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Biomedical subjects

H Stark

Publications and source records attributed to H Stark.

At least 19 recordsLinked to original sources
PubMed2000

Structure of a molluscan hemocyanin didecamer (HtH1 from Haliotis tuberculata) at 12 A resolution by cryoelectron microscopy.

A 12 A resolution three-dimensional density map of the Haliotis tuberculata hemocyanin type 1 (HtH1) didecamer has been obtained by cryoelectron microscopy of unstained molecules and angular reconstitution. The dyad symmetry of the 8 MDa D5 HtH1 didecamer, formed by the pairing of two asymmetric 4 MDa ring-like C5 decamers, is emphasised. The major and minor surface helical grooves of the didecamer are well defined, in agreement with earlier data on molluscan hemocyanins. The location of the obliquely orientated repeating unit, a subunit dimer, within the decamer has been defined. Following interactive extraction of this dimer, several new structural features of the dimer and of the subunit have now emerged with improved detail. The subunit dimer possesses pseudo 2-fold symmetry, resulting from the steric arrangement of the wall domains/functional units (FUs-abcdef) of the two subunits. The arc and collar FUs (g and h) depart from this inherent 2-fold symmetry and are thereby responsible for the asymmetry of the C5 decamer, with the internalised collar/arc complex at one edge of the decamer. The FU heterodimers forming the wall morphological units have a hollow centre, and thus create a series of repeating channels that extend within the wall through all three tiers of the decamer. The connections between the wall and the arc are defined with improved clarity, and evidence is provided to indicate that the arc and collar FU pairs have a homodimeric composition (gg and hh, respectively). Two possibilities for the subunit path within the subunit dimer are presented, which correlate with the available structural, immunolabelling and protease cleavage data from HtH1 and other molluscan hemocyanins.

Animals↗
PubMed2000

The inhibitory influence of an acquired escape strategy on subsequent avoidance learning in gerbils.

In gerbils an acquired non-avoidance strategy as pre-experience in a shuttle-box was studied in its influence on the learning of a standard avoidance paradigm. Thereby the same cue stimuli, frequency modulated tones (CS) and electric footshocks (US) were used in different behavioral paradigms. In the preexperience sessions the interstimulus interval between CS and US and on the other hand the escapability or inescapability of the US was varied. It was found that the experience with a relatively long interstimulus interval led to a prolonged maintenance of an acquired escape strategy during subsequent standard avoidance learning. This effect increased with the preexperience of an inescapable US. Additional insights into the temporal inhibition of avoidance learning by the pre-experience were provided by behavioral events like attention responses and orienting responses reflecting components of information processing.

Acoustic Stimulation↗
PubMed2000

Large-scale movement of elongation factor G and extensive conformational change of the ribosome during translocation.

Elongation factor (EF) G promotes tRNA translocation on the ribosome. We present three-dimensional reconstructions, obtained by cryo-electron microscopy, of EF-G-ribosome complexes before and after translocation. In the pretranslocation state, domain 1 of EF-G interacts with the L7/12 stalk on the 50S subunit, while domain 4 contacts the shoulder of the 30S subunit in the region where protein S4 is located. During translocation, EF-G experiences an extensive reorientation, such that, after translocation, domain 4 reaches into the decoding center. The factor assumes different conformations before and after translocation. The structure of the ribosome is changed substantially in the pretranslocation state, in particular at the head-to-body junction in the 30S subunit, suggesting a possible mechanism of translocation.

Fusidic Acid↗
PubMed2000

Histamine H(3)-receptor antagonists inhibit gastroprotection by (R)-alpha-methylhistamine in the rat.

