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Subunit Composition (subunit + composition)
Selected AbstractsCharacteristics and physiological role of hyperpolarization activated currents in mouse cold thermoreceptorsTHE JOURNAL OF PHYSIOLOGY, Issue 9 2009Patricio Orio Hyperpolarization-activated currents (Ih) are mediated by the expression of combinations of hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel subunits (HCN1,4). These cation currents are key regulators of cellular excitability in the heart and many neurons in the nervous system. Subunit composition determines the gating properties and cAMP sensitivity of native Ih currents. We investigated the functional properties of Ih in adult mouse cold thermoreceptor neurons from the trigeminal ganglion, identified by their high sensitivity to moderate cooling and responsiveness to menthol. All cultured cold-sensitive (CS) neurons expressed a fast activating Ih, which was fully blocked by extracellular Cs+ or ZD7288 and had biophysical properties consistent with those of heteromeric HCN1,HCN2 channels. In CS neurons from HCN1(,/,) animals, Ih was greatly reduced but not abolished. We find that Ih activity is not essential for the transduction of cold stimuli in CS neurons. Nevertheless, Ih has the potential to shape the excitability of CS neurons. First, Ih blockade caused a membrane hyperpolarization in CS neurons of about 5 mV. Furthermore, impedance power analysis showed that all CS neurons had a prominent subthreshold membrane resonance in the 5,7 Hz range, completely abolished upon blockade of Ih and absent in HCN1 null mice. This frequency range matches the spontaneous firing frequency of cold thermoreceptor terminals in vivo. Behavioural responses to cooling were reduced in HCN1 null mice and after peripheral pharmacological blockade of Ih with ZD7288, suggesting that Ih plays an important role in peripheral sensitivity to cold. [source] The impact of diazepam's discovery on the treatment and understanding of status epilepticusEPILEPSIA, Issue 9 2009Howard P. Goodkin Summary The fortuitous discovery of the benzodiazepines and the subsequent application of these agents to the treatment of status epilepticus (SE) heralds in the modern age of treating this neurologic emergency. More than 50 years after their discovery, the benzodiazepines remain the drugs of first choice in the treatment of SE. However, the benzodiazepines can be ineffective, especially in those patients whose seizures are the most prolonged. The benzodiazepines act by increasing the affinity of ,-aminobutyric acid (GABA) for GABAA receptors. A receptor's subunit composition affects its functional and pharmacologic properties, trafficking, and cellular localization. The GABAA receptors that mediate synaptic inhibition typically contain a ,2 subunit and are diazepam-sensitive. Among the GABAA receptors that mediate tonic inhibition are the benzodiazepine-insensitive , subunit,containing receptors. The initial studies investigating the pathogenesis of SE demonstrated that a reduction in GABA-mediated inhibition within the hippocampus was important in maintenance of SE, and this reduction correlated with a rapid modification in the postsynaptic GABAA receptor population expressed on the surface of the hippocampal principal neurons. Subsequent studies found that this rapid modification is, in part, mediated by an activity-dependent, subunit-specific trafficking of the receptors that resulted in the reduction in the surface expression of the benzodiazepine-sensitive ,2 subunit,containing receptors and the preserved surface expression of the benzodiazepine-insensitive , subunit-containing receptors. This improved understanding of the changes in the trafficking of GABAA receptors during SE partially accounts for the development of benzodiazepine-pharmacoresistance and has implications for the current and future treatment of benzodiazepine-refractory SE. [source] Characterization of Neuronal Migration Disorders in Neocortical Structures: Loss or Preservation of Inhibitory Interneurons?EPILEPSIA, Issue 7 2000Petra Schwarz Summary: Purpose: Neuronal migration disorders (NMD) are often associated with therapy-resistant epilepsy. In human cerebral cortex, this hyperexcitability has been correlated with a loss of inhibitory interneurons. We used a rat model of focal cortical NMD (microgyria) to determine whether the expression of epileptiform activity in this model coincides with a decrease in inhibitory interneurons. Methods: In 2- to 4-month-old rats, the density of interneurons immunoreactive for ,-aminobutyric acid (GABA), cal-bindin, and parvalbumin was determined in fronto-parietal cortex in nine 200-,m-wide sectors located up to 2.5 mm lateral and 2.0 mm medial from the lesion center in primary parietal cortex (Par 1). Quantitative measurements in homotopic areas of age-matched sham-operated rats served as controls. Results: The freeze lesion performed in newborn rat cortex resulted in adult rats with a microgyrus extending in a rostro-caudal direction from frontal to occipital cortex. The density of GABA- and parvalbumin-positive neurons in fronto-parietal cortex was not significantly different between lesioned and control animals. Only the density of calbindin-immunoreactive neurons located 1.0 mm lateral and 0.5 mm medial from the lesion was significantly (Student t test, p > 0.05) larger in freeze-lesioned rats (5.817 ± 562 and 6,400 ± 795 cells per mm3, respectively; n = 12) compared with measurements in homotopic regions in Parl cortex of controls (4,507 ± 281 and 4,061 ± 319 cells per mm3, respectively; n = 5). Conclusions: The previously reported widespread functional changes in this model of cortical NMD are not related to a general