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Biomedical subjects

U Heinemann

Publications and source records attributed to U Heinemann.

At least 271 records · Page 15Linked to original sources
PubMed1991

X-ray analysis of cubic crystals of the complex formed between ribonuclease T1 and guanosine-3',5'-bisphosphate.

The complex formed between ribonuclease T1 (RNase T1) and guanosine-3',5'-bisphosphate (3',5'-pGp) crystallizes in the cubic space group I23 with alpha = 86.47 (4) A. X-ray data were collected on a four-circle diffractometer to 3.2 A resolution and the structure was determined by molecular-replacement methods [ULTIMA; Rabinovich & Shakked (1984). Acta Cryst. A40, 195-200] based on the RNase T1 coordinates taken from the complex with guanosine-2'-phosphate. Refinement converged at 16.6% for 1540 data with Fo greater than 1 sigma (Fo) with acceptable stereochemistry. The RNase T1 conformation is comparable to that in other complexes which crystallize preferentially in space group P2(1)2(1)2(1) except for side chains that interact intermolecularly. The guanine of 3',5'-pGp is bound to the recognition site in the same way as in other guanine-containing complexes except for its interaction with Glu46. The side-chain carboxylate of this amino acid does not form hydrogen bonds to N1H and N2H of guanine but is rotated so as to permit insertion of two water molecules which replace its acceptor functions. In contrast to other guanosine derivatives which are bound to RNase T1 in the syn form, 3',5'-pGp is anti. This conformation positions the two phosphate groups 'outside' the protein, with hydrogen-bonding contacts only to water molecules; the active site is filled by water. The RNase T1-3',5'-pGp complex probably has biological significance as it may represent the enzyme-product complex before dissociation.

Aspergillus oryzae↗
PubMed1991

Evidence for a substrate-binding subsite in ribonuclease T1. Crystal structure of the complex with two guanosines, and model building of the complex with the substrate guanylyl-3',5'-guanosine.

The enzyme ribonuclease T1 cleaves single-stranded RNA at the 3'-side of guanosine. The structure of the complex with two guanosines has been analyzed at 1.8-A resolution and refined to a crystallographic R value of 14.0%. One guanosine occupies the guanosine recognition site as observed in previously analyzed complexes of ribonuclease T1 with guanosine phosphates. The other is bound to a base-unspecific subsite marking the binding locus of the nucleoside 3'-proximal to guanosine in a cleavable RNA chain. The positions of the guanosine bound to the recognition site and of the guanine base at the subsite were used to guide model building of the substrate guanylyl-3',5'-guanosine bound to the active site of ribonuclease T1. After energy minimization and a 7-ps stochastic dynamics simulation, a plausible model of the enzyme-substrate complex was obtained which may serve as a reference point in consideration of the mechanisms of RNA hydrolysis by ribonuclease T1.

Crystallization↗
PubMed1991

Crystallization of the hybrid Bacillus (1-3,1-4)-beta-glucanase H(A16-M).

The extremely thermostable hybrid Bacillus (1-3,1-4)-beta-glucanase H(A16-M) has been crystallized with polyethylene glycol by vapour diffusion. The single crystals diffract to a resolution of 2.2 A. The protein crystallizes in the orthorhombic space group P2(1)2(1)2(1) with a = 70.22(7) A, b = 72.56(1) A, c = 49.97(1) A, and has one molecule per asymmetric unit.

Bacillus↗
PubMed1991

Effect of a single 3'-methylene phosphonate linkage on the conformation of an A-DNA octamer double helix.

The three-dimensional structure of the self-complementary DNA octamer d(GCCCGpGGC) has been determined in the crystalline state using X-ray diffraction data to a nominal resolutoin of 2.12 measured from a very small crystal at DESY, Hamburg. The structure was refined with stereochemical restraints to an R value of 17.1%. d(GCCCGpGGC), containing one single 3'-methylene phosphonate linkage (denoted p), forms an A-DNA double helix with strict dyad symmetry, that is distinct from canonical A-DNA by a wide open major groove and a small average base-pair inclination against the helix axis. The conformation of the unmodified control d(GCCCGGGC) is known from an X-ray analysis of isomorphous crystals (Heinemann et al. (1987) Nucleic Acids Res. 15, 9531-9550). Comparison of the two structures reveals only minor conformational differences, most notably in the pucker of the reduced deoxyribose. It is suggested that oligonucleotides with charged 3'-methylene phosphonate groups may form stable duplexes with complementary DNA or RNA strands rendering them candidates for use as gene-regulatory antisense probes.

