Constraining the Mass of the Graviton with the Planetary Ephemeris INPOP.

We use the planetary ephemeris INPOP17b to constrain the existence of a Yukawa suppression to the Newtonian potential, generically associated with the graviton's mass. We also give an interpretation of this result for a specific case of fifth force framework. We find that the residuals for the Cassini spacecraft significantly (90% C.L.) degrade for Compton wavelengths of the graviton smaller than 1.83×10^{13} km, which correspond to a graviton mass bigger than 6.76×10^{-23} eV/c^{2}. This limit is comparable in magnitude to the one obtained by the LIGO-Virgo Collaboration in the radiative regime. We also use this specific example to defend that constraints on alternative theories of gravity obtained from postfit residuals may be generically overestimated.

Association of MR relaxation and cartilage deformation in knee osteoarthritis.

We assessed the relationship between cartilage MR relaxation times and biomechanical response of tibiofemoral articular cartilage to physiological loading in healthy subjects and patients with osteoarthritis (OA). Female subjects above 40 years of age with (N(1) = 20) and without (N(2) = 10) OA were imaged on a 3T MR scanner using a custom made loading device. MR images were acquired with the knee flexed at 20° with and without a compressive load of 50% of the subject's bodyweight. The subjects were categorized based on the clinical MRI scoring of medial and lateral cartilage surfaces. Data were stratified twice into two equal groups (low and high) at the median value of T(1ρ) and T(2) relaxation time. The change in contact area and cartilage deformation was measured within these groups. Paired Student's t-test (α = 0.05) was used to analyze the effect of loading on contact area and deformation. The average area of the contact region in the medial compartment was significantly higher in OA subjects compared with normal subjects in both unloaded (314 ± 112 mm(2) vs. 227 ± 106 mm(2), p = 0.023) and loaded (425 ± 128 mm(2) vs. 316 ± 107 mm(2), p = 0.01) conditions. The overall relative change of cartilage thickness in the medial compartment was significantly higher than the lateral compartment (-5.3 ± 9.9% vs. -1.9 ± 9.2%, p = 0.042). When cartilage was divided into deep and superficial layers, superficial layers showed higher changes in relaxation time (T(1ρ) and T(2)) than the changes in relaxation time of whole cartilage (Normal: 12.5% vs. 6.9%; OA: 10.9% vs. 4.6%). The average T(1ρ) and T(2) times, change in area of contact region, and change in cartilage thickness in subjects with OA were higher when compared to normal subjects. This study provides support for a relationship between the mechanical response of cartilage to physiological loading (cartilage-on-cartilage contact area and cartilage deformation) and MR relaxation times (T(1ρ) and T(2)) in both OA patients and normal subjects.

Existence of collisional trajectories of Mercury, Mars and Venus with the Earth.

It has been established that, owing to the proximity of a resonance with Jupiter, Mercury's eccentricity can be pumped to values large enough to allow collision with Venus within 5 Gyr (refs 1-3). This conclusion, however, was established either with averaged equations that are not appropriate near the collisions or with non-relativistic models in which the resonance effect is greatly enhanced by a decrease of the perihelion velocity of Mercury. In these previous studies, the Earth's orbit was essentially unaffected. Here we report numerical simulations of the evolution of the Solar System over 5 Gyr, including contributions from the Moon and general relativity. In a set of 2,501 orbits with initial conditions that are in agreement with our present knowledge of the parameters of the Solar System, we found, as in previous studies, that one per cent of the solutions lead to a large increase in Mercury's eccentricity-an increase large enough to allow collisions with Venus or the Sun. More surprisingly, in one of these high-eccentricity solutions, a subsequent decrease in Mercury's eccentricity induces a transfer of angular momentum from the giant planets that destabilizes all the terrestrial planets approximately 3.34 Gyr from now, with possible collisions of Mercury, Mars or Venus with the Earth.

OA clinical trials: current targets and trials for OA. Choosing molecular targets: what have we learned and where we are headed?