loss of inhibitory interneurons. Other factors, such as a decrease in GABA receptor density, modifications in GABAA receptor subunit composition, or alterations in the excitatory network, e.g., an increase in the density of calbindin-immunoreactive pyramidal cells, more likely contribute to the global disinhibition and widespread expression of pathophysiological activity in this model of cortical NMD. [source] Differential responses to NMDA receptor activation in rat hippocampal interneurons and pyramidal cells may underlie enhanced pyramidal cell vulnerabilityEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2005E. Avignone Abstract Hippocampal interneurons are generally more resistant than pyramidal cells to excitotoxic insults. Because NMDA receptors play a crucial role in neurodegeneration, we have compared the response to exogenous NMDA in CA1 pyramidal cells and interneurons of the stratum oriens using combined whole-cell patch-clamp recording and ratiometric Ca2+ imaging. In voltage-clamp, current-clamp or in nominally Mg2+ -free medium, NMDA (10 µm; 3,5 min exposure in the presence of tetrodotoxin) induced a markedly larger inward current and Ca2+ rise in pyramidal cells than in interneurons. Pyramidal cells also showed a more pronounced voltage dependence in their response to NMDA. We hypothesized that this enhanced response to NMDA receptor activation in pyramidal cells could underlie their increased vulnerability to excitotoxicity. Using loss of dye as an indicator of degenerative membrane disruption, interneurons tolerated continuous exposure to a high concentration of NMDA (30 µm) for longer periods than pyramidal cells. This acute neurodegeneration in pyramidal cells was independent of intracellular Ca2+, because high intracellular BAPTA (20 mm) did not prolong survival time. Thus, a plausible explanation for the enhanced sensitivity of pyramidal neurons to excitotoxic insults associated with cerebral ischemia is their greater response to NMDA receptor activation, which may reflect differences in NMDA receptor expression and/or subunit composition. [source] GluR- and TrkB-mediated maturation of GABAA receptor function during the period of eye openingEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2005Christian Henneberger Abstract Synapse maturation includes the shortening of postsynaptic currents, due to changes in the subunit composition of respective transmitter receptors. Patch clamp experiments revealed that GABAergic inhibitory postsynaptic currents (ISPCs) of superior colliculus neurons significantly shorten from postnatal day (P)1 to P21. The change started after P6 and was steepest between P12 and P15, i.e. around eye opening. It was accompanied by enhanced sensitivity to zolpidem and increased expression of GABAAR ,1 mRNA, whereas the level of ,3 mRNA decreased. This result is consistent with the hypothesis that the IPSC kinetics of developing collicular neurons is determined by the level of ,1/,3. As ,1/,3 peaked when N -methyl-D-aspartate receptor (NMDAR)-mediated synaptic currents reached their maximum (P12) it was asked whether NMDAR activity can shape the kinetics of GABAergic IPSCs. Cultured collicular neurons were treated with NMDA or NMDAR block, and it was found that the former resulted in faster and the latter in slower IPSC decay. Group I mGluR blockade had no effect. Experiments with bdnf,/, mice revealed that, with some delay, the increase of ,1/,3 mRNA also occurred in the chronic absence of brain-derived neurotrophic factor (BDNF) and, again, this was accompanied by the shortening of IPSCs. In addition, there was an age-dependent depression of IPSC amplitudes by endogenous BDNF, which might reflect the developmental increase in the expression of GABAAR ,2L, as opposed to ,2S. Together, these experiments suggest that the GABAAR , subunit switch and the associated change in the IPSC kinetics were specifically controlled by NMDAR activity and independent on the signalling through group I mGluRs or TrkB. [source] Synaptic localization of GABAA receptor subunits in the substantia nigra of the rat: effects of quinolinic acid lesions of the striatumEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2002Fumino Fujiyama Abstract The inhibitory amino acid, ,-aminobutyric acid (GABA), plays a critical role in the substantia nigra (SN) in health and disease. GABA transmission is controlled in part by the type(s) of GABA receptor expressed, their subunit composition and their location in relation to GABA release sites. In order to define the subcellular localization of GABAA receptors in the SN in normal and pathological conditions, sections of SN from control rats and rats that had received quinolinic acid lesions of the striatum were immunolabelled using the postembedding immunogold technique with antibodies against subunits of the GABAA receptor. Immunolabelling for ,1, ,2/3 and ,2 subunits was primarily located at symmetrical synapses. Double-labelling revealed that ,2/3 subunit-positive synapses were formed by terminals that were enriched in GABA. Colocalization of ,1, ,2/3 and ,2 subunits occurred at individual symmetrical synapses, some of which were identified as degenerating terminals derived from the striatum. In the SN ipsilateral to the striatal lesion there was a significant elevation of immunolabelling for ,2/3 subunits of the GABAA receptor at symmetrical synapses, but not of GluR2/3 subunits of the AMPA receptor at asymmetrical synapses. It was concluded that fast GABAA -mediated transmission occurs primarily at symmetrical synapses within the SN, that different receptor subunits coexist at individual synapses and that the upregulation of GABAA receptors following striatal lesions is expressed as increased receptor density