binding modes are distinguished by a defined shift of the CD maximum and the presence of characteristic isodichroic points in the long wavelength range from 313 nm to 325 nm. The first binding mode is independent of ionic strength and is primarily determined by specific interaction to dA.dT base pairs. Employing a netropsin derivative and different salt conditions it is demonstrated that ionic contacts are essential for the second binding mode. Other alternating duplexes and natural DNA also exhibit more or less a second step in the interaction with netropsin observable at high ratio of ligand per nucleotide. The second binding mode is absent for poly(dA).poly(dT). The presence of a two-step binding mechanism is also demonstrated in the complex formation of poly(dA-dT).poly(dA-dT) with the distamycin analog consisting of pentamethylpyrrolecarboxamide. While the binding mode I of netropsin is identical with its localization in the minor groove, for binding mode II we consider two alternative interpretations.

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.

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.

[An information system for a department of nuclear medicine (author's transl)]. / Ein Abteilungs-Informationssystem für die Nuklearmedizin.

A computer system is connected on-line to each working place of a department for nuclear medicine. It is assisting the daily work such as the measuring and processing of the measured data, the medical interpretation of the results and the finding of the diagnosis, and finally the composing and writing of medical reports. Additionally this system supports the organization of the department in many ways. The organisation and functions of the system are described.