Search PubMed⌕ Search

Biomedical subjects

S Diekmann

Publications and source records attributed to S Diekmann.

At least 55 records · Page 3Linked to original sources
PubMed1996

Long-lasting transneuronal changes in rat dentate granule cell dendrites after entorhinal cortex lesion. A combined intracellular injection and electron microscopy study.

Following entorhinal cortex lesion, inhibitory hippocampal neurons show a persistent rarefication of those dendrites formally receiving entorhinal input. Physiological data indicate a long lasting disequilibrium of inhibition and excitation in the de-entorhinated hippocampus. We analyzed the intracellularly-stained dendritic tree of de-entorhinated excitatory rat granule cells. Granule cells of controls and animals surviving 2, 8, 60 and 270 days after unilateral entorhinal cortex lesion were impaled. Dendrites of control cells were of typical shape, traced to the hippocampal fissure and a complete dye filling of dendrites was ascertained by EM-analysis. Conversely, 60 and 270 days following lesioning, dendrites were only rarely seen to extend into the outer portions of the molecular layer and the dendritic architecture became significantly rarefied. Sixty days post-lesion, intracellularly filled dendrites extending to the middle molecular layer were surrounded by cell clusters resembling glia. Some of these contained the neuronally applied dye, suggesting a close association of the cytosolic compartments with the altered dendrites. These observed alterations exceed the process of sprouting and de novo synaptogenesis of remaining afference for long periods of time. The dendritic morphology of both inhibitory and excitatory neurons seems to require specific input from the entorhinal cortex. Moreover, sprouting of remaining afferents is apparently not sufficient to compensate for this loss of input.

Animals↗
PubMed1995

Hepatocyte nitric oxide biosynthesis inhibits glucose output and competes with urea synthesis for L-arginine.

Inflammatory stimulation of the liver is known to induce nitric oxide (NO) biosynthesis. NO can interfere with the activity of a number of enzymes important to cellular metabolism. This study was carried out to investigate the influence of NO on rat hepatocyte glucose output and urea production. Induction of NO synthesis by incubation with a combination of cytokines and lipopolysaccharide led to a 48.8 +/- 2.4% inhibition of glucose output and to a 45.0 +/- 6.4% suppression of urea production. Inhibition of NO synthesis with NG-monomethyl-L-arginine was able to totally prevent these effects. High concentrations of L-arginine overcame the inhibition of urea production caused by endogenous NO synthesis. Exposure of HC to NO donors resulted in a concentration-dependent inhibition of glucose output, without having any effect on urea production. Hepatocellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity was also found to be inhibited by endogenously produced NO (33.5 +/- 5.2%), as well as by exogenously applied NO. However, an exact correlation between GAPDH activity and glucose output could not be established. These data indicate that NO biosynthesis may contribute to the development of hepatic dysfunction in chronic sepsis.

Animals↗
PubMed1994

Immobilized residue-specific endoproteinases for protein sequencing.

Before proteins can be sequenced, the peptide chain has to be cut into small fragments of less than about 50 amino acids using residue-specific endoproteinases. These enzymes can be immobilized in a highly active form. Using immobilized endoproteinases for protein sequencing results in a series of advantages: (1) the high enzyme activity in the column results in short reaction times; (2) the protein fragments are easily eluted from the column whilst the endoproteinase is completely retained on the column; the protein fragments are clean yielding in low sequencing background; (3) the protein sample to be sequenced is free of exogenous enzymes; (4) endoproteinase self-digestion is prevented by immobilization; therefore, the sample solution does not contain any endoproteinase fragments; (5) enzymes are especially stable when immobilized. Columns with immobilized endoproteinases can be applied repeatedly and stored for many months.

Amino Acid Sequence↗
PubMed1994

The organotypic entorhinal-hippocampal complex slice culture of adolescent rats. A model to study transcellular changes in a circuit particularly vulnerable in neurodegenerative disorders.

