Genome-wide evaluation and discovery of vertebrate A-to-I RNA editing sites.

RNA editing by adenosine deamination, catalyzed by adenosine deaminases acting on RNA (ADAR), is a post-transcriptional modification that contributes to transcriptome and proteome diversity and is widespread in mammals. Here we administer a bioinformatics search strategy to the human and mouse genomes to explore the landscape of A-to-I RNA editing. In both organisms we find evidence for high excess of A/G-type discrepancies (inosine appears as a guanosine in cloned cDNA) at non-polymorphic, non-synonymous codon sites over other types of discrepancies, suggesting the existence of several thousand recoding editing sites in the human and mouse genomes. We experimentally validate recoding-type A-to-I RNA editing in a number of human genes with high scoring positions including the coatomer protein complex subunit alpha (COPA) as well as cyclin dependent kinase CDK13.

On the influence of pore structure on the free-imbibition of sessile drops into nanoporous substrates.

We report here a model experimental study on the influence of pore structure on the free-imbibition of sessile drops into nanoporous substrates. The work takes advantage of the existence of distinct pore structures on the two sides of a nanoporous alumina membrane: straight parallel channels versus a denser and tortous network. We show first that the spreading which coexists with the free-imbibition predominates in the early stage well follows on both sides the power-law scaling with time predicted by the universal Tanner's law. More interestingly, we found also that the imbibition rate scales in a similar way with the time on both sides of the membrane, showing that the pore structure does not affect qualitatively the free-imbibition kinetics. On the other hand, our results clearly show that the pore structure has a quantitative impact on the imbibition rate, which increases markedly from the A side (dense network of short and tortuous pores) to the side B (straight vertical channels). This latter result shows that, as regards the free-imbibition, the topology of the pores has a preeminent impact on their volume, which is here comparable for both sides of the membrane. More unexpectedly, this quantitative impact of the pore structure on the imbibition rate seems to display a certain sensitivity to the viscosity of the liquid.

Wetting-induced formation of quasiperiodical wrinkling patterns in alginate-based coatings.

We present the results of systematic model experimental investigations on wrinkling instabilities which develop on alginate-based coatings when they are wetted by swelling electrolyte drops. The wrinkles first appear randomly within the wet spot, before they selectively protrude out around the periphery in a quasiperiodical wrinkle pattern. We discuss the critical parameters that drive the emergence (ionic strength and swelling rate) and spatial feature (size and periodicity) of these swelling-induced structures on such complex functional coatings. Beyond their relative aesthetics and their fundamental interest related to morphological instabilities, these reconstruction structures which are invisible to the naked eye can develop in a variety of technological processes (inkjet printing for instance), affecting irreversibly the quality of the products.

Precision and accuracy of joint space width measurements of the medial compartment of the knee using standardized MTP semi-flexed radiographs.

OBJECTIVE: To quantify the precision and accuracy of measurements of joint space width (JSW) and joint space narrowing (JSN) from the medial tibiofemoral compartment of knee radiographs using a simple and easily adaptable protocol. METHODS: Radiographs of a caliper (a surrogate for JSW) were obtained to determine the precision limits of the system under ideal conditions. Bilateral knee radiographs from 10 healthy volunteers were obtained at three different times using the metatarsophalangeal (MTP) semi-flexed view posterior-anterior position without fluoroscopy. A backlit digitizing tablet and three manual methods were used to measure JSW and analyses of precision were performed. The accuracy of measuring change in JSW (a measure of JSN) was estimated from radiographs of cadaver knees that were placed in a servo-hydraulic device that moved the femur relative to the tibia through known intervals. RESULTS: Radiographic measurements of the caliper inter-blade distance were comparable to the resolution limits of the backlit digitizing tablet (0.025 mm). Repeated radiography of healthy subject knees produced JSW standard deviation (SD) measurements of 0.08 mm by the median SD method, and 0.11 mm by repeated measures analysis. The accuracy of JSN measurements in the cadaver knees as a mean difference from the known reference value was 0.09 mm. CONCLUSION: The results indicate a high level of precision in measurements of JSW from MTP semi-flexed view knee radiographs of normal volunteers. Reproducibility was attained through careful subject positioning without fluoroscopy and the use of a backlit digitizing tablet. From the cadaver study we can predict that greater than 0.13 mm of measured JSN represents actual or true change in JSN. This radiographic technique can be used as a primary measure for early knee osteoarthritis (OA) when cartilage thickness is decreasing and limited bony remodeling has occurred.