(R)-alpha-methylhistamine, a selective agonist of histamine H(3) receptors, is capable of protecting the gastric mucosa against differently acting damaging agents. The objective of the present study was to determine whether H(3) receptors mediate its protective action in the rat. Gastric mucosal lesions were induced intragastrically (i.g.) by 0.6 N HCl, 1 ml rat(-1). (R)-alpha-methylhistamine, 100 mg kg(-1) i.g., substantially reduced the severity of macroscopically and histologically assessed damage caused by concentrated acid. Prior treatment with highly selective H(3)-receptor antagonists, ciproxifan (0.3, 1 and 3 mg kg(-1) i.g.) and clobenpropit (3, 10 and 30 mg kg(-1) i.g.), dose-dependently inhibited the protection exerted by (R)-alpha-methylhistamine up to a complete reversal. When given alone at high doses, both antagonists tended to worsen the HCl-induced histologic damage. During basal conditions, (R)-alpha-methylhistamine, 100 mg kg(-1) i. g., caused a significant increase in titratable acidity of the gastric juice. Prior treatment with ciproxifan (3 mg kg(-1) i.g.) and clobenpropit (30 mg kg(-1) i.g.) did not alter the secretory response to (R)-alpha-methylhistamine. Clobenpropit alone, but not ciproxifan, increased the volume of gastric juice, and both compounds alone had no effect on titratable acid. Present findings support evidence that H(3) receptors are actively involved in the maintenance of gastric mucosal integrity, with no apparent role in the regulation of basal gastric acid secretion.

Animals↗
PubMed2000

High-diffraction-efficiency pseudorandom encoding.

Pseudorandom encoding (PRE) is a statistics-based procedure in which a pure-phase spatial light modulator (SLM) can yield, on the average, the prescribed diffraction pattern specified by the user. We seek to combine PRE with the optimization of an aperture-based target function. The target function is a fully complex input transmittance, unrealizable by a phase-only SLM, that generates a prescribed light intensity. The optimization is done to increase the diffraction efficiency of the overall process. We compare three optimization methods-Monte Carlo simulation, a genetic algorithm, and a gradient search-for maximizing the diffraction efficiency of a spot-array generator. Calculated solutions are then encoded by PRE, and the resulting diffraction patterns are computer simulated. Details on the complexity of each procedure are furnished, as well as comparisons on the quality, such as uniformity of the output spot array.

Computer Simulation↗
PubMed2000

Encapsulation of plasmid DNA in stabilized plasmid-lipid particles composed of different cationic lipid concentration for optimal transfection activity.

In previous work (Wheeler et al. (1999) Gene Therapy 6, 271-281) we have shown that plasmid DNA can be entrapped in "stabilized plasmid lipid particles" (SPLP) using low levels (5-10 mol%) of cationic lipid, the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), and a polyethyleneglycol (PEG) coating for stabilization. The PEG moieties are attached to a ceramide anchor containing an arachidoyl acyl group (PEG-CerC20). However, these SPLP exhibit low transfection potencies in vitro as compared to plasmid/cationic lipid complexes formed with liposomes composed of cationic and neutral lipid at a 1:1 lipid ratio. The objective of this study was to construct SPLPs with increased cationic lipid contents that result in maximum transfection levels. A phosphate buffer detergent dialysis technique is described resulting in formation of SPLP containing 7-42.5 mol% DODAC with reproducible encapsulation efficiency of up to 80%. An octanoyl acyl group was used as anchor for the PEG moiety (PEG-CerC8) permitting a quick exchange out of the SPLP to further optimize the in vitro and in vivo transfection. We have demonstrated that this technique can be used to encapsulate either linearized DNA or supercoiled plasmids ranging from 3-20 kb. The SPLP formed could be isolated from empty vesicles by sucrose density gradient centrifugation, and exhibited a narrow size distribution of approximately 75 +/- 6 nm as determined by cryo-electron microscopy. The high plasmid-to-lipid ratio observed corresponded to one plasmid per particle. The SPLP consist of a lipid bilayer surrounding the plasmid DNA as visualized by cryo-electron microscopy. SPLP containing a range of DODAC concentrations were tested for in vitro and in vivo transfection. In vitro, in COS-7 cells transfection reached a maximum after 48 h. The transfection efficiency increased when the DODAC concentration in the SPLP was decreased from 42.5 to 24 mol% DODAC. Decreasing the cationic lipid concentration improved transfection in part due to decreased toxicity. In vivo studies using an intraperitoneal B16 tumor model and intraperitoneal administration of SPLP showed maximum transfection activity for SPLP containing 24 mol% DODAC. Gene expression observed in tumor cells was increased by approximately one magnitude as compared to cationic lipid/DNA complexes. The SPLP were stable and upon storage at 4 degrees C no significant change in the transfection activity was observed over a one-year period. Thus this phosphate buffer detergent dialysis technique can be used to generate SPLP formulations containing a wide range of cationic lipid concentrations to determine optimal SPLP composition for high transfection activity and low toxicity.

