Biomedical subjects

U Heinemann

Publications and source records attributed to U Heinemann.

At least 307 records · Page 17Linked to original sources

PubMed1989

The role of excitatory amino acids in synaptic transmission in the hippocampus.

We have highlighted some aspects of the action of excitatory amino acid transmission in the hippocampus. Fast epsps can be blocked by CNQX to reveal a component of synaptic transmission which is mediated by NMDA receptors. Extracellular recordings of ionic activities show that NMDA and non-NMDA ionophores are permeable to the major monovalent cations, while NMDA ionophores also appear to be permeable to Ca2+. Interactions of agonists applied by iontophoresis may be correlates of phenomena such as LTP, which can be evoked by appropriate synaptic stimulation.

Amino Acids↗

PubMed1988

Three-dimensional structure of the ribonuclease T1 2'-GMP complex at 1.9-A resolution.

The complex formed between the enzyme ribonuclease T1 (EC 3.1.27.3) and its specific inhibitor 2'-guanylic acid (2'-GMP) has been refined to R = 0.180 using x-ray diffraction data to 1.9-A resolution. The protein molecule displays a compact fold; a 4.5 turn alpha-helix packed over an antiparallel beta-pleated sheet shields most of the hydrophobic interior of the protein against the solvent. The extended pleated sheet structure of ribonuclease T1 is composed of three long and four short strands building up a two-stranded minor beta-sheet near the amino terminus and a five-stranded major sheet in the interior of the protein molecule. In the complex with ribonuclease T1, the inhibitor 2'-guanylic acid adopts the syn-conformation and C2'-endo sugar pucker. Binding of the nucleotide is mainly achieved through amino acid residues 38-46 of the protein. The catalytically active amino acid residues of ribonuclease T1 (His40, Glu58, Arg77, and His92) are located within the major beta-sheet which, as evident from the analysis of atomic temperature factors, provides an environment of minimal local mobility. The geometry of the active site is consistent with a mechanism for phosphodiester hydrolysis where, in the transesterification step, His40 and/or Glu58 act as a general base toward the ribose 2'-hydroxyl group and His92, as a general acid, donates a proton to the leaving 5'-hydroxyl group.

Endoribonucleases↗

PubMed1988

Verapamil blocks the afterhyperpolarization but not the spike frequency accommodation of rat CA1 pyramidal cells in vitro.

The effects of prolonged periods (up to 4 h) of perfusion with verapamil (100 microM) or D600 (100 microM) on synaptically and directly evoked responses of rat CA1 pyramidal cells were determined in vitro. The slow depolarization underlying burst generation and the slow afterhyperpolarization following directly evoked repetitive firing were blocked, but spike frequency accommodation was not. There was an increase in threshold for evoking synaptic responses and the amplitude of evoked inhibitory postsynaptic potentials (IPSPs) was decreased slightly. The results suggest that verapamil can partially block voltage dependent Ca influx into CA1 cells and that currents underlying accommodation and the slow afterhyperpolarization (AHP) may differ.

Action Potentials↗

PubMed1988

Three-dimensional structure of d(GGGATCCC) in the crystalline state.

The structure of the self-complementary octamer d(GGGATCCC) has been analysed by single crystal X-ray diffraction methods at a nominal resolution of 2.5 A. With acceptable stereochemistry of the model the crystallographic R factor was 16.6% after restrained least-squares refinement. In the crystal, d(GGGATCCC) forms an A-DNA double helix with slightly varying conformation of the two strands. The average displacement of the base pairs from the helix axis is unusually large and is accompanied by pronounced sliding of the base pairs along their long axes at all dinucleotide steps except for the central AT. With 12 base pairs per complete turn the helix is considerably underwound. As observed with most oligodeoxyribonucleotides analysed by X-ray crystallography so far, the octamer displays reduced base pair tilt, increased rise per base pair and a more open major groove compared with canonical A-DNA. We propose that, based on these parameters, three A-helical sub-families may be defined; d(GGGATCCC) then is a representative of the class with intermediate tilt, rise, and major groove width.