at synapses. The upregulation of GABAA receptors in Huntington's disease and its models is thus likely to lead to an increased efficiency of transmission at intact GABAergic synapses in the SN and may partly underlie the motor abnormalities of this disorder. [source] Differential sensitivity of medium- and large-sized striatal neurons to NMDA but not kainate receptor activation in the ratEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2001Carlos Cepeda Abstract Infrared videomicroscopy and differential interference contrast optics were used to identify medium- and large-sized neurons in striatal slices from young rats. Whole-cell patch-clamp recordings were obtained to compare membrane currents evoked by application of N -methyl- d -aspartate (NMDA) and kainate. Inward currents and current densities induced by NMDA were significantly smaller in large- than in medium-sized striatal neurons. The negative slope conductance for NMDA currents was greater in medium- than in large-sized neurons and more depolarization was required to remove the Mg2+ blockade. In contrast, currents induced by kainate were significantly greater in large-sized neurons whilst current densities were approximately equal in both cell types. Spontaneous excitatory postsynaptic currents occurred frequently in medium-sized neurons but were relatively infrequent in large-sized neurons. Excitatory postsynaptic currents evoked by electrical stimulation were smaller in large- than in medium-sized neurons. A final set of experiments assessed a functional consequence of the differential sensitivity of medium- and large-sized neurons to NMDA. Cell swelling was used to examine changes in somatic area in both neuronal types after prolonged application of NMDA or kainate. NMDA produced a time-dependent increase in somatic area in medium-sized neurons whilst it produced only minimal changes in large interneurons. In contrast, application of kainate produced significant swelling in both medium- and large-sized cells. We hypothesize that reduced sensitivity to NMDA may be due to variations in receptor subunit composition and/or the relative density of receptors in the two cell types. These findings help define the conditions that put neurons at risk for excitotoxic damage in neurological disorders. [source] Differential modulation of AMPA receptors by cyclothiazide in two types of striatal neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2000Vladimir S. Vorobjev Abstract The modulation of ,-amino-3-hydroxy-5-methyl-4-isoxazol-propionate (AMPA) receptor-mediated currents by cyclothiazide was investigated in acutely isolated cells from rat striatum with whole-cell patch-clamp recording. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) was used to identify medium spiny and giant aspiny neurons and to determine their AMPA receptor subunit composition mostly in separate experiments. After pretreatment with cyclothiazide, kainate-induced AMPA responses were more strongly potentiated in medium spiny than in giant aspiny neurons; cyclothiazide induced a ninefold leftward shift in the kainate concentration,response curve for medium spiny neurons (not giant aspiny neurons). The EC50s for the cyclothiazide potentiation did not differ substantially between medium spiny neurons and giant aspiny neurons. The recovery of kainate-activated currents from modulation by cyclothiazide was slower for medium spiny neurons than for giant aspiny neurons. Medium spiny neurons expressed GluR-A, GluR-B and GluR-C, but not GluR-D subunits in both flip and flop splice variants. All giant aspiny neurons expressed GluR-A and GluR-D, exclusively in the flop form, half of them also expressed GluR-B and GluR-C. This is in keeping with slow and fast desensitization kinetics in medium spiny neurons and giant aspiny neurons, respectively, and differences in cyclothiazide modulation. The rate of cyclothiazide dissociation from the AMPA receptor, activated by glutamate, was ,,90 times slower in medium spiny neurons than in giant aspiny neurons. In giant aspiny neurons (not medium spiny neurons) this rate was strongly dependent on the presence of an agonist; 1 m m glutamate increased it 30-fold. Thus, two major cell groups in the striatum display distinct AMPA receptor compositions carrying specific properties of glutamate responses. Excitatory transmission will thus be differentially affected by cyclothiazide-type compounds. [source] Isolation and structural characterization of the Ndh complex from mesophyll and bundle sheath chloroplasts of Zea maysFEBS JOURNAL, Issue 11 2005Costel C. Darie Complex I (NADH: ubiquinone oxidoreductase) is the first complex in the respiratory electron transport chain. Homologs of this complex exist in bacteria, mitochondria and chloroplasts. The minimal complex I from mitochondria and bacteria contains 14 different subunits grouped into three modules: membrane, connecting, and soluble subcomplexes. The complex I homolog (NADH dehydrogenase or Ndh complex) from chloroplasts from higher plants contains genes for two out of three modules: the membrane and connecting subcomplexes. However, there is not much information about the existence of the soluble subcomplex (which is the electron input device in bacterial complex I) in the composition of the Ndh complex. Furthermore, there are contrasting reports regarding the subunit composition of the Ndh complex and its molecular mass. By using blue native (BN)/PAGE and Tricine/PAGE or colorless-native (CN)/PAGE, BN/PAGE and Tricine/PAGE, combined with mass spectrometry, we attempted to obtain more information about the plastidal Ndh complex from maize (Zea mays). Using antibodies, we detected the expression of a new ndh gene (ndhE) in mesophyll (MS) and bundle sheath (BS) chloroplasts and in ethioplasts (ET). We determined the molecular mass of the Ndh complex (550 kDa) and observed that it splits into a 300 kDa membrane subcomplex (containing NdhE) and a 250 kDa subcomplex (containing NdhH, -J and -K). The Ndh complex forms dimers at 1000,1100 kDa in both MS and BS chloroplasts. Native/PAGE of the MS and BS chloroplasts allowed us to determine that the Ndh complex contains at least 14 different subunits. The native gel electrophoresis, western blotting and mass spectrometry allowed us to identify five of the Ndh subunits. We also provide a method that allows the purification of large amounts of Ndh complex for further structural, as well as functional studies. [source] An architectural perspective on signaling by the pre-, ,, and ,, T cell receptorsIMMUNOLOGICAL REVIEWS, Issue 1 2003Sandra M. Hayes Summary: The T cell antigen receptor (TCR) is a multimeric complex composed of an antigen-binding clonotypic heterodimer and a signal transducing complex consisting of the CD3 dimers (CD3,, and CD3,,) and a TCR-, homodimer. In all jawed vertebrates there are two T cell lineages, ,, and ,,, distinguished by the clonotypic subunits contained within their TCRs (TCR-, and -, or TCR-, and -,, respectively). A third receptor complex, the preTCR, is only expressed on immature T cells. The preTCR, which contains the invariant pre-T, (pT,) chain in lieu of TCR-,, plays a critical role in the early development of ,, lineage cells. The subunit composition of the signal transducing complexes of the pre-, ,,- and ,,TCRs was previously thought to be identical. However, recent data demonstrate that there are significant differences in the signal transducing complexes of these three TCRs. For example, ,,TCRs contain both CD3,, and CD3,, dimers, whereas ,,TCRs contain only CD3,, dimers. Moreover, preTCR function appears to be unaffected in the absence of CD3,, suggesting that CD3,, dimers are dispensable for pre-TCR assembly. In this review, we summarize current data relating to the subunit composition of the pre-, ,,- and ,,TCRs and discuss how these structural differences may impact receptor signaling and ,,/,, lineage determination. [source] NF- ,B in liver diseases: a target for drug therapyJOURNAL OF APPLIED TOXICOLOGY, Issue 2 2009Pablo Muriel Abstract There are five nuclear factor- ,B (NF- ,B) transcription factors with important roles in innate immunity, liver inflammation, fibrosis and apoptosis prevention. Several inhibitors of NF- ,B, like caffeic acid, captopril, curcumin, pyrrolidine dithiocarbamate, resveratrol, silymarin and thalidomide, have demonstrated antinecrotic, anticholestatic, antifibrotic and anticancer activities in the liver. A link between inflammation and hepatocellular carcinoma through the NF- ,B pathway has been observed, providing ample experimental support for the tumor-promoting function of NF- ,B in various models of cancer. NF- ,B has been associated with the induction of proinflammatory gene expression and has attracted interest as a target for the treatment of inflammatory disease. However, despite much attention being focused on the deleterious effects of NF- ,B, activation of this factor during the resolution of inflammation is associated with the production of antiinflammatory molecules like interleukin (IL)-10 and the onset of apoptosis. This suggests that NF- ,B has an antiinflammatory role in vivo involving the regulation of the resolution of inflammation. Also, NF- ,B promotes liver regeneration by upregulating IL-6 and other molecules like hepatocyte growth factor. It has been postulated that the beneficial properties of NF- ,B are due to p50 homodimers, whose activation prevents cholestatic and chronic liver injury. More basic understanding on the function of the diverse NF- ,B factors is urgently needed in different physiological and pathological conditions, because depending on the subunit composition of the dimmer, the disease and the stage of the illness, inhibition of the factor may result in a beneficial or in a deleterious response. Copyright © 2008 John Wiley & Sons, Ltd. [source] GABAA receptors in aging and Alzheimer's diseaseJOURNAL OF NEUROCHEMISTRY, Issue 4 2007Robert A. Rissman Abstract In this article we present a comprehensive review of relevant research and reports on the GABAA receptor in the aged and Alzheimer's disease (AD) brain. In comparison to glutamatergic and cholinergic systems, the GABAergic system is relatively spared in AD, but the precise mechanisms underlying differential vulnerability are not well understood. Using several methods, investigations demonstrate that despite resistance of the GABAergic system to neurodegeneration, particular subunits of the GABAA receptor are altered with age and AD, which can induce compensatory increases in GABAA receptor subunits within surrounding cells. We conclude that although aging- and disease-related changes in GABAA receptor subunits may be modest, the mechanisms that compensate for these changes may alter the pharmacokinetic and physiological properties of the receptor. It is therefore crucial to understand the subunit composition of individual GABAA receptors in the diseased brain when developing therapeutics that act at these receptors. [source] Subcellular segregation of distinct heteromeric NMDA glutamate receptors in the striatumJOURNAL OF NEUROCHEMISTRY, Issue 4 2003Anthone W. Dunah Abstract Functional N -methyl- d -aspartate (NMDA) glutamate receptors are composed of heteromeric complexes of NR1, the obligatory subunit for channel activity, and NR2 or NR3 family members, which confer variability in the properties of the receptors. Recent studies have provided evidence for the existence of both binary (containing NR1 and either