OBJECTIVE: The purpose of this article is to review the current status of drug development as it relates to both molecular targets and clinical trials for osteoarthritis (OA). METHODS: A review of the literature in the context of currently what is known of the pathophysiology of OA and the learnings from past clinical trials is provided. Also discussed is the challenge of demonstrating efficacy and clinical benefit for pharmacologic interventions for OA in the context of current regulatory guidance documents for therapies for the treatment of OA. RESULTS: There is a large unmet medical need for pharmacologic therapeutic interventions that modify the progression of OA and treat the symptoms associated with OA. The development of Disease Modifying OA Drugs (DMOADs) should take into account the current status of therapeutic interventions, as well as the various tissues that constitute the joint and contribute to joint mechanics, and the symptoms associated with structural changes. There is much to be learned about the pathophysiology of the joint that is currently poorly understood particularly as it relates to tissues other than hyaline articular cartilage. Improving our understanding that these tissues play in OA pathophysiology will likely yield treatment breakthroughs. Recently, tremendous progress has been made in the understanding of pain pathways with an emerging diversity of pain mechanisms and biology suggesting heterogeneity in pain etiology in OA. A multitude of new targets have been identified at the level of neuronal transduction/excitability, conduction, sensitization and transmission with multiple emerging compounds in development. CONCLUSIONS: The development of symptom modifying OA drug is exploding with a plethora of pain pathways being pursued and multiple candidates in advanced stages of clinical development. Structure modification in OA remains complex with significant development challenges.

The feasibility of characterizing the spatial distribution of cartilage T(2) using texture analysis.

OBJECTIVE: The purpose of this study was (1) to characterize the spatial distribution of cartilage T(2) in postmenopausal osteoarthritis (OA) patients and age-matched healthy subjects using second order texture measures at baseline, and (2) to analyze changes in the texture of cartilage T(2) after 9 months. METHODS: 3.0T-MRI of the knee was performed in 8 mild OA patients and 10 age-matched controls at baseline and after 9 months. Cartilage T(2), volume, and average thickness were calculated in all patients. Texture analysis, based on the gray level co-occurrence matrix, was performed on the cartilage T(2) maps. Texture parameters, including entropy and angular second moment, were calculated at 0 degrees (corresponding to the anterior-posterior axis) and at 90 degrees (corresponding to the superior-inferior axis), with pixel offsets ranging from 1 to 3 pixels. RESULTS: Least square means analysis showed that mean T(2) values, their standard deviation (SD), and their entropy were greater (P<0.05) in OA patients than in controls. Over 9 months, the SD and entropy of cartilage T(2) significantly (P<0.05) decreased in OA patients, while no significant changes were evident in cartilage thickness or volume. CONCLUSION: The mean cartilage T(2) values, their SD, and their entropy were greater in OA patients than in controls, indicating that the T(2) values in osteoarthritic cartilage are not only elevated, but also more heterogeneous than those in healthy cartilage. The longitudinal results demonstrate that changes in texture parameters of cartilage T(2) may precede morphological changes in thickness and volume in the progression of OA.

Optimization of the fixed-flexion knee radiograph.

PURPOSE: To develop a user-friendly method of achieving optimal radiographs for measurement of joint space width of the knee with minimal radiation exposure. In order to accomplish this the X-ray technologist must (1) be able to identify the anterior and posterior rims of the tibial plateau at a variety of X-ray head angles and (2) be able to choose the direction to adjust the head angle to get a better view based on the criteria for acceptable radiographs. METHODS: We have developed a training manual and materials to instruct investigators and radiology technologists in a method that uses a commercially available Plexiglas positioning frame (Synaflexer) and standard X-ray equipment to achieve optimal X-rays with regard to tibial plateau alignment of the knee. This should be accomplished with four or fewer radiographs. RESULTS: Optimized radiographs for joint space width measurements are achieved without the need for fluoroscopy or foot maps. CONCLUSIONS: This method is readily understood and instituted by radiology technologists in the field.

MRI-derived T2 relaxation times and cartilage morphometry of the tibio-femoral joint in subjects with and without osteoarthritis during a 1-year follow-up.

OBJECTIVE: To assess differences in magnetic resonance imaging (MRI)-based compositional (T2) and morphometric (volume and thickness) parameters of the tibio-femoral joint cartilage in subjects with and without osteoarthritis (OA) and compare these with clinical assessment tools during a 1-year follow-up. METHOD: Three Tesla MRI of the knee joint was performed in eight female patients (body mass index [BMI]>30) with early OA and 10 age-matched female controls (BMI<30) at baseline (BL) and after 3, 6 and 12 months. Cartilage T2 maps, volume and average thickness were calculated in five compartments (medial/lateral femoral condyle, medial/lateral tibia and trochlea). These data were correlated with changes in clinical parameters and joint space width determined in standardized knee radiographs using a mixed random effects model. RESULTS: At BL, T2 was significantly higher (P<0.05) across the cartilage in patients (45.68+/-5.17ms) compared to controls (41.75+/-4.33ms). Patients had significantly (P<0.05) less cartilage volume and less average cartilage thickness in the tibia than controls (2.10+/-0.53cm(3) vs 2.91+/-0.49cm(3) and 1.59+/-0.24mm vs 1.90+/-0.29mm, respectively). A significant change in clinical parameters of OA, cartilage T2 values or a decrease of volume and average thickness could not be demonstrated within both groups. CONCLUSION: Significant differences between the groups indicate that both T2 and morphometric parameters may be useful in quantifying early OA related changes. In a 12-month follow-up, however, no significant alterations of the studied parameters were found, which may be due to the length of the observation interval.