DNA↗

PubMed1988

Lowering extracellular calcium reverses paired pulse habituation into facilitation in dentate granule cells and removes a late IPSP.

Simultaneous measurements of cellular responses and extracellular field potentials as well as extracellular Ca2+ concentration [( Ca2+]0) were performed in the dentate gyrus granule cell layer during paired pulse stimulation of the lateral perforant path at resting [Ca2+]o and during washout of calcium. At resting [Ca2+]o the second response to a paired stimulus was smaller than the first response. This frequency habituation reversed into frequency potentiation (second response larger than the first one) during lowering of [Ca2+]o at about the same time when a late presumed inhibitory postsynaptic potential (IPSP) was abolished. This suggests that a slow inhibition can account for part of frequency habituation in the dentate gyrus.

Action Potentials↗

PubMed1988

Developmental changes in neuronal sensitivity to excitatory amino acids in area CA1 of the rat hippocampus.

The laminar distribution of decreases in extracellular free calcium and concomitant field potentials induced by repetitive orthodromic stimulation, ionophoretic application of N-methyl-D-aspartate and quisqualate, was studied in the CA1 field of rat hippocampal slices, at two different stages during postnatal development. While stimulation-elicited and quisqualate-induced signals remain maximal in stratum pyramidale during the first postnatal month, the laminar profiles of responses to N-methyl-D-aspartate (NMDA) depend on age: the responses to this agonist are maximal in stratum pyramidale in 5-9-day-old rats and in stratum radiatum in 12-30-day-old rats. Our findings suggest that, during the second postnatal week, the apical dendrites of pyramidal neurons in area CAl become more sensitive to NMDA, which is expressed by big influxes of calcium at this level.

Action Potentials↗

PubMed1988

Differences in magnesium and calcium effects on N-methyl-D-aspartate- and quisqualate-induced decreases in extracellular sodium concentration in rat hippocampal slices.

Decreases in extracellular sodium concentration [( Na+]o) and associated slow negative field potentials (fp's) were monitored with double barreled sodium sensitive/reference microelectrodes in area CA1 of rat hippocampal slices during iontophoretic application of the glutamate receptor agonists N-methyl-D-aspartate (NMDA) and quisqualate (quis). The effects of lowering [Ca2+]o on these signals were compared to those of lowering [Mg2+]o. Both NMDA- and quis-induced decreases in [Na+]o of up to 60 mM and in the fp's of up to 8 mV. Decreasing [Mg2+]o enhanced NMDA-induced signals, whereas quis-induced signals were unaffected. Lowering [Ca2+]o also enhanced NMDA signals, although somewhat less than lowering [Mg2+]o. This effect was still present, even when voltage dependent Na+ currents were blocked by 10(-7) tetrodotoxin. Interestingly, quis-induced signals could be enhanced in a low Ca2+ medium as well, but only when high quis concentrations were used. The results suggest that, during the sorts of large decreases of [Ca2+]o observed during seizure activity, activation of NMDA receptors is facilitated.

Action Potentials↗

PubMed1988

Adenosine antagonists combined with 4-aminopyridine cause partial recovery of synaptic transmission in low Ca media.