The entorhinal-hippocampal system is severely altered in many neurodegenerative disorders with mnemonic malfunction, e.g. Alzheimer's, Parkinson's and Huntington's disease. The present approach characterizes an organotypic complex slice culture comprising both the entorhinal cortex and the hippocampal formation in order to establish a tool for experimental studies of the entorhinal-hippocampal interaction and its presumed neurodegenerative alterations in vitro. Slices were obtained from rats at about postnatal day 15 and maintained in culture using the interface technique. Thus, also structures known to be developed gradually during the first weeks postnatally are in accord to structures seen in adult rats. After two-three weeks in vitro, slices in the culture dish still revealed the typical morphological features of the entorhinal-hippocampal formation as visible with the dissecting microscope. Biocytin, which is taken up by and transported within living cells, labeled typical cell bodies, dendrites and axons of stellate neurons in layer II and pyramidal cells in layer III when applied to the outer layers of the entorhinal cortex. Small injections of biocytin within the dentate gyrus displayed living granule cells and the maintenance of their projection to the pyramidal cells in CA3, i.e., a typical suprapyramidal plexus of mossy fibers. The presence of axons of entorhinal neurons traveling towards the hippocampus and growth cones traversing the deep layers of the entorhinal cortex indicate that both brain regions are still interacting. Immunocytochemistry for calbindin D-28K revealed labeled neurons in layer II of the entorhinal cortex and dentate granule cells which are known to contain this calcium-binding protein.

Alzheimer Disease↗
PubMed1994

The use of Lucifer Yellow and Mini-Ruby for intracellular staining in fixed brain tissue: methodological considerations evaluated in rat and human autopsy brains.

The quality of intracellular filling of Mini-Ruby (MR) is comparable to that of Lucifer Yellow (LY) in both perfusion-fixed rat and immersion-fixed autopsy human tissue. In adult human brain, the use of MR avoids the co-conversion of the typical intracellular lipofuscin deposits as is invariably the case during the photoconversion of LY. In autopsy studies (n = 35), the quality of intracellular staining does not correlate with age (range: 25-91 years) or the postmortem delay (range: 7-50 h). A short agonal status, however, is advantageous.

Adult↗
PubMed1994

Human 170 kDa and 180 kDa topoisomerases II bind preferentially to curved and left-handed linear DNA.

The binding activities of the 170 kDa and the 180 kDa human topoisomerases II (topo II alpha and topo II beta) to linear DNA fragments with different degrees of curvature were characterized. In gel retardation experiments it was shown that both forms of the enzyme bind preferentially to a curved 287 bp fragment, forming a detectable stable complex. The affinity for straight DNA fragments of similar length is significantly lower. Both a commercially available topo II alpha, isolated from placenta, and topo II alpha and topo II beta purified from nuclear extracts of the Namalwa lymphoma tissue culture line gave similar results. The effects of double-stranded poly[d(A-T)], poly[d(G-C)], supercoiled plasmid DNA and linear Z-DNA on the topo II-complex with curved DNA were analyzed in competition experiments. The hierarchy of affinities of the 180 kDa topo II beta for these DNAs has the order: linear left-handed DNA > supercoiled DNA > or = curved DNA >> poly[d(A-T)] > poly[d(G-C)]. The 170 kDa topo II alpha binds with similar affinity to curved DNA and linear Z-DNA > or = supercoiled DNA >> linear B-DNA. The data imply that human topoisomerase II binding is more sensitive to DNA secondary structure than to DNA sequence per se. The ability of the enzyme to preferentially recognize a wide variety of sequences in unusual secondary structures suggests a mode of targeting the enzyme in vivo to regions of high negative supercoiling.

Animals↗
PubMed1993

DNA curvature influences the internal motions of supercoiled DNA.