Marginally specified generalized linear mixed models: a robust approach.

Longitudinal data modeling is complicated by the necessity to deal appropriately with the correlation between observations made on the same individual. Building on an earlier nonrobust version proposed by Heagerty (1999, Biometrics 55, 688-698), our robust marginally specified generalized linear mixed model (ROBMS-GLMM) provides an effective method for dealing with such data. This model is one of the first to allow both population-averaged and individual-specific inference. As well, it adopts the flexibility and interpretability of generalized linear mixed models for introducing dependence but builds a regression structure for the marginal mean, allowing valid application with time-dependent (exogenous) and time-independent covariates. These new estimators are obtained as solutions of a robustified likelihood equation involving Huber's least favorable distribution and a collection of weights. Huber's least favorable distribution produces estimates that are resistant to certain deviations from the random effects distributional assumptions. Innovative weighting strategies enable the ROBMS-GLMM to perform well when faced with outlying observations both in the response and covariates. We illustrate the methodology with an analysis of a prospective longitudinal study of laryngoscopic endotracheal intubation, a skill that numerous health-care professionals are expected to acquire. The principal goal of our research is to achieve robust inference in longitudinal analyses.

An overview on functional receptor autoradiography using [35S]GTPgammaS.

[35S]GTPgammaS binding autoradiography is a novel method to study the distribution and function of neurotransmitter receptors in tissue sections. This technique unifies the advantages of receptor-autoradiography and [35S]GTPgammaS binding, providing anatomical and functional information at the same time. Due to these two main features, it can also be called 'functional autoradiography'. [35S]GTPgammaS binding has long been used to study the first step of the intracellular signaling pathway, but until the mid 1990s it has only been performed on cell membrane extracts. Functional autoradiography evolved from this biochemical assay and ligand autoradiography, and is based on the increase in guanine nucleotide exchange at G-proteins upon agonist stimulation. With the technique, activation of G-protein-coupled receptors upon agonist binding can be detected, and, at the same time, the location of activated receptors can also be visualized. Thus only those presumably active G-protein-coupled receptors are visualized that can be involved in signal transduction. In the past 5 years the technique has become more and more frequently used in neuroscience, and it has been adapted to several receptors in different species, including also the human brain. [35S]GTPgammaS binding autoradiography can be used to describe the distribution of G-protein-coupled receptors. Some inferences on their coupling efficiency can also be drawn. Besides the localization of ligand binding sites, it provides information on the action of the ligand on the receptor: agonists, antagonists, and inverse agonists can clearly be distinguished. Moreover, [35S]GTPgammaS binding autoradiography can successfully be combined with other in vitro assays, like receptor autoradiography, in situ hybridization histochemistry, or even with biochemical and electrophysiological experiments. This review presents an overview on the history and the development of this technique. Its main advantages and limitations are summarized, together with a few basic technical questions. A number of experiments performed with [35S]GTPgammaS binding autoradiography so far, and some possible applications for the future, are also reviewed.

Modulation of 5-HT(1A) receptor activation by its interaction with wild-type and mutant g(alphai3) proteins.