Orthodromically and antidromically evoked field potentials, as well as changes in the extracellular calcium concentration [Ca2+]o were measured with ion selective/reference electrodes in area CA1 of rat hippocampal slices. Synaptic transmission was blocked by a low calcium, high magnesium medium. After cutting through the alveus, stratum pyramidale (Spyr) and part of stratum radiatum (Srad), repetitive electrical stimulation of Schaffer collaterals and commissural fibers elicited decreases of [Ca2+]o in Srad, the synaptic area, but not in Spyr, the soma layer of the pyramidal neurons. This indicates the absence of a measurable somatic Ca2+ influx due to postsynaptic activation and therefore, the decrease of [Ca2+]o in Scrad presumably reflect presynaptic Ca2+ entry. Antagonists of adenosine action such as adenosine deaminase and theophylline had no effects Ca2+ entry whereas 4-AP enhanced calcium signals in Scrad considerably. In some cases small [Ca2+]o decreases in Spyr appeared after treatment with 4-AP although field potentials did not reveal postsynaptic components. When 4-AP and antagonists of adenosine action were combined, a partial recovery of synaptic transmission was consistently seen during the course of repetitive stimulation. This was indicated by large decreases of [Ca2+]o in Spyr as well as by the generation of postsynaptic field potentials. The latter appeared late during the train pointing to frequency potentiation and a presynaptic site of action. The findings indicate that physiological levels of adenosine in the order of 1 microM have a powerful modulatory role on synaptic transmission by depressing presynaptic transmitter release. This seems to result not from an influence on presynaptic calcium uptake, but rather from changing the intracellular level of calcium or its coupling to the secretory process.

4-Aminopyridine↗

PubMed1988

Carbachol effects on hippocampal neurons in vitro: dependence on the rate of rise of carbachol tissue concentration.

Nominally K-sensitive microelectrodes were used to measure carbachol (CCh) in order to study the dependence of muscarinic effects on CCh concentration and exposure time in guinea pig hippocampal slices. Interference presumably originating from tissue choline-compounds was neutralized by pre-equilibration of the slices with 500 microM choline and calibration of the CCh-sensitive microelectrodes in the presence of the same choline-concentration. Muscarinic depolarization and reduction of the afterhyperpolarization (AHP) following a train of action potentials by bath applied CCh were monitored in granule cells and CA3 pyramidal neurons by intracellular recording. A fast bath application mode of CCh was designed, by which CCh tissue concentration reached a peak after 2-3 min and was washed out with a half time of about 8 min. After application of 30 nmol CCh in this way, the AHP was reduced according to the variation of CCh concentration over time. Neurons depolarized with some delay after the reduction of the AHP and started to repolarize 1 min before the peak of tissue CCh concentration (0.6 microM) was reached. Pirenzepine (1-10 microM) blocked only the depolarization, while atropine (1-10 microM) blocked both the depolarization and the reduction of the AHP. When superfusing with CCh containing saline, 80% of the final concentration was reached in the bath after 12 min, but in the tissue only after 45 min. The slow increase of tissue CCh concentration was concurrent with the slow decrease of the AHP. No effect on the membrane potential was observed. Atropine, but not pirenzepine, blocked the reduction of the AHP. Superfusion with a high CCh concentration (100-300 microM) containing saline depolarized neurons and reduced the AHP. Then pirenzepine repolarized neurons, whereas atropine both repolarized the cells and restored the AHP. It is concluded tha the muscarinic depolarization depends not only on the CCh concentration, but also on the rate of rise of CCh, while the reduction of the AHP depends solely on the concentration. This result is discussed in terms of the possibility that the depolarization is mediated by a short term desensitizing M1 muscarinic receptor subtype and the reduction of the AHP is mediated by a M2 muscarinic receptor subtype.

Animals↗

PubMed1988

Molecular dynamics simulations of ribonuclease T1. Effect of solvent on the interaction with 2'GMP.

Molecular dynamics simulations in vacuum and with a water sphere around the active site were performed on the 2'GMP-RNase T1 complex. The presence of water led to the maintenance of the 2'-GMP-RNase T1 interactions as compared to the X-ray structure, including the hydrogen bonds implicated in the enzyme-inhibitor recognition process. The sidechain of His92 in the molecular dynamics water simulation, however, hydrogen bonds directly to the phosphate of 2'GMP in contrast to the X-ray structure but in support of the role of that residue in the enzyme's catalytic mechanism. Fluctuations of active-site residues are not strongly influenced by water, possibly owing to the exclusion of water by the bound 2'GMP, which did show an increase in mobility. Analysis of the 2'GMP-RNase T1 interactions versus time reveal an equilibrium fluctuation in the presence of water, leading to a less favorable 2'GMP-RNase T1 interaction energy, suggesting a possible relationship between picosecond fluctuations and inhibitor dissociation occurring in the millisecond time domain.

