Nobel laureates' letter to President Bush.
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Embryo Disposition↗
Biomedical subjects
J Karle
Publications and source records attributed to J Karle.
Intrahippocampal infusion of antisense oligodeoxynucleotide to the GABA(A) receptor gamma2 subunit enhances neuropeptide Y gene expression.
The effects of hippocampal treatment with a phosphorothioate oligodeoxynucleotide (ODN) antisense to the gamma-aminobutyric acid (GABA)A receptor gamma2 subunit on neuropeptide Y (NPY) were studied. Adult male Wistar rats were treated with unilateral intrahippocampal infusion of gamma2 subunit antisense ODN for 5 days. Rats infused with mismatch ODN and naïve rats served as controls. Brain sections were analysed for levels of NPY mRNA by in situ hybridisation, NPY-immunoreactivity (NPY-ir) by means of immunocytochemistry, and specific NPY binding sites by in vitro receptor autoradiography. Following infusion of antisense ODN, a marked increase in cytoplasmic NPY-ir was observed in hilar neurones of the fascia dentata. Further, intense NPY-ir was visualised in the mossy fibres and in cell bodies of the entorhinal cortex and throughout the neocortex. High levels of NPY mRNA were detected in the same cortical areas of antisense treated rats. A very large increase was observed in the piriform and parietal areas. NPY gene expression also occurred in the granular cell layer, in which no NPY mRNA could be detected in normal animals. The level and distribution of cells displaying high levels of NPY mRNA differed among animals, perhaps as a result of the distinct anatomical location of ODN infusion. Finally, hippocampal levels of NPY specific binding increased, suggesting that NPY neurotransmission is markedly increased. These findings are reminiscent of reported changes in the expression of NPY mRNA and immunoreactivity in conditions of increased neuronal excitation and support the usefulness of the present animal model for the study of epileptic phenomena.
Animals↗
GABAA receptor antisense epilepsy: histological changes following infusion of antisense oligodeoxynucleotide to GABAA receptor gamma 2 subunit into rat hippocampus.
A deficiency of neuronal inhibition mediated by gamma-aminobutyric acid (GABA) via the GABAA receptor complex has been hypothesised to be a central factor in epileptogenesis. Intrahippocampal infusion of antisense oligodeoxynucleotide (ODN) to the GABAA receptor gamma 2 subunit in rats leads to electrographic limbic status epilepticus. In this model, epileptic phenomena are accompanied by loss of hippocampal neurones. The purpose of the present study was to investigate the time-course of morphological changes following hippocampal antisense 'knockdown' of the GABAA receptor gamma 2 subunit. gamma 2 subunit antisense ODN was infused continuously into the right hippocampus for periods between 1 and 5 days. After about 4 days of infusion, pronounced neurodegenerative changes were consistently observed within the ipsilateral hippocampus. In general, marked loss of CA3 pyramidal cells was found. The notion that the histological changes induced by the antisense ODN were specific to the applied ODN sequence was supported by the finding that a mismatch control ODN did not induce neurodegenerative changes, except for a small lesion in the immediate vicinity of the infusion site. Extensive ipsilateral hippocampal infiltration with monocytes and macrophages was a feature of antisense ODN infusion, but was considerably less pronounced after the infusion of control ODN. Immunocytochemistry using an antibody labeling glial fibrillary acidic protein (GFAP), revealed marked astroglial hypertrophy/proliferation after 4 days of antisense treatment, i.e., coincident with the development of neurodegeneration, in the ipsilateral hippocampus. At this time GFAP-immunoreactivity was also evident in the contralateral hippocampus, indicating contralateral spread of seizure activity.
Animals↗
Targeting brain GABAA receptors with antisense oligonucleotides: implications for epilepsy.
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Animals↗
Isomorphous replacement combined with anomalous dispersion in the linear equations: application to a crystal containing four nonapeptide conformers.
The investigation of the structure of the four conformers of the nonapeptide described here has an additional purpose: to illustrate a method for combining isomorphous replacement information with anomalous dispersion information within the linear equations that have found use in the analysis of multiple-wavelength anomalous dispersion data. In the present application, isomorphous replacement data were obtained from the replacement of naturally occurring S atoms in the nonapeptide with Se atoms. Only one wavelength was used for the analysis: Cu Kalpha radiation. Details of the analysis are presented, as well as the structural results obtained. It was found that the four independent molecules in the structure have similar, but not identical, conformations. The backbones fold into predominantly alpha-helices with one or two 310-type hydrogen bonds and have extended side chains. Three to four water molecules are associated with each of the four head-to-tail regions between the peptides. Optimal packing between hydrophobic surfaces may account for the existence of four molecules in an asymmetric unit.