Crystallography↗
PubMed1991

Molecular dynamics simulation of the hydration shell of a B-DNA decamer reveals two main types of minor-groove hydration depending on groove width.

The conformation of the self-complementary B-DNA decamer C-C-A-A-C-G-T-T-G-G is known from a high-resolution x-ray crystal structure analysis. Molecular dynamics simulation of the hydration shell of the decamer has revealed two main types of minor-groove hydration, depending on groove width. The narrow part of the minor groove has a spine of hydration analogous to that described for the A + T-rich center of the minor groove in the dodecamer C-G-C-G-A-A-T-T-C-G-C-G [Drew, H. R. & Dickerson, R. E. (1981) J. Mol. Biol. 151, 535-556], the first hydration layer of which contains one water molecular per base pair. In contrast, in the wide part of the minor groove, each base is hydrated individually, water molecules lying predominantly in the base plane. In intermediate-width regions, preferred water-molecule sites are shifted away from the base plane in a 3'-to-5' direction. This shift becomes more pronounced as the minor groove narrows, until the two water molecules lie approximately midway between base pairs. If the minor groove is narrowed still further, it accommodates only one water molecule, and the hydration transforms to the well-known water spine. The observed pattern agrees with available crystallographic data and with our earlier calculations. The results confirm the assumption that preferred positions of water oxygens in the minor groove depend predominantly on groove width rather than on base sequence. However, the location of water hydrogens, and the network of hydrogen bonding, can depend on base sequence. We suggest a simple explanation of water-spine formation in the narrow minor groove of a random DNA sequence. The spine of hydration may be a property of the minor groove of overwound variants of B-DNA, the C and D forms, for which the middle part of the decamer C-C-A-A-C-G-T-T-G-G can serve as a model.

Base Sequence↗
PubMed1991

The conformation of a B-DNA decamer is mainly determined by its sequence and not by crystal environment.

By comparing the conformations adopted by a double-stranded decameric B-DNA fragment in different crystal environments, we address the question of the degree of deformability of DNA helices. The three-dimensional structure of the self-complementary DNA decamer CCAGGCmeCTGG has been determined from crystals of space group P6 at 2.25 A resolution with an R value of 17.2% for 2407 1 sigma structure amplitudes. The oligonucleotide forms a B-type double helix with a characteristic sequence-dependent conformation closely resembling that of the corresponding unmethylated decamer, the structure of which is known from a high-resolution analysis of crystals of space group C2. Evidently, both the effects of single-site methylation and altered crystal environment on the DNA conformation are small. Therefore, double-helical DNA may possess sequence-determined conformational features that are less deformable than previously thought.

Base Sequence↗
PubMed1991

Regional and time dependent variations of low Mg2+ induced epileptiform activity in rat temporal cortex slices.

In order to study spatial interactions during low magnesium induced epileptiform activity, changes in extracellular potassium concentration ([K+]0) and associated slow field potentials (f.p.'s) were recorded in thin rat temporal cortex slices (400 microns) containing the neocortical temporal area 3 (Te3), the entorhinal cortex (EC) and the hippocampal formation with the dentate gyrus, area CA3 and CA1 and the subiculum (Sub). The epileptiform activity was characterized by short recurrent epileptiform discharges (40 to 80 ms, 20/min) in areas CA3 and CA1 and by interictal discharges and tonic and clonic seizure like events (SLE's) (13-88s) in the EC, Te3 and Sub. While interictal discharges occurred independent of each other in the different subfields, the three areas became synchronized during the course of a SLE. The EC, Te3 and Sub all could represent the "focus" for generation of the SLE's. This initiation site for SLE's sometimes changed from one area to another. The characteristics of the rises in [K+]0 and subsequent undershoots were comparable to previous observations in in vivo preparations. Interestingly, rises in [K+]0 could start before actual onset of seizure like activity in secondarily recruited areas. The epileptiform activity could change its characteristics to either a state of recurrent tonic discharge episodes or to a continuous clonic discharge state reminiscent of various forms of status epilepticus. We did not observe, in any of these states, active participation by area CA3 in the epileptiform activity of the EC in spite of clear projected activity to the dentate gyrus. Even after application of picrotoxin (20 microM), area CA3 did not actively participate in the SLE's generated in the entorhinal cortex. When baclofen (2 microM) was added to the picrotoxin containing medium, SLE's occurred both in the entorhinal cortex and in area CA3, suggesting that inhibition of inhibitory interneurons by baclofen could overcome the "filtering" of projected activity from the entorhinal cortex to the hippocampus.