Endoribonucleases↗

PubMed1988

Activation of N-methyl-D-aspartate receptors parallels changes in cellular and synaptic properties of dentate gyrus granule cells after kindling.

1. The cellular and synaptic properties of rat dentate gyrus granule cells (GCs) were examined using intra-/extracellular and Ca2+-sensitive microelectrode recordings following epilepsy induced by kindling of the hippocampal commissures or amygdala. 2. The recordings were made in hippocampal slices prepared from sham-stimulated controls and animals that have received daily stimuli to reach stage IV-V of kindling. The average number of stimulation trials (60 Hz/1 s, 100-150 microA) required to reach full motor seizures (stage V) was 23 +/- 2 for commissural kindling and 14 +/- 1 for amygdala kindling. 3. The resting membrane potential of GCs following kindling (RMP; -72 +/- 3 mV) was not significantly different from the RMP of control GCs (-70 +/- 2 mV). Similarly, action potential height and threshold were unaffected by kindling. However, kindling altered other cellular properties of GCs regardless of the site of stimulation (hippocampal commissures or amygdala), the stage of kindling reached (IV or V), or the time elapsed between the last kindling stimulus and preparation of the hippocampal slices (24 h-6 wk). The input resistance of kindled GCs (55 +/- 4 M omega) was significantly higher than that of controls (40 +/- 3 M omega). In contrast to most control GCs, the slope conductance (GS) of kindled neurons, measured with constant-amplitude current injections at various membrane potentials, generally increased at membrane potentials more negative than rest. Furthermore, other voltage-dependent ionic conductances (see below), that were not normally encountered in control GCs, were present in kindled neurons. 4. The intracellularly recorded monosynaptic excitatory postsynaptic potentials (EPSPs) of kindled GCs, evoked through the stimulation of the lateral perforant pathway, differed significantly from the EPSPs of control GCs. The amplitudes of control EPSPs increased upon hyperpolarizations and decreased following depolarizations of the membrane, as expected for conventional EPSPs without contribution from voltage-dependent conductances. In contrast, the EPSPs of kindled GCs invariably increased in amplitude and duration at membrane potentials 5-20 mV depolarized from rest, indicating the presence of a characteristic voltage-dependent component. Frequently, following the synaptically triggered action potentials, kindled GCs displayed depolarizing afterpotentials. 5. Perfusion of the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV; 30 microM) had no effect on the EPSPs of control GCs, but consistently reduced the amplitude and duration of EPSPs in kindled GCs.(ABSTRACT TRUNCATED AT 400 WORDS)

Amygdala↗

PubMed1988

Synaptic and intrinsic responses of medical entorhinal cortical cells in normal and magnesium-free medium in vitro.

1. Extracellular recordings were made from slices of hippocampus plus parahippocampal regions maintained in vitro. Field potentials, recorded in the entorhinal cortex after stimulation in the subiculum, resembled those observed in vivo. 2. Washout of magnesium from the slices resulted in paroxysmal events which resembled those occurring during sustained seizures in vivo. These events were greatest in amplitude and duration in layers IV/V of the medial entorhinal cortex and could occur both spontaneously and in response to subicular stimulation. Spontaneous seizure-like events were not prevented by severing the connections between the hippocampus and entorhinal cortex, but much smaller and shorter events occurring in the dentate gyrus were stopped by this manipulation. Both spontaneous and evoked paroxysmal events were blocked by perfusion with the N-methyl-D-aspartate (NMDA) receptor antagonist, DL-2-amino-5-phosphonovalerate (2-AP5). 3. Neurons in layers IV/V were characterized by intracellular recording. Injection of depolarizing current in most cells evoked a train of nondecrementing action potentials with only weak spike frequency accommodation and little or no posttrain after hyperpolarization. 4. A small number of cells displayed burst response when depolarized by positive current. The burst consisted of a slow depolarization with superimposed action potentials which decreased in amplitude and increased in duration during the discharge. The burst was terminated by a strong after hyperpolarization and thereafter, during prolonged current pulses a train of nondecrementing spikes occurred. The burst response remained if the cell was held at hyperpolarized levels but was inactivated by holding the cell at a depolarized level. 5. Depolarizing synaptic potentials could be evoked by stimulation in the subiculum. A delayed and prolonged depolarization clearly decremented with membrane hyperpolarization and, occasionally, increased with depolarization. 6. Washout of magnesium from the slices resulted in an enhancement of the late depolarization and a reversal of its voltage dependence. Eventually a single shock to the subiculum evoked a large all-or-none paroxysmal depolarization associated with a massive increase in membrane conductance. Similar events occurred spontaneously in all cells tested. The paroxysmal depolarizations, both spontaneous and evoked, were rapidly blocked by 2-AP5. 7. It is concluded that medial entorhinal cortical cells possess several intrinsic and synaptic properties which confer an extreme susceptibility to generation of sustained seizure activity.(ABSTRACT TRUNCATED AT 400 WORDS)