We present evidence that short curved DNA segments can act as mediators for the ordering of large domains in superhelical DNA. Using a non-invasive solution method (dynamic light scattering), we investigated the effect of permanently curved inserts on the solution structure and on the internal motions of superhelical plasmid DNA. We find that the dynamics of superhelical DNA are strongly influenced by sequence- or protein-induced bending: in superhelical plasmids containing curved inserts the amplitude of the internal motion is lower than that of non-curved controls. Furthermore, the relative arrangement of curved sequences in the plasmids can influence the overall shape of the superhelical DNA. On linearized forms of the plasmids, these effects are not observed.

Base Sequence↗
PubMed1992

DNA curvature does not require bifurcated hydrogen bonds or pyrimidine methyl groups.

Short tracts of the homopolymer dA.dT confer intrinsic curvature on the axis of the DNA double helix. This phenomenon is assumed to be a consequence of such tracts adopting a stable B'-DNA conformation that is distinct from B-form structure normally assumed by other DNA sequences. The more stable B' structure of dA.dT tracts has been attributed to several possible stabilizing factors: (1) optimal base stacking interactions consequent upon the high propeller twist, (2) bifurcated hydrogen bonds between adjacent dA.dT base-pairs, (3) stacking interactions involving the dT methyl groups, and finally (4) a putative spine of ordered water molecules in the minor groove. DNA oligodeoxynucleotides have been synthesized that enable these hypotheses to be tested; of particular interest is the combination of effects due to bifurcation (2) and methylation of the pyrimidines nucleotides (3). The data indicate that neither bifurcated hydrogen bonds nor pyrimidine methyl groups nor both are essential for DNA curvature. The data further suggest that the influence of the minor groove spine of hydration on the B'-formation is small. The experiments favor the hypothesis that base stacking interactions are the dominant force in stabilizing the B'-form structure.

Base Sequence↗
PubMed1992

Fluorescence resonance energy transfer analysis of the structure of the four-way DNA junction.

We have carried out fluorescence resonance energy transfer (FRET) measurements on four-way DNA junctions in order to analyze the global structure and its dependence on the concentration of several types of ions. A knowledge of the structure and its sensitivity to the solution environment is important for a full understanding of recombination events in DNA. The stereochemical arrangement of the four DNA helices that make up the four-way junction was established by a global comparison of the efficiency of FRET between donor and acceptor molecules attached pairwise in all possible permutations to the 5' termini of the duplex arms of the four-way structure. The conclusions are based upon a comparison between a series of many identical DNA molecules which have been labeled on different positions, rather than a determination of a few absolute distances. Details of the FRET analysis are presented; features of the analysis with particular relevance to DNA structures are emphasized. Three methods were employed to determine the efficiency of FRET: (1) enhancement of the acceptor fluorescence, (2) decrease of the donor quantum yield, and (3) shortening of the donor fluorescence lifetime. The FRET results indicate that the arms of the four-way junction are arranged in an antiparallel stacked X-structure when salt is added to the solution. The ion-related conformational change upon addition of salt to a solution originally at low ionic strength progresses in a continuous noncooperative manner as the ionic strength of the solution increases. The mode of ion interaction at the strand exchange site of the junction is discussed.

Base Sequence↗
PubMed1991

Model for the interaction of DNA junctions and resolving enzymes.

Four-way DNA junctions are thought to be important intermediates in a number of recombination processes. Resolution of these junctions occurs by cleavage of two strands of DNA to generate two duplex molecules. The interaction between DNA junctions and resolving enzymes appears to be largely structure-specific, reflecting a molecular recognition on a significant scale. We propose a working model for this interaction that takes account of the present state of knowledge of the structure of the DNA junction, and the substrate requirements of the enzymes. We note that three different enzymes introduce cleavages at phosphodiester bonds that are presented on one side of the molecule, suggesting that the enzymes selectively interact with this face of the junction. By forcing a junction of constant sequence to adopt one or other of the two possible antiparallel isomers, we show that the junction is cleaved in such a way as to suggest a constant mode of interaction with the protein that is dependent on structure rather than sequence. We propose that the feature that is recognized is a mutual inclination of two DNA helices at approximately 120 degrees. We show that a number of DNA substrates that contain similar inclined helices, such as a three-way junction, bulged duplexes and a duplex that is curved because of repeated runs of oligoadenine sequences, are each cleaved by phage T4 endonuclease VII. This mode of DNA-protein interaction could be significant in either recombination or DNA repair processes.