Constitutive and agonist-dependent activation of the recombinant human 5-HT(1A) receptor (RC: 2.1.5HT.01A) was investigated by co-expression with a rat G(alphai3) protein in Cos-7 cells. The interaction between the 5-HT(1A) receptor and rat G(alphai3) protein was modulated by substitution of the G(alphai3) protein site for pertussis toxin-catalysed ADP-ribosylation (cysteine(351)) by each of the natural amino acids. Enhanced basal [(35)S]GTPgammaS binding responses (+24 to +189%) were observed with the mutant G(alphai3) proteins containing at position 351 either a histidine, glutamine, serine, tyrosine or a nonpolar amino acid with the exception of a proline. With each of these mutant G(alphai3) proteins, spiperone (10 microM), but not WAY 100635 (10 microM), reduced (-22 to -60%, p<0.05) the enhanced basal [(35)S]GTPgammaS binding response. 5-HT (10 microM)-mediated [(35)S]GTPgammaS binding responses attained for some of the mutant G(alphai3)Cys(351) proteins (Phe, Met, Val and Ala) more than 300% of that obtained with the wt G(alphai3) protein. Similar results were also obtained with the prototypical 5-HT(1A) agonist 8-OH-DPAT and the partial agonist (-)-pindolol. Fusion proteins assembled from the 5-HT(1A) receptor and either the wt G(alphai3)Cys(351), mutant G(alphai3)Cys(351)Gly or G(alphai3)Cys(351)Ile protein displayed similar observations for these ligands as obtained by co-expression of the 5-HT(1A) receptor with each of these G(alphai3) proteins. Both the degree of 5-HT(1A) receptor activation by 8-OH-DPAT and (-)-pindolol, and its inhibition by spiperone, strongly correlate (r(2): 0.78-0.81) with the octanol/water partition coefficients of the mutated amino acid at position 351 of the G(alphai3) protein. The present data also suggest the wt G(alphai3) protein does not result in maximal activation of the 5-HT(1A) receptor by the agonists being investigated.

Memory of an operant response and of depressed mood retained in activation states of 5-HT(1A) receptors: evidence from rodent models.

Three series of studies were conducted to specify the role of 5-HT(1A) receptors in memory; using selective ligands that differentially activate 5-HT(1A) receptors, it was determined whether a change in the activation state of these receptors can lead to deficient retrieval, and whether a so-produced deficit can occur in an animal model of depression. First, in vitro studies of [35S]GTPgammaS binding responses identified ligands that differentially activate 5-HT(1A) receptors in rat hippocampus. WAY 100635, 8-OH-DPAT and flesinoxan induced 5-HT(1A) receptor activation that amounted to -2, +50 and +63%, respectively, of that produced by 5-HT. Second, we determined whether changes in the activation state of 5-HT(1A) receptors could impair the retrieval of an operant response in vivo. Rats treated with either a 5-HT(1A) receptor ligand or saline were trained to lever press for milk reward, and were then tested for retrieval with either the same or another treatment. Animals trained with 8-OH-DPAT retrieved the response when tested in the same state, but not when tested in the saline state, and vice versa. Rats trained with 0.16 mg/kg of 8-OH-DPAT also retrieved the response when tested with the other intermediate-efficacy ligand flesinoxan (0.63 mg/kg), but not when tested in a state of lower-magnitude activation (i.e. with 0.16 mg/kg of WAY 100635). Animals trained with 0.16 mg/kg of WAY 100635 retrieved the response when tested in this same state or with saline, but not when tested in a state of intermediate-magnitude activation (i.e. with 0.16 mg/kg of 8-OH-DPAT). Finally, studies using the forced swimming paradigm indicated that the retrieval of learned immobility was similarly dependent upon the activation state of 5-HT(1A) receptors. The findings indicate that changes in activation states of 5-HT(1A) receptors can impair the retrieval of learned responses. It is suggested that depression may in part be acquired in the course of ontogeny and may be available for retrieval in the same but not in other states; various biological rhythms conceivably define such states.

Kinetic differences at the single molecule level account for the functional diversity of rabbit cardiac myosin isoforms.

