316 research outputs found
Effects of Antiepileptic Drugs on GABA Responses and on Reduction of GABA Responses by PTZ and DMCM on Mouse Neurons in Cell Culture
The mechanisms of action of antiepileptic drugs effective against generalized absence seizures (antiabsence AEDs) remain uncertain. Antiabsence AEDs are generally effective against seizures induced in experimental animals by pentylenĂtĂtrazol (PTZ) and methyl-6,7-dimethoxy-4-ethyl-Î-carboline-3-carboxylate (DMCM), drugs which reduce GABAergic inhibition. Thus, antiabsence AEDs have been suggested to enhance GABAergic inhibition. We studied the effects of several AEDs on GABA responses recorded from mouse spinal cord neurons grown in primary dissociated cell culture. Four antiabsence AEDs were included: ethosuximide (ESM), dimethadione (DMO), sodium valproate (VPA), and diazepam (DZP). Two experimental AEDs, CGS 9896 and ZK 91296, with anticonvulsant action against PTZ- or DMCM-induced seizures were also included. Possible effects of the antiabsence and experimental AEDS on PTZ- and DMCM-induced inhibition of GABA responses were also evaluated. PTZ and DMCM revers-ibly reduced GABA responses in a concentration-dependent manner. PTZ complĂtĂly inhibited GABA responses at 10 mM (IC 50 of 1.1 mM), whereas DMCM-induced inhibition of GABA responses reached a plateau level of 39% of control values at 1 p.M (IC 50 of 33 nM). ESM (1,200 ÎM), DMO (6 mM), VPA (200 u.M), CGS 9896 (2 ÎM), and ZK 98% (2 Î M ) did not alter GABA responses. DZP enhanced GABA responses in a concentration-dependent manner. The inhibition of GABA responses produced by PTZ 1 mM was unaltered by ESM (600 Î M ), DMO (6 mM), CGS 9896 (1 Î M), or ZK 9896 (1 ÎM)- Coapplication of VPA (200 ÎM) and PTZ (1 mM) slightly enhanced the PTZ effect. DZP (> 10 nM), however, reversed the PTZ-induced reduction of GABA responses. The DMCM (250 nM) inhibition of GABA-responses was unaltered by ESM (600 Î.M), DMO (2 mM), or VPA (200 ÎM). CGS 9896 (2 Î M ) and ZK 91296 (2 ÎM), however, antagonized the DMCM effect. DZP (> 10 nM) significantly reversed the DMCM-induced inhibition of GABA responses. The lack of effect of VPA, ESM, and DMO on postsynaptic GABA responses suggests that direct enhancement of postsynaptic GABA action is not a common mechanism of action of antiabsence AEDs. The AEDs DZP, CGS 98%, and ZK 912% all reversed DMCM, but not PTZ, reduction of GABA responses, suggesting that these AEDs blocked DMCM seizures by acting at benzodiazepine receptors. However, since only DZP enhanced GABA responses, it is unclear how CGS 98% and ZK 912% blocked PTZ seizures. Key Words: AnticonvulsantsâGABAâNeuron cultureâCell cultureâSpinal cord neuronsâConvulsants. RESUMEN Los mecanismos de accidn de las medicaciones antiepilĂpticas eficaces contra los ataques generalizados de ausencia (AEDs antiausencia) permanecen inciertos. Los AEDs antiausencia son, generalmente, eficaces contra ataques experimentales inducidos por el pentilentetrazol (PTZ) y el metil-6,7-dimetoxy-4-etil-Pcarbolina-3-carboxilato (DMCM) en animates, medicaciones que reducen la inhibiciĂn GABAĂrgica. Hemos estudiado los efectos de varios AEDs sobre respuestas-GABA registradas en las neuronas de la mĂdula espinal de ratones que habian crecido en cultivos de cĂlulas primarieas disociadas. Cuatro AEDs antiausencia fueron incluidos: etoxusimida (ESM), dimetadiona (DMO), valproato sĂdico (VPA) y diazepan (DZP). TambtĂn se incluyeron dos AEDs experimentales, CGS 9896 y ZK 912%, con acciĂn anticonvulsiva contra los ataques inducidos por PTZ o DMCM. TambiĂn se valoraron los posibles efectos de los AEDs antiausencia y experimentales sobre el PTZ y la inhibiciĂn de las respuestas-GABA inducidas por el DMCM. El PTZ y el DMCM redujeron las respuestas-GABA de modo reversible y dependiendo de sus concentraciones. El PTZ inhibiĂ cmpleta-mente las respuestas-GABA a 10 mM (IC 50 de 1.1 mM) mientras que la inhibitiĂn de las respuestas GABA inducida por el DMCM alcanzĂ un nivel estable del 39% de los valores control con 1 Î. M (IC 50 de 33 mM). La ESM (1200 Î.M), la DMO (6 mM), el VPA (200 Î M ), el CGS 98% (2 Î M) y el ZK 98% (2 Î M) no alteraron las respuestas-GABA. El DZP aumentĂ las respuestas GABA de una manera concentraciĂn-dependiente. La inhibition de las respuestas-GABA producidas por el PTZ (1 mM), no se altero con las ESM (600 Î M), la DMO (6 mM), el CGS 98% (1 Î M) o el ZK 98% (1 Î .M). La co-aplicacion de VPA (200 Î M) y el PTZ (1 mM) aument6 ligeramente los efectos del PTZ. Sin embargo el DZP (10 nM) revirtiĂ significativamente la inhibition de las