Animals↗
PubMed1991

Binding of Hoechst 33258 and 4',6'-diamidino-2-phenylindole to self-complementary decadeoxynucleotides with modified exocyclic base substituents.

Fluorescence titrations have been carried out to determine the association constants (Ka) for binding of the dyes Hoechst 33258 and DAPI to the self-complementary decamer d(CTGAATTCAG) and nine duplex derivatives with exocyclic substituent changes in the six central base pairs. Many Ka values are in the range (2-5) x 10(8) (duplex M)-1 at 5.5 degrees C. Replacement of the leftmost adenine by 2-aminopurine in the sequence decreases Ka for Hoechst 33258 by a factor of 170. When the centermost adenine is replaced by 2-aminopurine, Ka for Hoechst 33258 and DAPI is too small to be evaluated. When the centermost adenine is replaced by purine, Ka for both dyes increases, but this very stable duplex-Hoechst 33258 complex is nonfluorescent. The measured affinities are compared to expectations derived from X-ray studies with dodecamer-dye complexes having an identical central binding sequence (Pjura et al., 1987; Teng et al., 1988; Larsen et al., 1989).

2-Aminopurine↗
PubMed1990

A non-curved chicken lysozyme 5' matrix attachment site is 3' followed by a strongly curved DNA sequence.

Matrix attachment regions (MARs) partition the genome into functional and structural loop-domains. Here, we determined the relative matrix affinity of cloned fragments of the chicken lysozyme 5' MAR. We show that this region contains a non-curved high-affinity binding site, which is 3' followed by a strongly curved DNA sequence that exhibits weak matrix binding. DNA curvature is not a physical property required for strong matrix binding. Possible biological functions of this sequence arrangement, particularly of the strongly curved DNA, are discussed.

Animals↗
PubMed1990

The stereochemistry of a four-way DNA junction: a theoretical study.

The stereochemical conformation of the four-way helical junction in DNA (the Holliday junction; the postulated central intermediate of genetic recombination) has been analysed, using molecular mechanical computer modelling. A version of the AMBER program package was employed, that had been modified to include the influence of counterions and a global optimisation procedure. Starting from an extended planar structure, the conformation was varied in order to minimise the energy, and we discuss three structures obtained by this procedure. One structure is closely related to a square-planar cross, in which there is no stacking interaction between the four double helical stems. This structure is probably closely similar to that observed experimentally in the absence of cations. The remaining two structures are based on related, yet distinct, conformations, in which there is pairwise coaxial stacking of neighbouring stems. In these structures, the four DNA stems adopt the form of two quasi-continuous helices, in which base stacking is very similar to that found in standard B-DNA geometry. The two stacked helices so formed are not aligned parallel to each other, but subtend an angle of approximately 60 degrees. The strands that exchange between one stacked helix and the other are disposed about the smaller angle of the cross (i.e. 60 degrees rather than 120 degrees), generating an approximately antiparallel alignment of DNA sequences. This structure is precisely the stacked X-structure proposed on the basis of experimental data. The calculations indicate distortions from standard B-DNA conformation that are required to adopt the stacked X-structure; a widening of the minor groove at the junction, and reorientation of the central phosphate groups of the exchanging strands. An important feature of the stacked X-structure is that it presents two structurally distinct sides. These may be recognised differently by enzymes, providing a rationalisation for the points of cleavage by Holliday resolvases.

Base Sequence↗