1. Cardiac V3 myosin generates slower actin filament velocities and higher average isometric forces (in an in vitro motility assay) when compared with the V1 isoform. 2. To account for differences in V1 and V3 force and motion generation at the molecular level, we characterized the mechanics and kinetics of single V1 and V3 myosin molecules using a dual laser trap setup. 3. No differences in either unitary displacement (approximately 7 nm) or force (approximately 0.8 pN) were observed between isoforms; however, the duration of unitary displacement events was significantly longer for the V3 isoform at MgATP concentrations > 10 microM. 4. Our results were interpreted on the basis of a cross-bridge model in which displacement event durations were determined by the rates of MgADP release from, and MgATP binding to, myosin. 5. We propose that the release rate of MgADP from V3 myosin is half that of V1 myosin without any difference in their rates of MgATP binding; thus, kinetic differences between the two cardiac myosin isoforms are sufficient to account for their functional diversity.

Modulation of 5-HT1A receptor signalling by point-mutation of cysteine351 in the rat Galpha(o) protein.

The activity state of G proteins is involved in the ligands' maximal responses that can be produced by activating the 5-HT1A receptor (Pauwels et al., 1997). The present study investigated the ligand responses at the recombinant h 5-HT1A receptor (RC: 2.1.5HT.01A) as mediated by the Galpha(o) protein. Therefore, a fusion protein was constructed between the 5-HT1A receptor and a pertussis toxin resistant rat Galpha(o)Cys351Gly mutant protein to define its pharmacological properties at a receptor: Galpha(o) protein density ratio of 1. Pertussis toxin treatment (100 ng/ml) affected neither the expression of the 5-HT1A receptor fusion protein as measured by [3H] MPPF (3.0+/-0.7 pmol/mg protein) nor the 5-HT-mediated [35S]GTPgammaS binding response (146+/-34 fmol/mg protein) in Cos-7 cells. 8-OH-DPAT (Emax: 55+/-7%) and buspirone (Emax: 22+/-4%) yielded partial agonist activity as compared to 5-HT, whereas WAY 100635 acted as a competitive antagonist (pK(B): 9.75+/-0.17). The magnitude of the 8-OH-DPAT response (Emax, %) was highly dependent on the nature of the amino acid 351 in the C-terminus of the Galpha(o) protein: Ile351 (93+/-4) > Cys351 (79+/-3) > Gly351 (55+/-7). The Emax values (%) of buspirone displayed the following gradient: 69+/-5 approximately/= 62+/-8 > 22+/-4. For comparison, maximal responses of 8-OH-DPAT and buspirone were enhanced versus 5-HT upon co-expression of the 5-HT1A receptor with the respective Galpha(o) proteins, probably due to an altered receptor: Galpha(o) protein density ratio. In conclusion, residue 351 of the rat Galpha(o) protein is involved in determining the magnitude of 5-HT1A receptor activation that ligands can produce at these receptors. Moreover, the fusion protein approach allows quantitative comparisons of the intrinsic activities of ligands between one single receptor subtype with different Galpha protein subtypes.

Autoradiographic studies of 5-HT1A-receptor-stimulated [35S]GTPgammaS-binding responses in the human and monkey brain.

G-protein activation mediated by serotonin 5-HT1A receptors in human and monkey brain was investigated by using quantitative autoradiography of agonist-stimulated [35S]GTPgammaS binding to whole-hemisphere brain sections. [35S]GTPgammaS binding was stimulated by the mixed 5-HT1A/1B/1D agonist L 694247 (10 microm) in human brain regions enriched in 5-HT1A binding sites [e.g. hippocampus (132-137%), superficial layers of the neocortex (37-61%), and cingulate and entorhinal cortex (34 and 32%, respectively)]. L 694247 caused virtually no stimulation in regions with 5-HT1B/1D receptors, such as substantia nigra, caudate nucleus and putamen. Similar results were obtained with monkey brain sections. The L 694247-mediated [35S]GTPgammaS-binding responses in human and monkey brain sections were antagonized by the selective, silent 5-HT1A antagonist WAY 100635 (10 microm). The 5-HT1B inverse agonist SB 224289 (10 microm) did not affect the [35S]GTPgammaS-binding response of L 694247. The distribution pattern of the [35S]GTPgammaS-binding response and the antagonist profile suggest the L 694247-induced response in human and monkey brain is mediated by 5-HT1A receptors. A weak stimulation of [35S]GTPgammaS binding was also observed in human hippocampus with either 10 microm 8-OH-DPAT (25 +/- 4%) or naratriptan (42 +/- 2%) compared with that obtained with L 694247. In conclusion, G-protein activation by 5-HT1A receptors can be measured in human and monkey brain sections. L 694247 appears to possess higher efficacy at 5-HT1A receptors compared with 8-OH-DPAT and naratriptan.