NR2A or NR2B) and ternary (containing NR1, NR2A, and NR2B) receptor complexes in the adult mammalian brain. However, the mechanisms regulating subunit assembly and receptor localization are not well understood. In the CNS, NMDA subunits are present both at intracellular sites and the post-synaptic membrane of neurons. Using biochemical protein fractionation and co-immunoprecipitation approaches we have found that in rat striatum binary NMDA receptors are widely distributed, and can be identified in the light membrane, synaptosomal membrane, and synaptic vesicle-enriched subcellular compartments. In contrast, ternary receptors are found exclusively in the synaptosomal membranes. When striatal proteins are chemically cross-linked prior to subcellular fractionation, ternary NMDA receptors can be precipitated from the light membrane and synaptic vesicle-enriched fractions where this type of receptor complex is not detectable under normal conditions. These findings suggest differential targeting of distinct types of NMDA receptor assemblies between intracellular and post-synaptic sites based on subunit composition. This targeting may underlie important differences in the regulation of the transport pathways involved in both normal as well as pathological receptor functions. [source] N -methyl-d-aspartate Receptor Responses Are Differentially Modulated by Noncompetitive Receptor Antagonists and Ethanol in Inbred Long-Sleep and Short-Sleep Mice: Behavior and ElectrophysiologyALCOHOLISM, Issue 12 2000Taleen Hanania Background: Short-sleep (SS) mice exhibit higher locomotor activity than do long-sleep (LS) mice when injected with low doses of ethanol or the noncompetitive N -methyl-D-aspartate receptor (NMDAR) antagonist MK-801 (dizocilpine). SS mice also have higher densities of brain NMDARs. However, two strains of LS X SS recombinant inbred (RI) mice also show differential activation to ethanol and MK-801, but have similar numbers of NMDARs. Here we used inbred LS (ILS) and SS (ISS) mice to investigate further the relationship between NMDARs and sensitivity to the stimulant effects of low doses of ethanol. Methods: Open field activity and spontaneous alternations were measured after saline or drug injection. [3H]MK-801 binding parameters were determined in hippocampus, cortex, striatum, and nucleus accumbens. Extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 region of hippocampal slices. Results: Systemic injection of either ethanol or MK-801 increased locomotor activity to a greater extent in ISS mice than in ILS mice. The competitive NMDAR antagonist 2-carboxypiperazin-4-yl-propyl-1,1phosphonic acid (±CPP) depressed activity of ILS, but not ISS, mice. No strain differences were observed in spontaneous alternations or in the number or affinity of NMDARs in the brain regions examined. Likewise, the magnitudes of hippocampal NMDAR-mediated fEPSPs were similar in ILS and ISS mice and were inhibited to the same extent by a competitive NMDAR antagonist. However, both ethanol and the NMDAR NR2B receptor antagonist ifenprodil inhibited the late component of hippocampal NMDAR fEPSPs to a greater extent in ISS, than in ILS, mice. Conclusions: Differential ethanol- and MK-801-induced behavioral activation in ILS and ISS mice was not associated with differences in NMDAR number. Nonetheless, pharmacological differences in hippocampal NMDAR responsiveness suggest that ISS mice express NMDARs that have a greater sensitivity to noncompetitive, but not competitive, NMDAR antagonists. These differences, which may reflect differences in NMDAR subunit composition, could underlie the differential responsiveness to low doses of ethanol in ILS and ISS mice. [source] Quantification of monomeric and polymeric wheat proteins and the relationship of protein fractions to wheat quality,JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 10 2003Jerry Suchy Abstract Wheat protein composition is important for understanding the biochemical basis of wheat quality. The objective of this study was to design a simple protein fractionation protocol with low cross-contamination and to show that these protein fractions were associated with wheat quality. The protocol consists of three sequential extractions from 100 mg of flour with 7.5% propan-1-ol and 0.3 M sodium iodide (monomeric-rich protein), 50% propan-1-ol (soluble glutenin-rich protein) and 40% propan-1-ol and 0.2% dithiothreitol (insoluble glutenin-rich protein). Nitrogen content of protein solubility groups was determined from dry residues using an automated combustion nitrogen analyser. About 90% of the total protein in the flour was solubilised. Cross-contamination of protein fractions was evaluated by SDS-PAGE, SE-HPLC and RP-HPLC. Variation in nitrogen content of the protein solubility fractions was lowest for monomeric-rich protein (<2%) and insoluble glutenin-rich protein (<4%). Three wheats with similar high-molecular-weight (HMW) glutenin subunit composition, Alpha 16, Glenlea and Roblin, varied significantly (P , 0.05) in the proportion of monomeric-rich and insoluble glutenin-rich protein in the flour. Dough rheological properties were directly related to the proportion of insoluble glutenin-rich protein and inversely related to the proportion of monomeric-rich protein. The protocol was validated using an expanded set of 11 wheats which also showed that inter-cultivar differences in the proportion of monomeric-rich, insoluble glutenin-rich protein and glutenin-to-gliadin ratio in the flour governed dough rheological properties such as mixograph, farinograph and microextension tests. The protocol has merit for quality screening in wheat-breeding programmes when the sample size is too small or when time constraints limit the ability to