Animals↗
PubMed1991

A note on crystal packing and global helix structure in short A-DNA duplexes.

A simple relation exists between the packing density in crystals of short A-DNA duplexes and their global double-helical structure. The volume per nucleotide pair shows a linear inverse correlation with the mean displacement of base pairs from the best straight helix axis. The mean displacement is a measure of major groove depth and varies between -3.3 A and -4.9 A in A-form oligonucleotides analysed in the crystalline state. Since the mean displacement of base pairs from the helix axis determines other helical parameters such as base-pair longitudinal slide, its correlation with crystal packing is of considerable interest. The displacement-packing correlation is very clear for octamer duplexes which crystallize in three different lattices. Longer A-helical fragments sometimes deviate from the rule. It may be speculated whether A-form duplexes not completing a full helical turn are especially prone to distortions due to packing in crystals or arising from intermolecular contacts in solution.

Base Sequence↗
PubMed1991

Molecular cloning, nucleotide sequence, and characterization of the Bacillus subtilis gene encoding the DNA-binding protein HBsu.

A homologous class of histonelike proteins which are believed to wrap the DNA and to condense the chromosome into highly folded nucleoid structures has been identified in different bacterial species. Bacillus subtilis encodes a homodimeric DNA-binding protein called HBsu. We have cloned the corresponding gene (hbs) on a 3.8-kb fragment. The gene was subcloned to a 1-kb fragment, sequenced, and characterized. It encodes a 92-amino-acid protein with a predicted molecular mass of 9,884 Da. Fortunately, analysis of the DNA sequence downstream of the 3' end of hbs revealed the location of the first 19 amino acid residues of MtrA. This finding located the hbs gene unequivocally to the 5' end of the mtr operon at about 204 degrees on the standard genetic map of B. subtilis. Northern (RNA) blot analysis and primer extension studies indicated the presence of two distinct hbs transcripts, which were found to be initiated at two different sites located about 160 bases apart. Several attempts to replace the hbs gene in the B. subtilis chromosome with a cat-interrupted copy (hbs::cat) through marker replacement recombination were unsuccessful. In order to study whether hbs is an essential gene, we have constructed a strain containing a truncated copy of the gene behind its own promoter and another intact copy under control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible spac-1 promoter. In this strain (BM19), normal growth was found to depend on IPTG, whereas in the absence of IPTG, growth was severely affected. These results suggest an essential role for the hbs gene product for normal growth in B. subtilis.

Amino Acid Sequence↗
PubMed1990

Late low magnesium-induced epileptiform activity in rat entorhinal cortex slices becomes insensitive to the anticonvulsant valproic acid.

We investigated time-dependent changes in low magnesium-induced epileptiform activity in combined rat entorhinal cortex/hippocampal slices with extracellular recording techniques. While in area CA3 short interictal discharges are generated without any major changes in activity during prolonged recording periods, initial tonic clonic ictaform events in the entorhinal cortex may change with time. We observed often a transition into a state of recurrent tonic activity without any clonic afterdischarges. Alternatively, seizures could stay in the clonic discharge mode for the rest of the experiment. These different seizure states were not equally affected by the anticonvulsant valproic acid. While the early clonic tonic discharges in the entorhinal cortex and the interictal like activity in area CA3 were effectively suppressed by valproic acid (VPA) the late recurrent tonic seizure discharge state was unaffected by the drug. It was, however, still sensitive to the N-methyl-D-aspartate (NMDA) receptor antagonist 2-aminophosphonovalerate. These findings point to seizure-induced changes in neuronal interaction in rat entorhinal cortex.

2-Amino-5-phosphonovalerate↗
PubMed1990

Quisqualate-induced changes in extracellular sodium and calcium concentrations persist in the combined presence of NMDA and non-NMDA receptor antagonists in rat hippocampal slices.