Anti-SSA/Ro52 autoantibodies blocking the cardiac 5-HT4 serotoninergic receptor could explain neonatal lupus congenital heart block.

The 52-kDa SSA/Ro (Ro52) ribonucleoprotein is an antigenic target strongly associated with the autoimmune response in mothers whose children develop neonatal lupus and congenital heart block. When sera from patients with systemic lupus erythematosus were used as autoimmune controls in an enzyme immunoassay to screen for antibodies against the human serotoninergic 5-HT4-receptor, a high correlation was found between the presence of anti-Ro52 protein antibodies in such sera and antibodies reacting with a synthetic peptide, corresponding to the second extracellular loop of the human 5-HT4 receptor (amino acid residues 165-185). Homology scanning between the 5-HT4 peptide and the sequence of the Ro52 protein indicated two potential common epitopes located between residues 365 and 396 of the Ro52 protein. Cross-reactivity was found between the peptide derived from the 5-HT4 receptor, and a peptide corresponding to residues 365-382 of the Ro52 protein. Autoantibodies, affinity-purified on the 5-HT4 receptor peptide, specifically recognized both the Ro52 protein and the 5-HT4 receptor protein in immunoblots. The affinity-purified antibodies antagonized the serotonin-induced L-type Ca channel activation on human atrial cells. This effect could explain the electrophysiological abnormalities in neonatal lupus.

Isolation of the serotoninergic 5-HT4(e) receptor from human heart and comparative analysis of its pharmacological profile in C6-glial and CHO cell lines.

RT - PCR technique was used to clone the human 5-HT(4(e)) receptor (h5-HT(4(e))) from heart atrium. We showed that this h5-HT(4(e)) receptor splice variant is restricted to brain and heart atrium. Recombinant h5-HT(4(e)) receptor was stably expressed in CHO and C6-glial cell lines at 347 and 88 fmol mg(-1) protein, respectively. Expression of h5-HT(4(e)) receptors at the cell membrane was confirmed by immunoblotting. The receptor binding profile, determined by competition with [(3)H]-GR113808 of a number of 5-HT(4) ligands, was consistent with that previously reported for other 5-HT(4) receptor isoforms. Surprisingly, we found that the rank order of potencies (EC(50)) of 5-HT(4) agonists obtained from adenylyl cyclase functional assays was inversely correlated to their rank order of affinities (K(i)) obtained from binding assays. Furthermore, EC(50) values for 5-HT, renzapride and cisapride were 2 fold lower in C6-glial cells than in CHO cells. ML10302 and renzapride behaved like partial agonists on the h5-HT(4(e)) receptor. These results are in agreement with the reported low efficacy of the these two compounds on L-type Ca(2+) currents and myocyte contractility in human atrium. A constitutive activity of the h5-HT(4(e)) receptor was observed in CHO cells in the absence of any 5-HT(4) ligand and two 5-HT(4) antagonists, GR113808 and ML10375, behaved as inverse agonists. These data show that the h5-HT(4(e)) receptor has a pharmacological profile which is close to the native h5-HT(4) receptor in human atrium with a functional potency which is dependent on the cellular context in which the receptor is expressed.

The 5-HT4 receptor antagonist ML10375 inhibits the constitutive activity of human 5-HT4(c) receptor.

Transient expression in COS-7 cells of the recombinant human 5-hydroxytryptamine (5-HT) h5-HT4(c) receptor isoform led to constitutive activity of the receptor. The 5-HT4 receptor antagonist 2-(cis-3,5-dimethylpiperidino)ethyl 4-amino-5-chloro-2-methoxybenzoate (ML10375) at 1 microM completely abolished the 5-HT (1 microM)-mediated increase in adenylyl cyclase activity in COS-7 cells expressing the h5-HT4(c) receptor. Moreover, ML10375 also reduced basal cAMP levels in cells over-expressing the receptor, even in the absence of agonist. The inhibitory effect of ML10375 on basal adenylyl cyclase activity was not modified by pre-treatment of the cells with pertussis toxin, indicating that ML10375 acts through inactivation of spontaneously active h5-HT4(c) receptors rather than through a Gi/Go regulatory pathway. We conclude that ML10375 acts as an inverse agonist on the h5-HT4(c) receptor.