Two heads of myosin are better than one for generating force and motion.

Several classes of the myosin superfamily are distinguished by their "double-headed" structure, where each head is a molecular motor capable of hydrolyzing ATP and interacting with actin to generate force and motion. The functional significance of this dimeric structure, however, has eluded investigators since its discovery in the late 1960s. Using an optical-trap transducer, we have measured the unitary displacement and force produced by double-headed and single-headed smooth- and skeletal-muscle myosins. Single-headed myosin produces approximately half the displacement and force (approximately 6 nm; 0.7 pN) of double-headed myosin (approximately 10 nm; 1.4 pN) during a unitary interaction with actin. These data suggest that muscle myosins require both heads to generate maximal force and motion.

Magnitude of 5-HT1B and 5-HT1A receptor activation in guinea-pig and rat brain: evidence from sumatriptan dimer-mediated [35S]GTPgammaS binding responses.

The present study reports on G-protein activation by recombinant 5-HT receptors and by native 5-HT1A and 5-HT1B receptors in guinea-pig and rat brain using agonist-stimulated [35S]GTPgammaS binding responses mediated by a new 5-HT ligand, a dimer of sumatriptan. Dimerization of sumatriptan increased the binding affinity for h 5-HT1B (pKi: 9.22 vs. 7.79 for sumatriptan), h 5-HT1D (9.07 vs. 8.08) and also h 5-HT1A receptors (7.80 vs. 6.40), while the binding affinity for h 5-ht1E (6.67 vs. 6.19) and h 5-ht1F (7.37 vs. 7.78) receptors was not affected. Sumatriptan dimer (10 microM) stimulated [35S]GTPgammaS binding mainly in the superficial gray layer of the superior colliculi, hippocampus and substantia nigra of guinea-pig and rat coronal brain sections. This fits with the labelling by the 5-HT1B/1D receptor antagonist [3H] GR 125743. The observed [35S]GTPgammaS binding responses in the substantia nigra are likely to be mediated by stimulation of the 5-HT1B receptor subtype, since they were antagonized by the 5-HT1B inverse agonist SB 224289 (10 microM), and not by the 5-HT2A/1D antagonist ketanserin (10 microM). Quantitative assessment of the [35S]GTPgammaS binding responses in the substantia nigra of rat showed highly efficacious responses for both sumatriptan dimer and its monomer. In contrast, less efficacious agonist responses (51+/-10% and 35+/-13%, respectively) were measured in the guinea-pig substantia nigra. This may suggest that the G-protein coupling efficacy of 5-HT1B receptors is different between the substantia nigra of both species. In addition, the sumatriptan dimer also activated guinea-pig and rat hippocampal 5-HT1A receptors with high efficacy in contrast to sumatriptan. Therefore, dimerization of sumatriptan can be considered as a new approach to transform a partial 5-HT1A agonist into a more efficacious agonist. In conclusion, the sumatriptan dimer stimulates G-protein activation via 5-HT1B receptors besides 5-HT1A receptors in guinea-pig and rat brain. The magnitude of the 5-HT1B receptor responses is superior for sumatriptan and its dimer in rat compared to guinea-pig substantia nigra.

Clinical measurement of the static rear stability of occupied wheelchairs.