2-Amino-5-phosphonovalerate↗

PubMed1987

Extracellular K+ and Ca2+ changes during epileptiform discharges in the immature rat neocortex.

Picrotoxin-induced epileptiform activity was examined in neocortical slices prepared from 8- to 15-day-old rats. This activity consisted of spontaneous bursts of 3-5 discharges that resembled interictal spikes and were interspersed with ictal-like paroxysms lasting 10-30 s. Measurements of extracellular potassium ([K+]o) and calcium ([Ca2+]o) were made during these spontaneous epileptiform events, using ion-sensitive electrodes. Individual interictal spikes were associated with [Ca2+]o decreases of 0.1-0.2 mM, whereas sustained ictal-like discharges were accompanied by decreases of 0.3-0.4 mM. Measurement of [K+]o showed that individual interictal spikes were associated with increases in [K+]o up to 12 mM, whereas increases to more than 20 mM accompanied long-lasting ictal-like discharges. Maximum increases in [K+]o were observed ca. 600 microns below the pial surface. [K+]o increases were followed by undershoots of the resting [K+]o level. The unusually high [K+]o levels associated with epileptiform discharges in the immature neocortex suggest that disturbances in [K+]o regulation may contribute to the generation of the picrotoxin-induced, spontaneous, prolonged ictal-like discharges observed in the 8- to 15-day age group.

Animals↗

PubMed1987

Crystal structure analysis of an A-DNA fragment at 1.8 A resolution: d(GCCCGGGC).

Single crystals of the self-complementary octadeoxyribonucleotide d(GCCCGGGC) have been analysed by X-ray diffraction methods at a resolution of 1.8 A. The tetragonal unit cell of space group P4(3)2(1)2 has dimensions of a = 43.25 A and c = 24.61 A and contains eight strands of the oligonucleotide. The structure was refined by standard crystallographic techniques to an R factor of 17.1% using 1359 3 sigma structure factor observations. Two strands of the oligonucleotide are related by the crystallographic dyad axis to form a DNA helix in the A conformation. The d(GCCCGGGC) helix is characterized by a wide open major groove, a near perpendicular orientation of base pairs to the helix axis and an unusually small average helix twist angle of 31.3 degrees indicating a slightly underwound helix with 11.5 base pairs per turn. Extensive cross-strand stacking between guanine bases at the central cytosine-guanine step is made possible by a number of local conformational adjustments including a fully extended sugar-phosphate backbone of the central guanosine nucleotide.

Crystallization↗

PubMed1987

Helix geometry, hydration, and G.A mismatch in a B-DNA decamer.