Animals↗
PubMed2000

Substituted N-phenylcarbamates as histamine H3 receptor antagonists with improved in vivo potency.

Novel substituted N-phenylcarbamates as derivatives of 3-(1 H-imidazol-4-yl)propanol were prepared and tested for their antagonist potency in vitro and in vivo at histamine H3 receptors. Structural modifications with different alkyl and acetyl moieties were performed in an attempt to optimize pharmacodynamic and pharmacokinetic effects. Most compounds are active in a functional test for histamine H3 receptors on rat cerebral cortex synaptosomes as well as in a peripheral model on guinea pig ileum. But only carbamates without too bulky lipophilic residues showed pronounced to high antagonist potency on the enhancement of endogenous histamine in brain after p.o. administration to mice (ED50 values of 5.5 to 0.86 mg.kg-1). The tested compounds presented weak activities at histamine H1, H2, and muscarinic M3 receptors thus demonstrating their H3-receptor selectivity.

Animals↗
PubMed1999

Structure of influenza haemagglutinin at neutral and at fusogenic pH by electron cryo-microscopy.

The three-dimensional structures of the complete haemagglutinin (HA) of influenza virus A/Japan/305/57 (H2N2) in its native (neutral pH) and membrane fusion-competent (low pH) form by electron cryo-microscopy at a resolution of 10 A and 14 A, respectively, have been determined. In the fusion-competent form the subunits remain closely associated preserving typical overall features of the trimeric ectodomain at neutral pH. Rearrangements of the tertiary structure in the distal and the stem parts are associated with the formation of a central cavity through the entire ectodomain. We suggest that the cavity is essential for relocation of the so-called fusion sequence of HA towards the target membrane.

Cryoelectron Microscopy↗
PubMed1999

Novel partial agonists for the histamine H(3) receptor with high in vitro and in vivo activity.

Novel branched N-alkylcarbamates and aliphatic ethers derived from 3-(1H-imidazol-4-yl)propanol were prepared. The compounds were investigated on two functional histamine H(3)-receptor assays. Some compounds displayed partial agonist activity on synaptosomes of rat brain cortex but behaved as pure competitive antagonists on the guinea pig ileum. Under in vivo conditions after po application to mice, some of the compounds showed partial or full agonist activity. Highest in vivo potency was found for the 3,3-dimethylbutyl ether 10 (ED(50) = 0.29 mg/kg, full intrinsic activity). These novel agonists are structurally diverse from classical aminergic histamine H(3)-receptor agonists (e.g., (R)-alpha-methylhistamine, imetit) as they lack a basic moiety in the side chain, which is supposed to be important for the activation of the receptor protein. The selectivity for the histamine H(3) receptor was proven by determination of H(1)- and H(2)-receptor activity on functional assays of the guinea pig.

Animals↗
PubMed1999

Elucidating the medium-resolution structure of ribosomal particles: an interplay between electron cryo-microscopy and X-ray crystallograhy.