respuestas GABA inducidas por el DMCM. La falta de efectos de CPA, ESM y DMO sobre las respuestas GABA post-sinĂpticas sugiere que el incremento de la acciĂn GABA post-sinĂptica no es un mecanismo comĂn de actuatiĂn de las AEDs antiausencia. Todas las AEDs DZP, CGS 98% y ZK 912% revirtieron la reduction de las respuestas GABA producidas por el DMCM pero no las inducidas por el PTZ lo que sugiere que estos AEDs bloquean los ataques DMCM actuando sobre los receptores de la benzodiazepina. Sin embargo, puesto que el incremento de las respuestas GABA sĂlĂ se produce por el DZP, permanece todavia sin aclarar el por quĂ el CGS 98% y el ZK 912% bloquean los ataques producidos por el PTZ. ZUSAMMENFASSUNG Der Wirkmechansimus von Antiepileptika gegen generalisierte Absencen ist unklar. Antiabsencemittel sind generell wirkungs-voll gegen PTZ- und Methyl-6,7-Dimethoxy-4-Ăthyl-P-Carbolin-Î-Carboxylat (DMCM) induzierte tierexperimentelle AnfĂlle, also von Medikamenten, die die GABA-erge Inhibition reduzieren. Es wurde vermutet, daĂ Antiabsencemittel die GABA-erge Inhibition verstĂrken. Wir untersuchten die Wirkung von verschiedenen Antiepileptika auf GABA-Antworten in spinalen MĂuseneuronen, die in Zellkulturen gew-achsen waren. Es wurden 4 Absencemittel untersucht: Ethosux-imid (ESM), Dimethadion (DMD), Sodium Valproat (VPA) und Diazepam (DZP). ZusĂtzlich wurden 2 experimentelle Antiepileptika, CGS 98% und ZK 912%, die gegen PTZ0 oder DMCM-induzierte AnfĂlle wirkungsvoll sind, eingeschlossen. Mogliche Wirkungen der Antiabsence- und experimentellen Antiepileptika auf PTZ- und DMCM-induzierte Hemmung der GABA-Antworten wurden ebenfalls ausgewertet. PTZ und DMCM zeigten eine konzentrationsabhĂngige reversible Reduktion der GABA-Antworten. PTZ zeigte eine komplette Hemmung der GABA-Antworten bei 10 mM (IC 50 1,1 mM), DMCM-Hemmung der GABA-Antworten zeigte ein Plateau von 39% der Kontroll-werte bei 1 uJtf (ICJO von 33 mAfl. ESM (1200 uJtf), DMD (6 mM), VPA (200 Î M), CGS 98% (2 Î M) und ZK 98% (2 Î M) anderten nicht die GABA-Antworten. DZP verstarkte die GABA-Antworten konzentrationsabhangig. Die durch PTZ (1 mM) hervorgerufene Hemmung der GABA-Antworten war bei ESM (600 Î M), DMD (6 mM), CGS 98% (1 mAO und ZK 3836 (1 mM) unverĂndert. ZusĂtliche Anwendung von VPA (200 mM) und PTZ (1 mM) verstĂrkten geringfĂgig den PTZ-Effekt. DZP (10 nM) kehrte die durch PTZ hervorgerufene Reduktion der GABA-Antworten um. Die durch DMCM (250 nM) hervorgerufene Hemmung der GABA-Antworten war durch ESM (600 Î .M), DMD (2 mM) und VPA (200 Î M ) unbeeinflusst. CGS 98% (2 Î M) und ZK 912% (2 Î M ) antagonisierten die DMCM-Wirkung. DZP (>10 nM) kehrte die durch DMCM-induzierte Hemmung der GABA-Antworten um. Das Fehlen einer Wirkung von VPA. ESM und DMD auf die postsynaptischen GABA-Antworten legen nahe, daĂ eine direkte VerstĂrkung der postsynaptischen GABA-Aktion kein gemeinsamer Mechanis-mus der Antiabsencemittel darstellt. Die Antiepileptika DZP, CGS 98% und ZK 912% kehrten die DMCM-Wirkung auf die GABA-Antworten um, jedoch nicht die von PTZ, was vermuten lapt, daĂ diese Antiepileptika die DMCM-AnfĂlle Ăber die Wirkung an den Benzodiazipin-Rezeptoren verhinderte. Da jedoch nur DZP GABA-Antworten verstarkte, ist unklar, in welcher Weise CGS 98% und ZK 912% die PTZ-AnfaUe ver-hinderten.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65188/1/j.1528-1157.1989.tb05275.x.pd
Aorto-ventricular tunnel
Aorto-ventricular tunnel is a congenital, extracardiac channel which connects the ascending aorta above the sinutubular junction to the cavity of the left, or (less commonly) right ventricle. The exact incidence is unknown, estimates ranging from 0.5% of fetal cardiac malformations to less than 0.1% of congenitally malformed hearts in clinico-pathological series. Approximately 130 cases have been reported in the literature, about twice as many cases in males as in females. Associated defects, usually involving the proximal coronary arteries, or the aortic or pulmonary valves, are present in nearly half the cases. Occasional patients present with an asymptomatic heart murmur and cardiac enlargement, but most suffer heart failure in the first year of life. The etiology of aorto-ventricular tunnel is uncertain. It appears to result from a combination of maldevelopment of the cushions which give rise to the pulmonary and aortic roots, and abnormal separation of these structures. Echocardiography is the diagnostic investigation of choice. Antenatal diagnosis by fetal echocardiography is reliable after 18 weeks gestation. Aorto-ventricular tunnel must be distinguished from other lesions which cause rapid run-off of blood from the aorta and produce cardiac failure. Optimal management of symptomatic aorto-ventricular tunnel consists of diagnosis by echocardiography, complimented with cardiac catheterization as needed to elucidate coronary arterial origins or associated defects, and prompt surgical repair. Observation of the exceedingly rare, asymptomatic patient with a small tunnel may be justified by occasional spontaneous closure. All patients require life-long follow-up for recurrence of the tunnel, aortic valve incompetence, left ventricular function, and aneurysmal enlargement of the ascending aorta