Cloning, expression, and pharmacology of four human 5-hydroxytryptamine 4 receptor isoforms produced by alternative splicing in the carboxyl terminus.

We report here the molecular cloning of three new splice variants of the human serotonin 5-hydroxytryptamine4 (h5-HT4) receptor, which we named h5-HT4(b), h5-HT4(c), and h5-HT4(d). The sequence following the splicing site at Leu358 in the C-terminal tail of h5-HT4(b) displays a 74% protein identity with the same region in the long form of the rat 5-HT4 receptor (r5-HT4L) but is shorter by 18 amino acids compared to its rat counterpart. The splice variants h5-HT4(c) and h5-HT4(d) are the first of their kind to be described in any animal species. The C terminus of h5-HT4(c) displays a high number of putative phosphorylation sites. The h5-HT4(d) isoform corresponds to an ultrashort form of the receptor, with a truncation two amino acids after the splicing site. Tissue distribution studies revealed some degree of specificity in the pattern of expression of the different isoforms within the human body. The four splice variants transiently expressed in COS-7 cells displayed an identical 5-HT4 pharmacological profile and showed a similar ability to stimulate adenylyl cyclase activity in the presence of 5-HT. The stimulatory pattern of cyclic AMP formation in response to the 5-HT4 agonist renzapride was found to be significantly different between h5-HT4(a) and the other h5-HT4 isoforms, indicating that the splice variants may differ in the way they trigger the signal transduction cascade following receptor activation.

Molecular and functional characterization of a 5-HT4 receptor cloned from human atrium.

5-Hydroxytryptamine (5-HT) has been shown to exert positive inotropic, chronotropic, and lusitropic effects and to stimulate the L-type calcium channel current (I(Ca)) in human atrial tissue through activation of the pharmacologically defined 5-HT4 receptor subtype. However, the molecular nature of the receptor(s) involved in these effects is still unknown. In the present study, we report the molecular nature of a 5-HT4 receptor cloned from human atrium, h5-HT4A. Sequence analysis reveals that h5-HT4A displays a 93% protein identity with the short form of the 5-HT4 receptor recently isolated from rat brain. h5-HT4A mRNA is expressed in human atrium but not ventricle, and is also found in brain and GI tract. h5-HT4A transiently expressed in COS-7 cells displays a classical 5-HT4 pharmacological profile. However, affinities of the h5-HT4A receptor for agonists such as ML10302, BIMU1, renzapride or zacopride were 4-10-fold lower than the ones found in brain. Moreover, the stimulatory patterns of cAMP formation by h5-HT4A in response to the 5-HT4 agonists ML10302 and renzapride were very similar to the patterns of stimulation of I(Ca) obtained in response to these compounds in human atrial myocytes. We conclude that h5-HT4A likely mediates the effects of 5-HT in human atrium and may differ from 5-HT4 receptor isoforms present in the brain and GI tract.

In young primary cultures of rabbit kidney cortical collecting ducts intercalated cells originate from principal or undifferentiated cells.

The evolution of a primary culture of rabbit kidney cortical collecting tubule was followed over a period of 10 to 11 days. The cell types of this segment were characterized by using monoclonal antibodies, specifically directed against principal (Mab 703) and intercalated (Mab 503) cells of the apical membrane. The activity of a H+ pump ATPase was revealed in Mab 503-labeled cells, confirming that these cultured cells present characteristics of intercalated cells. The primary culture was also stained with peanut agglutinin (PNA), a specific ligand of beta intercalated cells. During the first two days, some cells, mainly Mab 503-labeled cells, disappeared, and cell division did not occur. At 2 days, the culture showed 80% and 18% of Mab 703-labeled and Mab 503-labeled cells, respectively. The first mitoses were observed at 2 days. From two to four days, cell division was nestly in Mab 703-labeled cells and only rarely seen in Mab 503-labeled cells, although during this period the proportion of Mab 703-labeled cells decreased to 44% of cells and that of Mab 503-labeled cells increased to 30%. The labeling with PNA was curious. Up to 2 days, PNA stained Mab 503-labeled cells, but from 4 days it stained other cells, probably dedifferentiated ones. In our culture conditions types of cells other than Mab 703-labeled and Mab 503-labeled cells occurred. First, throughout the life of the culture, some cells were not recognized by any monoclonal antibody; their number varied between 10 and 28% of the total cell number.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxin sensitivity of the calcium-dependent rubidium efflux in Madin-Darby canine kidney cells.