perform traditional rheological tests. For the Department of Agriculture and Agri-Food, Government of Canada, Copyright © Minister of Public Works and Government Services Canada 2003. Published for SCI by John Wiley & Sons, Ltd. [source] Remodeling protein complexes: Insights from the AAA+ unfoldase ClpX and Mu transposasePROTEIN SCIENCE, Issue 8 2005Briana M. Burton Abstract Multiprotein complexes in the cell are dynamic entities that are constantly undergoing changes in subunit composition and conformation to carry out their functions. The protein,DNA complex that promotes recombination of the bacteriophage Mu is a prime example of a complex that must undergo specific changes to carry out its function. The Clp/Hsp100 family of AAA+ ATPases plays a critical role in mediating such changes. The Clp/Hsp100 unfolding enzymes have been extensively studied for the roles they play in protein degradation. However, degradation is not the only fate for proteins that come in contact with the ATP-dependent unfolding enzymes. The Clp/Hsp100 enzymes induce structural changes in their substrates. These structural changes, which we refer to as "remodeling," ultimately change the biological activity of the substrate. These biological changes include activation, inactivation (not associated with degradation), and relocation within the cell. Analysis of the interaction between Escherichia coli ClpX unfoldase and the Mu recombination complex, has provided molecular insight into the mechanisms of protein remodeling. We discuss the key mechanistic features of the remodeling reactions promoted by ClpX and possible implications of these findings for other biological reactions. [source] Nested allosteric interactions in the cytoplasmic chaperonin containing TCP-1PROTEIN SCIENCE, Issue 2 2001Galit Kafri Abstract Initial rates of ATP hydrolysis by the chaperonin containing TCP-1 (CCT) from bovine testis were measured as a function of ATP concentration. Two allosteric transitions are observed: one at relatively low concentrations of ATP (<100 ,M) and the second at higher concentrations of ATP. The data suggest that CCT has positive intra-ring cooperativity and negative inter-ring cooperativity in ATP hydrolysis, with respect to ATP, as previously observed in the case of GroEL. It is shown that the relatively weak positive intra-ring cooperativity found in the case of CCT may be due to heterogeneity in its subunit composition. Our results suggest that nested allosteric behavior may be common to chaperone double-ring systems. [source] Proteomic and functional alterations in brain mitochondria from Tg2576 mice occur before amyloid plaque depositionPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 4 2007Frank Gillardon Dr. Abstract Synaptic dysfunction is an early event in Alzheimer's disease patients and has also been detected in transgenic mouse models. In the present study, we analyzed proteomic changes in synaptosomal fractions from Tg2576 mice that overexpress mutant human amyloid precursor protein (K670N, M671L) and from their nontransgenic littermates. Cortical and hippocampal tissue was microdissected at the onset of cognitive impairment, but before deposition of amyloid plaques. Crude synaptosomal fractions were prepared by differential centrifugation, proteins were separated by 2-D DIGE and identified by MS/MS. Significant alterations were detected in mitochondrial heat shock protein 70 pointing to a mitochondrial stress response. Subsequently, synaptosomal versus nonsynaptic mitochondria were purified from Tg2576 mice brains by density gradient centrifugation. Mitochondrial proteins were separated by IEF or Blue-native gel electrophoresis in the first dimension and SDS-PAGE in the second dimension. Numerous changes in the protein subunit composition of the respiratory chain complexes I and III were identified. Levels of corresponding mRNAs remain unchanged as shown by Affymetrix oligonucleotide array analysis. Functional examination revealed impaired state 3 respiration and uncoupled respiration in brain mitochondria from young Tg2576 mice. By immunoblotting, amyloid-beta oligomers were detected in synaptosomal fractions from Tg2576 mice and reduced glucose metabolism was observed in Tg2576 mice brains by [14C]-2-deoxyglucose infusion. Taken together, we demonstrate alterations in the mitochondrial proteome and function that occur in Tg2576 mice brains before amyloid plaque deposition suggesting that mitochondria are early targets of amyloid-beta aggregates. [source] Functional NR2B- and NR2D-containing NMDA receptor channels in rat substantia nigra dopaminergic neuronesTHE JOURNAL OF PHYSIOLOGY, Issue 1 2005Susan Jones NMDA receptors regulate burst firing of dopaminergic neurones in the substantia nigra pars compacta (SNc) and may contribute to excitotoxic cell death in Parkinson's disease (PD). In order to investigate the subunit composition of functional NMDA receptors in identified rat SNc dopaminergic neurones, we have analysed the properties of individual NMDA receptor channels in outside-out patches. NMDA (100 nm) activated channels corresponding to four chord conductances of 18, 30, 41 and 54 pS. Direct transitions were observed between all conductance levels. Between 18 pS and 41 pS conductance levels, direct transitions were asymmetric, consistent with the presence of NR2D-containing NMDA receptors. Channel activity in response to 100 nm or 200 ,m NMDA was not affected by zinc or TPEN (N,N,N,,N,-tetrakis-[2-pyridylmethyl]-ethylenediamine), indicating that SNc dopaminergic neurones do not contain functional NR2A subunits. The effect of the NR2B antagonist ifenprodil was complex: 1 ,m ifenprodil reduced open probability, while 10 ,m reduced channel open time but had no effect on open