Shocked monazite chronometry: integrating microstructural and in situ isotopic age data for determining precise impact ages
Monazite is a robust geochronometer and occurs in a wide range of rock types. Monazite also records shock deformation from meteorite impact but the effects of impact-related microstructures on the UâThâPb systematics remain poorly constrained. We have, therefore, analyzed shock-deformed monazite grains from the central uplift of the Vredefort impact structure, South Africa, and impact melt from the Araguainha impact structure, Brazil, using electron backscatter diffraction, electron microprobe elemental mapping, and secondary ion mass spectrometry (SIMS). Crystallographic orientation mapping of monazite grains from both impact structures reveals a similar combination of crystal-plastic deformation features, including shock twins, planar deformation bands and neoblasts. Shock twins were documented in up to four different orientations within individual monazite grains, occurring as compound and/or type one twins in (001), (100), (10 1 ÂŻ) , {110}, { 212 } , and type two (irrational) twin planes with rational shear directions in [ 0 1 ÂŻ 1 ÂŻ ] and [ 1 ÂŻ 1 ÂŻ 0 ]. SIMS UâThâPb analyses of the plastically deformed parent domains reveal discordant age arrays, where discordance scales with increasing plastic strain. The correlation between discordance and strain is likely a result of the formation of fast diffusion pathways during the shock event. Neoblasts in granular monazite domains are strain-free, having grown during the impact events via consumption of strained parent grains. Neoblastic monazite from the Inlandsee leucogranofels at Vredefort records a 207Pb/206Pb age of 2010 ± 15 Ma (2Ï, n = 9), consistent with previous impact age estimates of 2020 Ma. Neoblastic monazite from Araguainha impact melt yield a Concordia age of 259 ± 5 Ma (2Ï, n = 7), which is consistent with previous impact age estimates of 255 ± 3 Ma. Our results demonstrate that targeting discrete microstructural domains in shocked monazite, as identified through orientation mapping, for in situ UâThâPb analysis can date impact-related deformation. Monazite is, therefore, one of the few high-temperature geochronometers that can be used for accurate and precise dating of meteorite impacts
Isotropic 3D Nuclear Morphometry of Normal, Fibrocystic and Malignant Breast Epithelial Cells Reveals New Structural Alterations
Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D) objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading criteria.We applied micro-optical computed tomographic imaging and automated 3D nuclear morphometry to quantify and compare morphological variations between human cell lines derived from normal, benign fibrocystic or malignant breast epithelium. To reproduce the appearance and contrast in clinical cytopathology images, we stained cells with hematoxylin and eosin and obtained 3D images of 150 individual stained cells of each cell type at sub-micron, isotropic resolution. Applying volumetric image analyses, we computed 42 3D morphological and textural descriptors of cellular and nuclear structure.We observed four distinct nuclear shape categories, the predominant being a mushroom cap shape. Cell and nuclear volumes increased from normal to fibrocystic to metastatic type, but there was little difference in the volume ratio of nucleus to cytoplasm (N/C ratio) between the lines. Abnormal cell nuclei had more nucleoli, markedly higher density and clumpier chromatin organization compared to normal. Nuclei of non-tumorigenic, fibrocystic cells exhibited larger textural variations than metastatic cell nuclei. At p<0.0025 by ANOVA and Kruskal-Wallis tests, 90% of our computed descriptors statistically differentiated control from abnormal cell populations, but only 69% of these features statistically differentiated the fibrocystic from the metastatic cell populations.Our results provide a new perspective on nuclear structure variations associated with malignancy and point to the value of automated quantitative 3D nuclear morphometry as an objective tool to enable development of sensitive and specific nuclear grade classification in breast cancer diagnosis