OBJECTIVE: To evaluate a new clinical test, platform testing of the static rear stability of wheelchairs occupied by their users (using methods adapted from the International Organization for Standardization [ISO]), from the perspective of its measurement properties, safety, and comfort. DESIGN: Within-subject comparisons. SETTING: Rehabilitation center. PATIENTS: Ninety-seven wheelchair users. MAIN OUTCOME MEASURES: Static stability (with the brakes locked and unlocked, the occupant leaning forward and back, and with antitip devices in place), dynamic stability (the criterion measure), reliability, validity, sensitivity, specificity, predictive values, and likelihood ratios. RESULTS: Test-retest reliabilities (n = 18 to 24) were all >.93. The test's construct validity was demonstrated by the finding that static stability was appropriately affected by locking the brakes, body position, and antitip devices (p < .0001). Spearman's rank correlations between static and dynamic stability ranged from .29 to .65. Sensitivity ranged from 46% to 85%, specificity from 59% to 78%, positive predictive values from 76% to 86%, negative predictive values from 42% to 69%, positive likelihood ratios from 1.56 to 2.95, and negative likelihood ratios from .22 to .71. There were no adverse events, and the subjects tolerated the tests well. CONCLUSIONS: In the clinical setting, the ISO platform test of static rear stability has good to excellent measurement properties, is safe, and is well tolerated. Static-stability testing in this setting should be performed in the context of a comprehensive evaluation of wheelchair safety and performance.

Dimerization of 8-OH-DPAT increases activity at serotonin 5-HT1A receptors.

[35S]GTPgammaS binding responses can be used to measure differences between the intrinsic activity of ligands at human 5-hydroxytrypamine-(1A) (h 5-HT1A) receptors expressed in recombinant cell lines. The maximal [35S]GTPgammaS binding response to 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was lower than that to 5-HT in a recombinant C6-glial membrane preparation and dependent on the GDP concentration: it was attenuated by about 60% vs 5-HT by increasing the concentration of GDP from 0.3 to 30 and 300 microM. Whereas dimerization of 8-OH-DPAT almost did not affect its potency at h 5-HT1A receptors (pEC50: 7.45 and 7.40 for 8-OH-DPAT and its dimer at 30 microM GDP), it increased efficacy at h 5-HT1A receptors. The maximal response to the 8-OH-DPAT dimer was systematically greater than the response to 8-OH-DPAT and identical to that to 5-HT; moreover in contrast to the 8-OH-DPAT monomer, the maximal response to the dimer was unaffected by increasing the GDP concentration. An enhanced 135S]GTPgammaS binding response (44 to 63% vs 8-OH-DPAT) was also observed in the hippocampus, lateral septum, dorsal raphe and cingulate cortex of guinea-pig brain sections using autoradiography of 5-HT1A receptor-activated G-proteins. Hence, the 8-OH-DPAT dimer shows increased efficacy at 5-HT1A receptors compared to 8-OH-DPAT. The differential regulation of the maximal agonist responses by GDP suggests that the [35S]GTPgammaS binding responses to these two ligands could be mediated by different G-protein subtypes upon activation of the 5-HT1A receptor.

Investigation of the secondary DNA-binding site of the bacterial recombinase RecA.

The L2 loop is a DNA-binding site of RecA protein, a recombinase from Eschericha coli. Two DNA-binding sites have been functionally defined in this protein. To determine whether the L2 loop of RecA protein is part of the primary or secondary binding site, we have constructed proteins with site-specific mutations in the loop and investigated their biological, biochemical, and DNA binding properties. The mutation E207Q inhibits DNA repair and homologous recombination in vivo and prevents DNA strand exchange in vitro (Larminat, F., Cazaux, C., Germanier, M., and Defais, M. (1992) J. Bacteriol. 174, 6264-6269; Cazaux, C., Larminat, F., Villani, G., Johnson, N. P., Schnarr, M., and Defais, M. (1994) J. Biol. Chem. 269, 8246-8254). We have found that mutant protein RecAE207Q lacked one of the two single stranded DNA-binding sites of wild type RecA. The remaining site was functional, and biochemical activities of the mutant protein were the same as wild type RecA with ssDNA in the primary binding site. The second mutation, E207K, reduced but did not eliminate DNA repair, SOS induction, and homologous recombination in vivo. In the presence of ATP, mutant protein RecAE207K catalyzed DNA strand exchange in vitro at a slower rate than wild type protein, and ssDNA binding at site I was competitively inhibited. These results show that the L2 loop is or is part of the functional secondary DNA-binding site of RecA protein.