probability of channels activated by 100 nm NMDA. When the concentration of NMDA was increased to 200 ,m, ifenprodil (10 ,m) produced the expected reduction in open probability. These results indicate that NR2B subunits are present in SNc dopaminergic neurones. Taken together, these findings indicate that NR2D and NR2B subunits form functional NMDA receptor channels in SNc dopaminergic neurones, and suggest that they may form a triheteromeric NMDA receptor composed of NR1/NR2B/NR2D subunits. [source] The ,1 and ,6 subunit subtypes of the mammalian GABAA receptor confer distinct channel gating kineticsTHE JOURNAL OF PHYSIOLOGY, Issue 2 2004Janet L. Fisher The GABAA receptors show a large degree of structural heterogeneity, with seven different subunit families, and 16 different subtypes in mammalian species. The , family is the largest, with six different subtypes. The ,1 and ,6 subtypes are among the most diverse within this family and confer distinct pharmacological properties to recombinant and neuronal receptors. To determine whether different single channel and macroscopic kinetic properties were also associated with these subtypes, the ,1 or ,6 subunit was expressed in mammalian cells along with ,3 and ,2L subunits and the kinetic properties examined with outside-out patch recordings. The ,1,3,2L receptors responded to GABA with long-duration openings organized into multi-opening bursts. In contrast, channel openings of the ,6,3,2L receptors were predominately short in duration and occurred as isolated, single openings. The subunit subtype also affected the deactivation rate of the receptor, which was almost 2-fold slower for ,6,3,2L, compared with the ,1,3,2L isoform. Onset of fast desensitization did not differ between the isoforms. To determine the structural domains responsible for these differences in kinetic properties, we constructed six chimeric subunits, combining different regions of the ,1 and ,6 subunits. The properties of the chimeric subunits indicated that structures within the third transmembrane domain (TM3) and the TM3,TM4 intracellular loop conferred differences in single channel gating kinetics that subsequently affected the deactivation rate and GABA EC50. The effect of agonist concentration on the rise time of the current showed that the extracellular N-terminal domain was largely responsible for binding characteristics, while the transmembrane domains determined the activation rate at saturating GABA concentrations. This suggests that subunit structures outside of the agonist binding and pore-lining domains are responsible for the kinetic differences conferred by the ,1 and ,6 subtypes. Structural heterogeneity within these transmembrane and intracellular regions can therefore influence the characteristics of the postsynaptic response of GABAA receptors with different subunit composition. [source] Analysis of tannins in seeds and skins of Shiraz grapes throughout berry developmentAUSTRALIAN JOURNAL OF GRAPE AND WINE RESEARCH, Issue 1 2003MARK O. DOWNEY Abstract The flavan-3-ol and proanthocyanidin composition of both seeds and skin of Vitis vinifera L. cv. Shiraz grapes was determined by reversed-phase HPLC after acetone extraction and acid-catalysis in the presence of excess phloroglucinol. Samples were taken at weekly intervals from fruit-set until commercial harvest. The main period of proanthocyanidin accumulation in grape seeds occurred immediately after fruit-set with maximum levels observed around veraison. Over two seasons there was variation in both the timing and content of proanthocyanidins in seeds. In skin, proanthocyanidin accumulation occurred from fruit set until 1,2 weeks after veraison. Proanthocyanidin subunit composition was different in seeds and skin and changed during berry development but the mean degree of polymerisation of the tannin polymers in skins was higher than in the seeds at all stages of berry development. Proanthocyanidin levels in both seeds and skin decreased between veraison and harvest. Additional proanthocyanidin subunits were released when the residues remaining after acetone extraction were subjected to direct acid-catalysis in the presence of phloroglucinol. In the seeds, these accounted for much of the post-veraison decrease, but not in grape skin. At harvest, 75% of extractable berry proanthocyanidin was in the seeds. Accumulation of proanthocyanidins in the seeds appears to be independent of that in the skins, but in both tissues synthesis occurs early in berry development and maximum levels are reached around veraison. [source] Expression and structure of interleukin 4 receptors in primary meningeal tumorsCANCER, Issue 10 2005Sachin Puri M.Sc. Abstract BACKGROUND It was reported previously that malignant human tumors, like glioma and medulloblastoma, express high-density interleukin (IL-4) receptor mRNA and protein. Because IL-4 receptors (R) are sensitive targets for targeted therapeutics, knowledge of the expression of these receptors in other central nervous system tumors is of great interest. In this study, the authors examined the expression and subunit composition of IL-4R complex in primary human meningiomas. METHODS Reverse transcription-polymerase chain reaction (RT-PCR) analysis for IL-13R,1, IL-4R, and IL-2R,c was performed on total RNA extracted from 35 meningiomas and a normal human brain tissue sample. Results were confirmed in nine randomly selected tumors by quantitative real-time PCR and in situ immunofluorescence assay. RESULTS Transcripts for the IL-4R, and IL-13R,1 chains were overexpressed in meningiomas compared with normal brain tissue. The levels of IL-4R, mRNA appeared to be higher compared with the levels of IL-13R,1 mRNA. The results also showed that tumors with higher