Tuning hardness in calcite by incorporation of amino acids
Structural biominerals are inorganic/organic composites that exhibit remarkable mechanical properties. However, the structureâproperty relationships of even the simplest building unitâmineral single crystals containing embedded macromoleculesâremain poorly understood. Here, by means of a model biomineral made from calcite single crystals containing glycine (0â7âmol%) or aspartic acid (0â4âmol%), we elucidate the origin of the superior hardness of biogenic calcite. We analysed lattice distortions in these model crystals by using X-ray diffraction and molecular dynamics simulations, and by means of solid-state nuclear magnetic resonance show that the amino acids are incorporated as individual molecules. We also demonstrate that nanoindentation hardness increased with amino acid content, reaching values equivalent to their biogenic counterparts. A dislocation pinning model reveals that the enhanced hardness is determined by the force required to cut covalent bonds in the molecules
In Silico Prediction and Analysis of Caenorhabditis EF-hand Containing Proteins
Calcium (Ca+2) is a ubiquitous messenger in eukaryotes including Caenorhabditis. Ca+2-mediated signalling processes are usually carried out through well characterized proteins like calmodulin (CaM) and other Ca+2 binding proteins (CaBP). These proteins interact with different targets and activate it by bringing conformational changes. Majority of the EF-hand proteins in Caenorhabditis contain Ca+2 binding motifs. Here, we have performed homology modelling of CaM-like proteins using the crystal structure of Drosophila melanogaster CaM as a template. Molecular docking was applied to explore the binding mechanism of CaM-like proteins and IQ1 motif which is a âŒ25 residues and conform to the consensus sequence (I, L, V)QXXXRXXXX(R,K) to serve as a binding site for different EF hand proteins. We made an attempt to identify all the EF-hand (a helix-loop-helix structure characterized by a 12 residues loop sequence involved in metal coordination) containing proteins and their Ca+2 binding affinity in Caenorhabditis by analysing the complete genome sequence. Docking studies revealed that F165, F169, L29, E33, F44, L57, M61, M96, M97, M108, G65, V115, F93, N104, E144 of CaM-like protein is involved in the interaction with IQ1 motif. A maximum of 170 EF-hand proteins and 39 non-EF-hand proteins with Ca+2/metal binding motif were identified. Diverse proteins including enzyme, transcription, translation and large number of unknown proteins have one or more putative EF-hands. Phylogenetic analysis revealed seven major classes/groups that contain some families of proteins. Various domains that we identified in the EF-hand proteins (uncharacterized) would help in elucidating their functions. It is the first report of its kind where calcium binding loop sequences of EF-hand proteins were analyzed to decipher their calcium affinities. Variation in Ca+2-binding affinity of EF-hand CaBP could be further used to study the behaviour of these proteins. Our analyses postulated that Ca+2 is likely to be key player in Caenorhabditis cell signalling
Direct observation of mineral-organic composite formation reveals occlusion mechanism
Manipulation of inorganic materials with organic macromolecules enables organisms to create biominerals such as bones and seashells, where occlusion of biomacromolecules within individual crystals generates superior mechanical properties. Current understanding of this process comes from entrapment of micron-size particles in cooling melts. Here, by studying micelle incorporation in calcite with atomic force microscopy (AFM) and micromechanical simulations, we show that different mechanisms govern nanoscale occlusion. By simultaneously visualizing the micelles and propagating step edges, we demonstrate that the micelles experience significant compression during occlusion, which is accompanied by cavity formation. This generates local lattice strain, leading to enhanced mechanical properties. These results give new insight into the formation of occlusions in natural and synthetic crystals, and will facilitate the synthesis of multifunctional nanocomposite crystals
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