Amino Acid Sequence↗
Antisense oligonucleotide to GABA(A) receptor gamma2 subunit induces limbic status epilepticus.
Gamma-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the brain. A deficiency of GABAergic inhibition mediated via the GABAA receptor complex has for a long time been suspected to be a central factor in epileptogenesis. Status epilepticus is a condition of sustained and prolonged excitation of neuronal circuits, as detected by epileptiform discharges in the electroencephalogram (EEG). Reduction of GABAA receptor-mediated hippocampal inhibition has been implicated in the development of status epilepticus. The present study provides direct evidence of a link between the GABAA receptor and epilepsy. We show that selective inhibition of the expression of the GABAA receptor gamma2 subunit in the rat hippocampus by means of antisense oligonucleotides leads to spontaneous electrographic seizures that evolve into profound limbic status epilepticus, ultimately resulting in severe neurodegenerative changes. Concurrent treatment with diazepam prevents the development of status epilepticus and markedly reduces neuronal cell loss. These findings strongly support the hypothesis that the GABAA receptor is critically involved in the pathogenesis of seizures and status epilepticus.
Animals↗
Differential changes in induced seizures after hippocampal treatment of rats with an antisense oligodeoxynucleotide to the GABA(A) receptor gamma2 subunit.
Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the brain. Impairment of GABAergic neurotransmission may be involved in the pathogenesis of epileptic phenomena. We have previously characterized biochemical and histological changes following unilateral intrahippocampal infusion of a phosphorothioate antisense oligodeoxynucleotide to the GABA(A) receptor gamma2 subunit in rats in vivo. The aim of the present study was to investigate the behavioral changes of rats following unilateral hippocampal antisense 'knockdown' of the GABA(A) receptor gamma2 subunit. Antisense, but not mismatch control oligodeoxynucleotide treated rats had a significant weight loss (10%) during 6 d of treatment. Antisense treated rats exhibited no changes in spontaneous behavior, including anxiety-like behavior as measured in the social interaction test, compared to mismatch oligodeoxynucleotide treated rats. However, antisense treated rats developed pronounced changes in induced seizure activity. Seizures induced by subcutaneously injected pentylenetetrazol were markedly accentuated in antisense treated rats compared to treatment naive rats, whereas mismatch treated rats showed a lower seizure score than that of naive rats. Antisense treated rats had a significantly elevated threshold for seizures induced by electrical stimulation in the maximal electroshock seizure threshold test. The results suggest that intrahippocampal infusion of antisense oligodeoxynucleotide to the GABA(A) receptor gamma2 subunit leads to specific alterations in the sensitivity to induced seizures. The results are viewed as consequences of selective down-regulation of GABA(A) receptors and diminished inhibitory neurotransmission in the hippocampus.
Animals↗
Diazepam protects against rat hippocampal neuronal cell death induced by antisense oligodeoxynucleotide to GABA(A) receptor gamma2 subunit.
Antisense oligodeoxynucleotides (ODNs) are used for the selective inhibition of gene expression. Antisense ODNs are promising tools for the investigation of physiological implications of proteins in the central nervous system of rodents in vivo. We have previously demonstrated that a phosphorothioate antisense ODN to the GABA(A) receptor gamma2 subunit, but not sense or mismatch control ODNs, induces a decrease in ex vivo benzodiazepine receptor radioligand binding in rat hippocampus when infused into the hippocampus in vivo [Karle et al., Neurosci. Lett., 202 (1995) 97-100]. This effect is parallelled by a decrease in the number of GABA(A) receptors and an extensive loss of hippocampal neurones. There is increasing awareness of risks of toxic 'non-antisense' effects induced by ODNs, and in particular phosphorothioate ODNs. The present experiments were designed to investigate the specificity of effects induced by the gamma2 subunit antisense ODN. The temporal development of changes in [3H]flunitrazepam and [3H]quinuclidinyl benzilate binding as well as in tissue protein levels supports the notion that the antisense ODN primarily acts by blocking the expression of the targeted receptor subunit protein. Furthermore, it is shown that a threshold for the elicitation of neurodegenerative changes exists. Finally, it is demonstrated that diazepam treatment of rats protects against the development of neuronal cell death induced by the antisense ODN. Collectively, the results support the hypothesis that the neurodegeneration induced by the antisense ODN is a consequence of diminished GABAergic inhibitory tonus following a selective down-regulation of gamma2 subunit-containing GABA(A) receptor complexes.
Animals↗
The use of in vivo antisense oligonucleotide technology for the investigation of brain GABA receptors.