The DNA double helix is not a regular, featureless barberpole molecule. Different base sequences have their own special signature, in the way that they influence groove width, helical twist, bending, and mechanical rigidity or resistance to bending. These special features probably help other molecules such as repressors to read and recognize one base sequence in preference to another. Single crystal x-ray structure analysis is beginning to show us the various structures possible in the B-DNA family. The DNA decamer C-C-A-A-G-A-T-T-G-G appears to be a better model for mixed-sequence B-DNA than was the earlier C-G-C-G-A-A-T-T-C-G-C-G, which is more akin to regions of poly(dA).poly(dT). The G.A mismatch base pairs at the center of the decamer are in the anti-anti conformation about their bonds from base to sugar, in agreement with nuclear magnetic resonance evidence on this and other sequences, and in contrast to the anti-syn geometry reported for G.A pairs in C-G-C-G-A-A-T-T-A-G-C-G. The ordered spine of hydration seen earlier in the narrow-grooved dodecamer has its counterpart, in this wide-grooved decamer, in two strings of water molecules lining the walls of the minor groove, bridging from purine N3 or pyrimidine O2, to the following sugar O4'. The same strings of hydration are present in the phosphorothioate analog of G-C-G-C-G-C. Unlike the spine, which is broken up by the intrusion of amine groups at guanines, these water strings are found in general, mixed-sequence DNA because they can pass by unimpeded to either side of a guanine N2 amine. The spine and strings are perceived as two extremes of a general pattern of hydration of the minor groove, which probably is the dominant factor in making B-DNA the preferred form at high hydration.

Base Composition↗

PubMed1987

DNA-binding properties and primary structure of HB protein from Bacillus globigii.

The binding of Bacillus globigii HB protein to synthetic deoxyoligonucleotides of different length and sequence has been studied by polyacrylamide gel electrophoresis. Without detectable sequence specificity the protein binds to single-stranded and double-stranded DNA. Under the conditions employed, binding of HB protein to deoxyoligonucleotides with six or less nucleotides per strand cannot be detected while eight or more nucleotide units per strand of single-stranded DNA or base pairs of double-stranded DNA are sufficient for binding. The complete amino acid sequence of HB protein has been determined by manual Edman degradation of tryptic peptides. Like most DNA-binding proteins of its class, HB protein does not contain cysteine, tyrosine or tryptophan residues. The primary structure of HB protein shows 84% homology with the sequence of the related DNA-binding protein II from Bacillus stearothermophilus.

Amino Acid Sequence↗

PubMed1987

An N-methyl-D-aspartate receptor-independent excitatory action of partial reduction of extracellular [Mg2+] in CA1-region of rat hippocampal slices.

Partial reduction of [Mg2+]o from 2 to 1 mM markedly enhanced neuronal responses evoked by Schaffer collateral-commissural fiber stimulation in the CA1-region of rat hippocampal slices. The amplitude of extracellular population potentials recorded in the CA1-pyramidal cell layer and maximum dV/dt of extracellular population EPSP's recorded in the CA1-pyramidal apical dendritic layer were both increased. However, unlike findings from slices where Mg2+ was completely removed from the bathing medium, there was no spontaneous or evoked epileptiform activity, and the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate (2-APV) did not antagonize the enhancement of evoked responses. These results indicate that, in addition to the participation of NMDA receptors in the epileptiform activity observed when Mg2+ is completely removed from the bathing medium, there is also an NMDA receptor-independent excitatory action of partial reduction of [Mg2+]o in hippocampal slices.

2-Amino-5-phosphonovalerate↗

PubMed1987

Abolition of the orthodromically evoked IPSP of CA1 pyramidal cells before the EPSP during washout of calcium from hippocampal slices.

The complex EPSP-IPSP response of CA1 pyramidal cells to orthodromic activation of Schaffer collaterals-commissural afferent fibres was monitored during washout of Ca from hippocampal slices. The IPSP was clearly abolished before the EPSP and this occurred when the extracellular Ca([Ca2+]o) had fallen to between 1.03 and 0.7 mM. The loss of the IPSP was usually associated with a temporary increase in size of the EPSP and sometimes a membrane depolarization and the appearance of spontaneous activity. As [Ca2+]o fell further these effects were reversed and eventually the EPSP was abolished when [Ca2+]o had reached between 0.78 and 0.26 mM.

Animals↗