Interaction of 5-HT1B/D ligands with recombinant h 5-HT1A receptors: intrinsic activity and modulation by G-protein activation state.

Many 5-HT1B/D receptor ligands have affinity for 5-HT1A receptors. In the present study, the intrinsic activity of a series of 5-HT1B/D ligands was investigated at human 5-HT1A (h 5-HT1A) receptors by measuring G-protein activation in recombinant C6-glial and HeLa membranes, using agonist-stimulated [35S]GTPgammaS binding. In these two membrane preparations, the density of h 5-HT1A receptors (i.e., 246 to 320 fmol mg(-1) protein) and of their G-proteins, and the receptor: G-protein density ratio (0.08 to 0.18) appeared to be similar. It was found that: (i) the maximal [35S]GTPgammaS binding responses induced by the 5-HT1B/D receptor ligands in the HeLa preparation at 30 microM GDP were comparable to that of the native agonist 5-HT; (ii) as compared to 5-HT (1.00), similar potencies but lower maximal responses were observed in the C6-glial preparation at 0.3 microM GDP for zolmitriptan (0.89), dihydroergotamine (0.81), rizatriptan (0.71), CP122638 (0.69), naratriptan (0.60) and sumatriptan (0.53); and that (iii) maximal [35S]GTPgammaS binding responses induced by 5-HT1B/D ligands in the C6-glial preparation were either unaffected or significantly enhanced by increasing the GDP concentration from 0.3 to 30 microM and higher concentrations. These features differ from those observed with 5-HT1A receptor agonists; the latter display the same rank order of potency and efficacy in both membrane preparations, and increasing the amount of GDP with C6-glial membranes results in an attenuation of both the agonist's maximal effect and the apparent potency of partial agonists. The differential regulation of 5-HT1A and 5-HT1B/D agonist responses by GDP suggests that different G-protein subtypes are involved upon 5-HT1A receptor activation by 5-HT1A and 5-HT1B/D agonists.

G-protein activation at 5-HT1A receptors by the 5-ht1F ligand LY334370 in guinea-pig brain sections and recombinant cell lines.

1. G-protein activation by the 5-ht1F receptor agonist 5-(4-fluorobenzoyl)amino-3-(1-methylpiperidin-4-yl)-1H-indole fumarate (LY334370) was investigated by use of autoradiography of receptor-activated G-proteins in guinea-pig brain sections and [35S]-GTPgammaS binding responses in cell lines stably expressing human 5-HT1A (h 5-HT1A) receptors. 2. LY334370 (10 microM) caused little or no stimulation of [35S]-GTPgammaS binding in guinea-pig brain regions enriched in 5-ht1F binding sites (e.g., claustrum, caudate/putamen and thalamic nuclei), as identified by labelling with 10 nM [3H]-sumatriptan plus 10 nM 5-carboxamidotryptamine (5-CT). 3. Application of LY334370 (10 microM) to guinea-pig brain sections resulted in an increase of [35S]-GTPgammaS binding in hippocampus (123+/-17%), lateral septum (58+/-14%), dorsal raphe (57+/-10%), entorhinal (37+/-11%) and cingulate cortex (28+/-10%). This distribution fits with the G-protein activation mediated by 5-HT1A receptors as found with lisuride (10 microM), and labelling of 5-HT1A receptors by 140 pM [125I]-4-(2'-methoxy-phenyl)- -[2'-(n-2"-pyridinyl)-p-iodobenzamido]-ethyl-piperazine (p-MPPI). 4. The LY334370-mediated [35S]-GTPgammaS response was antagonized by the selective, silent 5-HT1A receptor antagonist N-[2-[4-(2-methoxyphenyl)1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohex anecarboxa-mide (WAY100635, 1 microM) in each of the brain structures investigated. The distribution pattern of the [35S]-GTPgammaS binding response and the antagonist profile suggest that the LY334370-induced response in guinea-pig brain is mediated by 5-HT1A receptors. 5. The maximal LY334370-induced [35S]-GTPgammaS binding response (83 to 94%) in membranes of recombinant C6-glial/h 5-HT1A and HeLa/h 5-HT1A cells was close to that of 5-HT, suggesting LY334370 to exert high intrinsic activity at h 5-HT1A receptors. 6. In conclusion, in guinea-pig brain sections and recombinant cell lines the 5-ht1F receptor agonist LY334370 causes G-protein activation that is mediated by 5-HT1A receptors. Caution should be taken when employing this ligand as a putative selective 5-ht1F agonist.