Antisense oligodeoxynucleotides (ODN) can be used as selective inhibitors of in vivo gene expression in the central nervous system (CNS) of experimental animals. The gamma-aminobutyric acid type A (GABAA) receptor is a member of the ligand-gated ion channel superfamily of neurotransmitter receptors. GABAA receptor function is allosterically modulated by several clinically important compounds, e.g. 1,4-benzodiazepines, barbiturates and certain neurosteroids, which recognize binding sites within the receptor complex. GABAA receptor chloride channel complexes are probably pentamers of different polypeptide subunits. The number of known subunit families and isoforms (six alpha s, four beta s, three gamma s, one delta and two rho s) indicates an extensive heterogeneity of GABAA receptors. The gamma 2 subunit is a functionally integral part of the GABAA receptor, necessary for the high affinity binding of benzodiazepines. The infusion of phosphorothioate ODN antisense to the gamma 2 subunit mRNA, but not control sense or mismatch ODN, into the lateral cerebral ventricle or into the hippocampus of rats leads to significant decreases in benzodiazepine receptor radioligand binding. In the hippocampus this is accompanied by a decrease in the number of GABAA receptors and by a loss of neurones, the latter possibly being due to reduced GABAergic inhibitory neurotransmission. Autoradiographic analysis following continuous intrahippocampal infusion of antisense ODN shows the regional extent of the effect on [3H]flunitrazepam binding. The continuous infusion of antisense ODN, but not of mismatch control ODN, into the right lateral cerebral ventricle induced a significant decrease in benzodiazepine binding and [3H]muscimol binding to membranes of the right cortex. Antisense ODN infused into the striatum decreased benzodiazepine binding and binding to the GABA binding site of the GABAA receptor to an extent similar to that found in the hippocampus. It is concluded that the preferred route of administration of antisense ODN for in vivo studies of the GABAA receptor may be by infusion into defined rat brain regions. The reported data support the idea that antisense ODN can be used as a valuable tool for the investigation of the contribution of individual GABAA receptor subunits to the properties of the receptor complex and of mechanisms of receptor subunit assembly.
Animals↗
Antisense oligonucleotide to GABAA receptor gamma 2 subunit induces loss of neurones in rat hippocampus.
The binding site for 1,4-benzodiazepines in the brain is part of the hetero-oligomeric gamma-aminobutyric acid (GABA)A receptor complex which regulates a chloride ion channel. The presence of the gamma 2 subunit in the complex is necessary for the binding of benzodiazepines to their binding site. This study demonstrates a reduction of benzodiazepine receptor radioligand binding by 43% compared to control following infusion of phosphorothioate antisense oligodeoxynucleotide to gamma 2 subunit into rat hippocampus. Reduction of benzodiazepine binding sites was paralleled by a decrease in [35S]tert-butyl-bicyclo-phosphorothionate ([35S]TBPS) binding (51%) and [3H]muscimol binding (37%), indicating a reduction in the number of GABAA receptors. Changed macroscopic appearance, reduced protein content and severe loss of neurones in antisense-treated hippocampi suggests that the reduced formation of GABAA receptors leads to neuronal cell death.
Animals↗
Modest reduction of benzodiazepine binding in rat brain in vivo induced by antisense oligonucleotide to GABAA receptor gamma 2 subunit subtype.
The GABAA (gamma-aminobutyric acid-A) receptor gamma 2 subunit subtype is probably a functionally integral part of the benzodiazepine binding site of the GABAA receptor complex, important for benzodiazepine pharmacology. We have evaluated the possibility of specifically reducing benzodiazepine receptor binding properties in vivo using phosphorothioate antisense oligodeoxynucleotides to inhibit the expression of GABAA receptor gamma 2 subunit subtype. Intracerebroventricular infusions of an antisense oligonucleotide reduced benzodiazepine receptor radioligand binding by 9-15% in specific rat brain regions.
Animals↗
New approaches to structure analysis.
There are relationships among the phases and magnitudes of the structure factors that have not been extensively studied regarding their potential for enhancing procedures for structure determination. These relationships arise from a special way of writing the determinantal inequalities that form the necessary and sufficient conditions for a Fourier series to be non-negative. This particular form also lends itself readily to the development of probability measures by the use of the central limit theorem. Higher-order determinants are of interest. The relationships among the phases and magnitudes of the structure factors are algebraic relationships and the focus is on those which retain their reliability, even though the magnitudes of the structure factors contain experimental errors. The future utility of the algebraic relationships depends upon the development of suitable algorithms for solving them to obtain values for the unknown phases. One approach concerns a method for extending the range of least-squares calculations by modifying the defining equations without changing the global minima and by further altering the nature of the minimization function from time to time during the course of the least-squares calculation, while still preserving the global minima. The objective is to smooth the minimization function and alter the remaining false minima from time to time so that the minimization function is not trapped in a false minimum. Some calculations have been made that indicate the nature of the algebraic relationships among the phases and magnitudes and how the results of the calculations are benefited by having large values for the structure-factor magnitudes in the determinants.(ABSTRACT TRUNCATED AT 250 WORDS)
Mathematics↗
[Serotonergic syndrome--in combination therapy with lithium and fluoxetine].