BACKGROUND: Ribosomes are the universal cellular organelles that accomplish the translation of the genetic code into proteins. Electron cryo-microscopy (cryo-EM) has yielded fairly detailed three-dimensional reconstructions of ribosomes. These were used to assist in the determination of higher resolution structures by X-ray crystallography. RESULTS: Molecular replacement studies using cryo-EM reconstructions provided feasible packing schemes for crystals of ribosomes and their two subunits from Thermus thermophilus, and of the large subunits from Haloarcula marismortui. For the large subunits, these studies also confirmed the major heavy-atom sites obtained by single isomorphous replacement combined with anomalous diffraction (SIRAS) and by multiple isomorphous replacement combined with anomalous diffraction (MIRAS) at approximately 10 A. Although adequate starting phases could not be obtained for the small subunits, the crystals of which diffract to 3.0 A, cryo-EM reconstructions were indispensable for analyzing their 7.2 A multiple isomorphous replacement (MIR) map. This work indicated that the conformation of the crystallized small subunits resembles that seen within the 70S ribosomes. Subsequently, crystals of particles trapped in their functionally active state were grown. CONCLUSIONS: Single-particle cryo-EM can contribute to the progress of crystallography of non-symmetrical, large and flexible macromolecular assemblies. Besides confirming heavy-atom sites, obtained from flat or overcrowded difference Patterson maps, the cryo-EM reconstructions assisted in elucidating packing arrangements. They also provided tools for the identification of the conformation within the crystals and for the estimation of the level of inherent non-isomorphism.

Crystallography, X-Ray↗
PubMed1999

Increase of extracellular dopamine in prefrontal cortex of gerbils during acquisition of the avoidance strategy in the shuttle-box.

We examined whether extracellular dopamine (DA) increase in medial prefrontal cortex is correlated with the establishment or with the retrieval of an auditory avoidance strategy in a shuttle-box. Using microdialysis from right medial prefrontal cortex (mPFC) in combination with behavioral measures, gerbils were trained on a tone-footshock combination in two sessions on two successive days. Sessions in each animal involving avoidance strategy formation and avoidance strategy retrieval were differentiated post hoc by using the percentage of conditioned responses (CR) and other behavioral measures of training sessions. It was found that exclusively strategy formation correlated with high DA levels in mPFC.

Acoustic Stimulation↗
PubMed1999

Development of chiral N-alkylcarbamates as new leads for potent and selective H3-receptor antagonists: synthesis, capillary electrophoresis, and in vitro and oral in vivo activity.

Novel carbamates as derivatives of 3-(1H-imidazol-4-yl)propanol with an N-alkyl chain were prepared as histamine H3-receptor antagonists. Branching of the N-alkyl side chain with methyl groups led to chiral compounds which were synthesized stereospecifically by a Mitsunobu protocol adapted Gabriel synthesis. The optical purity of some of the chiral compounds was determined (ee > 95%) by capillary electrophoresis (CE). The investigated compounds showed pronounced to high antagonist activity (Ki values of 4.1-316 nM) in a functional test for histamine H3 receptors on rat cerebral cortex synaptosomes. Similar H3-receptor antagonist activities were observed in a peripheral model on guinea pig ileum. No stereoselective discrimination for the H3 receptor for the chiral antagonists was found with the in vitro assays. All compounds were also screened for central H3-receptor antagonist activity in vivo in mice after po administration. Most compounds were potent agents of the H3-receptor-mediated enhancement of brain Ntau-methylhistamine levels. The enantiomers of the N-2-heptylcarbamate showed a stereoselective differentiation in their pharmacological effect in vivo (ED50 of 0.39 mg/kg for the (S)-derivative vs 1.5 mg/kg for the (R)-derivative) most probably caused by differences in pharmacokinetic parameters. H1- and H2-receptor activities were determined for some of the novel carbamates, demonstrating that they have a highly selective action at the histamine H3 receptor.

Administration, Oral↗
PubMed1999

Radiosynthesis and biodistribution of 123I-labeled antagonists of the histamine H3 receptor as potential SPECT ligands.

We have synthesized three 123I-labeled histamine H3 receptor ligands, i.e., [123I]GR 190028, [123I]FUB 271, and [123I]iodoproxyfan, in moderate to good radiochemical yields via a Cu+-assisted I-for-123I exchange method. Biodistribution in the rat of these compounds revealed high hepatic and pulmonary uptake. Brain uptake was moderate, but for [123I]iodoproxyfan, brain uptake was high enough for a pilot single photon emission computed tomography (SPECT) study in the rabbit. However, for this compound, the cerebral uptake could not be blocked by a pretreatment with [R]-alpha-methylhistamine, a selective, high-affinity histamine H3 receptor agonist, both in the SPECT study in the rabbit and in the biodistribution study in the rat. Apparently, [123I]iodoproxyfan is binding to a non-H3 receptor binding site. None of the three investigated compounds is suitable for use as a SPECT ligand for the H3 receptor in the brain.