In the CA1 stratum pyramidale of rat hippocampal slices we have used Ca2(+)- and Na(+)-sensitive microelectrodes to measure the changes in [Ca2+]o, [Na+]o and associated slow field potentials elicited by the iontophoretic application of the excitatory amino acids N-methyl-D-aspartic acid (NMDA), quisqualic acid (Quis), alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) and glutamic acid (Glu) in the presence of the NMDA receptor antagonists 2-amino-5-phosphonovaleric acid (AP5) and ketamine and the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Although a combination of these antagonists blocked stimulation-induced synaptic field potentials as well as AMPA- and NMDA-induced ionic changes and associated field potentials, Quis-induced ionic changes and associated field potentials could still be observed after 60 min. These residual Quis signals were also resistant to 2-amino-3-phosphonopropionic acid (AP3) and 2-amino-4-phosphonobutyric acid (AP4) suggesting that metabotropic receptors do not play a role in the generation of these signals. We suggest that the receptor class activated under these conditions may play a role in CNQX/AP5-resistant epileptogenesis as well as other pathophysiological conditions.

2-Amino-5-phosphonovalerate↗
PubMed1990

Prolonged electrophysiological maturation of transplanted hippocampal neurons.

The CA1 region of the rat hippocampal formation was lesioned by transient forebrain ischemia and subsequently repopulated with dispersed fetal hippocampal neurons. Using the hippocampal slice preparation, 7-12 months post-transplantation, intracellular recordings were made during synaptic activation and extracellular calcium measurements were made during iontophoretic application of excitatory amino acids. The data indicate that the developmental period of the transplanted neurons is prolonged with respect to N-methyl-D-aspartate (NMDA) receptor-mediated responses and may be involved in maintaining calcium dependent developmental processes such as fiber outgrowth.

Animals↗
PubMed1990

Laminar profiles of N-methyl-D-aspartate- and quisqualate-induced [Na+]o changes in rat hippocampus.

Since plastic changes in neuronal tissue are often associated with changes in responses to excitatory amino acids (EAA), a method is required which permits mapping of functional EAA receptor sites. In this study the measurement of extracellular sodium concentration changes [( Na+]o) induced by iontophoretic application of N-methyl-D-aspartate (NMDA) and quisqualate (Quis) is used as a tool to estimate the density of functional NMDA- and Quis-receptor sites in area CA1 and dentate gyrus of rat hippocampus. Largest decreases of [Na+]o induced by NMDA were found in basal and apical dendritic fields of area CA1 and in stratum moleculare of dentate gyrus at a distance of 50-100 microns from stratum granulare. Peak decreases in [Na+]o induced by Quis occurred at similar positions. Bath application of tetrodotoxin (TTX) suppressed that part of the extracellular Na+ loss mediated by Na+ fluxes through voltage activated Na+ channels. However, the laminar profiles of Quis- and NMDA-induced [Na+]o changes were not affected by TTX. Thus, EAA induced decreases in [Na+]o can be used to detect changes in receptor density and in receptor functionality of hippocampus which had undergone plastic changes.

Animals↗
PubMed1990

Anticonvulsant effects of tetronic acid derivatives on picrotoxin induced epileptiform activity in rat hippocampal slices.

We have investigated the effects of a new class of anticonvulsants, the tetronic acid derivatives AO33 (generic name: losigame) and AO78, on field potentials, extracellular calcium concentration changes and intracellular potentials in rat hippocampal slices treated with the non-competitive GABAA antagonist picrotoxin (PTX). The tetronic acid derivatives reduced and eventually blocked spontaneous epileptiform events, induced by 10 to 30 microM PTX. Stimulus induced burst discharges were shortened in duration, but not blocked. Extracellular calcium concentration changes and associated slow negative field potentials were diminished in a dose dependent manner. Intracellular recordings revealed no effect of AO33 on resting membrane potential, little effect on input resistance, a small increase in the threshold of action potentials and an attenuation of stimulus induced paroxysmal depolarisation shifts (PDSs). Spontaneous PDSs initially decreased in duration until they were no longer observable.

Action Potentials↗
PubMed1990

Effects of lowering [Na+]o and [K+]o and of ouabain on quisqualate-induced ionic changes in area CA1 of rat hippocampal slices.

Changes in extracellular ([Na+]o) and calcium ([Ca2+]o) concentration evoked by quisqualate (Quis), applied iontophoretically, were measured in rat hippocampal slices. Following an initial decrease in [Ca2+]o, Quis causes a post-application overshoot, which may reflect Ca2+ extrusion from cells by the Na+/Ca2+ exchanger. To test this hypothesis, the effects of directly and indirectly reduced transmembrane Na+ gradients on these Quis-induced Ca2+ overshoots were investigated. In all cases the post-application overshoots were reduced. This suggests that the Na+/Ca2+ exchanger is involved in the production of these Quis-induced Ca2+ overshoots which may have important implications for understanding how various types of glutamate receptors differ in their capability to trigger Ca2(+)-activated processes.

Animals↗