Serotonin syndrome is expected to occur more frequently with the increased use of specific serotonin reuptake inhibitors in the treatment of depression. A case of serotonin syndrome after combined treatment with lithium and fluoxetine is presented. Caution is advised when treating affective disorders with combinations of serotonergic agents.
Bipolar Disorder↗
NNC 01-0687, a selective dopamine D1 receptor antagonist, in the treatment of schizophrenia.
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Adult↗
Direct calculation of atomic coordinates from diffraction intensities: space group P1.
A method is presented that can extend the range of convergence for the least-squares minimization technique in nonlinear systems. The problem of determining directly the positions of atomic coordinates in a crystal from the measured intensities of x-ray scattering is used to illustrate the method. The minimization is facilitated by altering the minimization function in a way that reduces the number of false minima and, in addition, by altering the character of the false minima from time to time by changing the particular deck of data that is used and the values of a variety of parameters that may occur in the defining equations. With a structure consisting of 30 equal atoms in space group P1 and the use of exact data with a Cray XMP/216 computer, convergence to a global minimum was generally obtained, from a random positioning of the 90 coordinates, in a few minutes. Convergence within an hour for a 40-atom problem was much more sporadic, showing a considerable decrease in success with the increase in complexity. The possibility of extending the method to other mathematical systems is apparent.
Fourier Analysis↗
Some additional features of one-wavelength anomalous dispersion.
By use of appropriate algebraic formulas, illustrations are given of several characteristics of one-wavelength anomalous-dispersion data, for the case that one predominant type of anomalous-scattering atom is present. It is shown that, when the structure of the anomalous scatterer is known, some simple algebraic formulas may be used to generate initial values of many phases associated with a macromolecular structure. In some cases, there may be enough phases determined to permit further refinement and extension by use of current techniques for doing so. Another calculation shows the virtue of including isomorphous-replacement data and how readily that is done in an algebraic system. It is also shown that there is an advantage in accuracy through the use of a particular statistical calculation of the magnitude of the structure factors for the anomalous scatters, even when the magnitudes are known accurately because the structure of the anomalous scatterers is known. This occurs because the statistical values are scaled to adjust to errors in the data and thus avoids a disparity in scale that would occur otherwise in the algebraic system. With highly accurate data, the advantage would disappear. It is also shown, however, that, even with accurate one-wavelength anomalous-dispersion data, the coupling of isomerophous-replacement data leads to greater accuracy in evaluating phase differences than coupling with known magnitudes for the structure factors associated with the anomalous scatterers.(ABSTRACT TRUNCATED AT 250 WORDS)
Cytochrome c Group↗
Direct methods in protein crystallography.
It is pointed out that the 'direct methods' of phase determination for small-structure crystallography do not have immediate applicability to macromolecular structures. The term 'direct methods in macromolecular crystallography' is suggested to categorize a spectrum of approaches to macromolecular structure determination in which the analyses are characterized by the use of two-phase and higher-order-phase invariants. The evaluation of the invariants is generally obtained by the use of heavy-atom techniques. The results of a number of the more recent algebraic and probabilistic studies involving isomorphous replacement and anomalous dispersion thus become valid subjects for discussion here. These studies are described and suggestions are also presented concerning future applicability. Additional discussion concerns the special techniques of filtering, the use of non-crystallographic symmetry, some features of maximum entropy and attempts to apply phase-determining formulas to the refinement of macromolecular structure. It is noted that, in addition to the continuing remarkable progress in macromolecular crystallography based on the traditional applications of isomorphous replacement and anomalous dispersion, recent valuable advances have been made in the application of non-crystallographic symmetry, in particular, to virus structures and in applications of filtering. Good progress has also been reported in the application of exact linear algebra to multiple-wavelength anomalous-dispersion investigations of structures containing anomalous scatterers of only moderate scattering power.
Crystallography↗
Linear algebraic analyses of structures with one predominant type of anomalous scatterer.
Further studies have been made of the information content of the exact linear equations for analyzing anomalous dispersion data in one-wavelength experiments. The case of interest concerns structures containing atoms that essentially do not scatter anomalously and one type of anomalously scattering atoms. For this case, there are three alternative ways of writing the equations. The alternative sets of equations and the transformations for transforming one set into the other are given explicitly. Comparison calculations were made with different sets of equations. Isomorphous replacement information is readily introduced into the calculations and the advantage of doing so is clearly illustrated by the results. Another aspect of the potential of the exact linear algebraic theory is its application to multiple-wavelength experiments. Successful applications of the latter have been made by several collaborative groups of investigators.
Cytochrome c Group↗