Animals↗
PubMed1998

4-Alkynylphenyl imidazolylpropyl ethers as selective histamine H3-receptor antagonists with high oral central nervous system activity.

In search for potent and therapeutically useful H3-receptor antagonists, we prepared novel 4-alkynylphenyl ether derivatives of 3-(1H-imidazol-4-yl)propanol in a convenient synthetic route. All compounds were tested for in vitro and in vivo H3-receptor antagonist activity as well as for H3-receptor selectivity versus H1- and H2-receptors. The presented 4-alkynylphenyl ethers are highly potent and selective H3 antagonists showing oral activity and improved brain penetration. Particularly 4-ethynylphenyl 3-(1H-imidazol-4-yl)propyl ether (14a) displays striking in vitro and in vivo activity with a -log Ki value of 8.6 and an ED50 value of 0.12 mg/kg. At present 14a is the most potent H3-receptor antagonist in vivo and may therefore be a potential drug for the therapy of H3-receptor-dependent diseases of the central nervous system (CNS).

Administration, Oral↗
PubMed1998

General construction pattern of histamine H3-receptor antagonists: change of a paradigm.

Novel omega-phenyl substituted and unsubstituted alkyl and alkenyl imidazole derivatives were prepared and tested for their antagonist activity in vitro and in vivo at histamine H3-receptors. Some compounds showed high in vitro and in vivo H3-receptor activity despite their structure bearing no polar moiety in the centre of the molecule which is a common structural feature of all other antagonists known. Quite probably there are further in vivo effects for some compounds resulting from other receptor interactions.

Animals↗
PubMed1998

Correlation of the expansion segments in mammalian rRNA with the fine structure of the 80 S ribosome; a cryoelectron microscopic reconstruction of the rabbit reticulocyte ribosome at 21 A resolution.

Samples of 80 S ribosomes from rabbit reticulocytes were subjected to electron cryomicroscopy combined with angular reconstitution. A three-dimensional reconstruction at 21 A resolution was obtained, which was compared with the corresponding (previously published) reconstruction of Escherichia coli 70 S ribosomes carrying tRNAs at the A and P sites. In the region of the intersubunit cavity, the principal features observed in the 70 S ribosome (such as the L1 protuberance, the central protuberance and A site finger in the large subunit) could all be clearly identified in the 80 S particle. On the other hand, significant additional features were observed in the 80 S ribosomes on the solvent sides and lower regions of both subunits. In the case of the small (40 S) subunit, the most prominent additions are two extensions at the base of the particle. By comparing the secondary structure of the rabbit 18 S rRNA with our model for the three-dimensional arrangement of E. coli 16 S rRNA, these two extensions could be correlated with the rabbit expansion segments (each totalling ca 170 bases) in the regions of helix 21, and of helices 8, 9 and 44, respectively. A similar comparison of the secondary structures of mammalian 28 S rRNA and E. coli 23 S rRNA, combined with preliminary modelling studies on the 23 S rRNA within the 50 S subunit, enabled the additional features in the 60 S subunit to be sub-divided into five groups. The first (corresponding to a total of ca 335 extra bases in helices 45, 98 and 101) is located on the solvent side of the 60 S subunit, close to the L7/L12 area. The second (820 bases in helices 25 and 38) is centrally placed on the solvent side of the subunit, whereas the third group (totaling 225 bases in helices 18/19, 27/29, 52 and 54) lies towards the L1 side of the subunit. The fourth feature (80 bases in helices 78 and 79) lies within or close to the L1 protuberance itself, and the fifth (560 bases in helix 63) is located underneath the L1 protuberance on the interface side of the 60 S subunit.

Animals↗