disease grade tended to have increased mRNA expression for the IL-4R, chain. This IL-4R, mRNA overexpression appeared to be more frequent in younger patients (age < 37 years). The transcripts for IL-2R,c chain were not detected in any of the tumor samples or in normal brain tissue. Quantitative real-time PCR confirmed the results of the RT-PCR analysis. Meningiomas also demonstrated a bright immunofluorescent staining for the IL-4R, and IL-13R,1 chains but no staining for IL-2R,c. CONCLUSIONS Expression of the IL-4R, and IL-13R,1 chains and absence of IL-2,c expression established that meningiomas expressed type II IL-4Rs. These receptors may serve as a target for cytotoxin/immunotoxin therapy in patients with meningioma who are not amenable to surgical resection or for recurrent tumors. Cancer 2005. © 2005 American Cancer Society. [source] Central Nicotinic Receptors: Structure, Function, Ligands, and Therapeutic PotentialCHEMMEDCHEM, Issue 6 2007Novella Romanelli Abstract The growing interest in nicotinic receptors, because of their wide expression in neuronal and non-neuronal tissues and their involvement in several important CNS pathologies, has stimulated the synthesis of a high number of ligands able to modulate their function. These membrane proteins appear to be highly heterogeneous, and still only incomplete information is available on their structure, subunit composition, and stoichiometry. This is due to the lack of selective ligands to study the role of nAChR under physiological or pathological conditions; so far, only compounds showing selectivity between ,4,2 and ,7 receptors have been obtained. The nicotinic receptor ligands have been designed starting from lead compounds from natural sources such as nicotine, cytisine, or epibatidine, and, more recently, through the high-throughput screening of chemical libraries. This review focuses on the structure of the new agonists, antagonists, and allosteric ligands of nicotinic receptors, it highlights the current knowledge on the binding site models as a molecular modeling approach to design new compounds, and it discusses the nAChR modulators which have entered clinical trials. [source] Distinctive nicotinic acetylcholine receptor functional phenotypes of rat ventral tegmental area dopaminergic neuronsTHE JOURNAL OF PHYSIOLOGY, Issue 2 2009Kechun Yang Dopaminergic (DAergic) neuronal activity in the ventral tegmental area (VTA) is thought to contribute generally to pleasure, reward, and drug reinforcement and has been implicated in nicotine dependence. nAChRs expressed in the VTA exhibit diverse subunit compositions, but the functional and pharmacological properties are largely unknown. Here, using patch-clamp recordings in single DAergic neurons freshly dissociated from rat VTA, we clarified three functional subtypes of nAChRs (termed ID, IID and IIID receptors) based on whole-cell current kinetics and pharmacology. Kinetic analysis demonstrated that comparing to ID, IID receptor-mediated current had faster activation and decay constant and IIID receptor-mediated current had larger current density. Pharmacologically, ID receptor-mediated current was sensitive to the ,4,2-nAChR agonist RJR-2403 and antagonist dihydro-,-erythroidine (DH,E); IID receptor-mediated current was sensitive to the selective ,7-nAChR agonist choline and antagonist methyllycaconitine (MLA); while IIID receptor-mediated current was sensitive to the ,4-containing nAChR agonist cytisine and antagonist mecamylamine (MEC). The agonist concentration,response relationships demonstrated that IID receptor-mediated current exhibited the highest EC50 value compared to ID and IIID receptors, suggesting a relatively low agonist affinity of type IID receptors. These results suggest that the type ID, IID and IIID nAChR-mediated currents are predominately mediated by activation of ,4,2-nAChR, ,7-nAChR and a novel nAChR subtype(s), respectively. Collectively, these findings indicate that the VTA DAergic neurons express diversity and multiplicity of functional nAChR subtypes. Interestingly, each DAergic neuron predominantly expresses only one particularly functional nAChR subtype, which may have distinct but important roles in regulation of VTA DA neuronal function, DA transmission and nicotine dependence. [source] RIC-3 and nicotinic acetylcholine receptors: Biogenesis, properties, and diversityBIOTECHNOLOGY JOURNAL, Issue 12 2008Millet Treinin Dr.Article first published online: 27 OCT 200 Abstract Nicotinic acetylcholine receptors (nAChRs) belong to a diverse and widely expressed family of ion channels. These receptors are pentamers assembled from multiple combinations of subunits, with different subunit compositions producing receptors having different properties and functions. The diverse functions of nAChRs include an essential role in excitation of skeletal muscles and many modulatory roles throughout the central nervous system. Nicotinic receptors are also implicated in a number of brain pathologies such as epilepsy, schizophrenia, and Alzheimer's disease. Thus, it is important to understand the cellular mechanisms controlling both the numbers and the properties of surface expressed nAChRs. Genetic analysis in Caenorhabditis elegans identified a number of proteins specifically needed for biogenesis of nAChRs. Among these proteins is RIC-3, a member of a family of proteins having conserved structure and function. RIC-3 influences both surface expression and properties of nAChRs and its effects are subtype specific. Here we suggest that receptor-specific chaperones such as RIC-3 may play important roles in controlling receptor diversity by selectively regulating surface expression of nAChRs having specific subunit compositions. [source] | ||||||||||||||||