Autoradiography of serotonin 5-HT1A receptor-activated G proteins in guinea pig brain sections by agonist-stimulated [35S]GTPgammaS binding.

G protein activation mediated by serotonin 5-HT1A and 5-HT(1B/D) receptors in guinea pig brain was investigated by using quantitative autoradiography of agonist-stimulated [35S]GTPgammaS binding to brain sections. [35S]GTPgammaS binding was stimulated by the mixed 5-HT1A/5-HT(1B/D) agonist L694247 in brain structures enriched in 5-HT1A binding sites, i.e., hippocampus (+140 +/- 14%), dorsal raphe (+70 +/- 8%), lateral septum (+52 +/- 12%), cingulate (+36 +/- 8%), and entorhinal cortex (+34 +/- 5%). L694247 caused little or no stimulation of [35S]GTPgammaS binding in brain regions with high densities of 5-HT(1B/D) binding sites (e.g., substantia nigra, striatum, central gray, and dorsal subiculum). The [35S]GTPgammaS binding response was antagonized by WAY100635 (10 microM) and methiothepin (10 microM). In contrast, the 5-HT1B inverse agonist SB224289 (10 microM) did not affect the L694247-mediated [35S]GTPgammaS binding response, and the mixed 5-HT(1B/D) antagonist GR127935 (10 microM) yielded a partial blockade. The distribution pattern of the [35S]GTPgammaS binding response and the antagonist profile suggest the L694247-mediated response in guinea pig brain to be mediated by 5-HT1A receptors. In addition to L694247, 8-hydroxy-2-(di-n-propylamino)tetralin, and flesinoxan also stimulated [35S]GTPgammaS binding; their maximal responses varied between 46 and 52% compared with L694247, irrespective of the brain structure being considered. Sumatriptan, rizatriptan, and zolmitriptan (10 microM) stimulated [35S]GTPgammaS binding in the hippocampus by 20-50%. Naratriptan, CP122638, and dihydroergotamine stimulated [35S]GTPgammaS binding to a similar level as L694247 in hippocampus, lateral septum, and dorsal raphe. It appears that under the present experimental conditions, G protein activation through 5-HT1A but not 5-HT(1B/D) receptors can be measured in guinea pig brain sections.

Smooth muscle and skeletal muscle myosins produce similar unitary forces and displacements in the laser trap.

Purified smooth muscle myosin in the in vitro motility assay propels actin filaments at 1/10 the velocity, yet produces 3-4 times more force than skeletal muscle myosin. At the level of a single myosin molecule, these differences in force and actin filament velocity may be reflected in the size and duration of single motion and force-generating events, or in the kinetics of the cross-bridge cycle. Specifically, an increase in either unitary force or duty cycle may explain the enhanced force-generating capacity of smooth muscle myosin. Similarly, an increase in attached time or decrease in unitary displacement may explain the reduced actin filament velocity of smooth muscle myosin. To discriminate between these possibilities, we used a laser trap to measure unitary forces and displacements from single smooth and skeletal muscle myosin molecules. We analyzed our data using mean-variance analysis, which does not rely on scoring individual events by eye, and emphasizes periods in the data with constant properties. Both myosins demonstrated multiple but similar event populations with discrete peaks at approximately +11 and -11 nm in displacement, and 1.5 and 3.5 pN in force. Mean attached times for smooth muscle myosin were longer than for skeletal-muscle myosin. These results explain much of the difference in actin filament velocity between these myosins, and suggest that an increased duty cycle is responsible for the enhanced force-generating capacity of smooth over skeletal-muscle myosin.