86Rb+ efflux was measured on polarized Madin-Darby canine kidney cells under A23187 or ATP stimulation. This efflux, inhibited by barium, Leiurus quinquestriatus hebraeus venom and charybdotoxin was attributed to the stimulation of Ca(++)-activated maxi K+ channels. Snake venom from Dendroaspis polylepis did not alter the stimulation as well as did apamine. ATP was effective on both the apical and basolateral membranes and the Ca(++)-activated maxi K+ channels were predominantly found on the basolateral membrane. This study presents the physiological evidence that dendrotoxin is ineffective on the epithelial Ca(++)-activated maxi K+ channel present in MDCK cells.

Effect of calcitonin on the regulation of intracellular pH in primary cultures of rabbit early distal tubule.

To examine the intracellular pH (pHi) regulation in primary cultures of rabbit distal convoluted tubules (DCTb) we used the pH-sensitive dye 2,7-bis-carboxyethyl-5(6)-carboxyfluorescein (BCECF/AM) and a video-microscopy technique. DCTb segments were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The culture epithelia were grown on semi-transparent permeable supports. Before pHi measurement, DCTb primary cultures were maintained for 48-96 h in growth-factor-free medium to obtain quiescent cells. We had previously shown that two mechanisms are involved in the regulation of intracellular pH: a basolateral Na+/H+ exchanger and an apical Cl-/HCO3- exchanger. The pHi of DCTb cells was significantly decreased by the addition of 60 nM human calcitonin (from 7.30 +/- 0.04 to 7.08 +/- 0.04). This response to calcitonin was dose-dependent and mimicked by both forskolin and permeant cyclic AMP derivatives. An initial acidification (of 0.25 pH unit in 7-8 min) was observed after the addition of basolateral amiloride (1 mM). The persistence of the effect induced by human calcitonin in these conditions, suggests that the Na+/H+ exchanger is not involved in the response. However, the acidification response was blocked in both the absence of chloride at the apical side and by the apical addition of 0.1 mM 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). These experiments suggest that the target for the human calcitonin effect on pHi is the Cl-/HCO3- exchanger. This study confirms the importance of this transporter in pHi regulation within the physiological pHi range and the influence of calcitonin in the regulation of DCTb cell function.

Toxin pharmacology of the ATP-induced hyperpolarization in Madin-Darby canine kidney cells.

The effects of Leiurus quinquestriatus hebraeus (LQH) venom, mamba venom, Buthus tamulus (BT) venom, purified apamin and synthetic charybdotoxin on the membrane hyperpolarization induced by extracellular ATP were examined in Madin-Darby canine kidney cells. For this we used a membrane potential probe (bisoxonol) to determine the potential variations. The relation between bisoxonal fluorescence and membrane potential was established by treating Madin-Darby canine kidney cells suspended in solutions containing various external sodium concentrations with gramicidin. Extracellular ATP induced a rapid hyperpolarization that was blocked by LQH venom and synthetic charybdotoxin. BT venom also blocked the response but at a much higher concentration than that of LQH. Mamba venom (Dendroaspis polylepis) and apamin did not modify the ATP-induced hyperpolarization. We concluded that the ATP induced hyperpolarization was due to the augmentation of the potassium conductance probably through Ca(2+)-activated K+ channels sensitive to charybdotoxin but not to mamba venom. The interaction previously described between charybdotoxin and dendrotoxin (the main toxin of mamba venom) was not observed in our case.

Principal and intercalated cells in primary cultures of rabbit renal collecting tubules revealed by monoclonal antibodies.

In this study we describe the production and characterization of two monoclonal antibodies (mAb 503 and mAb 703) raised against the apical membrane of rabbit cortical collecting tubule (CCT) cells. The specificity of the two monoclonal antibodies was studied by immunoelectron microscopy on kidney sections. These antibodies were used to identify principal and intercalated cells in primary cultures of CCT. To assess the maintenance of the basic characteristics of the cortical collecting cells during the growth process we determined the biochemical and electrophysiological properties of cultured CCT. Of the monoclonal antibodies produced mAb 503 was specifically directed against the luminal membrane of intercalated cells as shown by immunoelectron microscopy. mAb 703 bound specifically the apical membrane of the principal cells. In primary cultures of CCT mAb 503 and mAb 703 bound antigens present on the apical membrane of different cells and permitted the study of the distribution of the two cell types. Results showed the maintenance of the epithelial polarity of cultured CCT and the expression of specific antigens.

Antigenic expression of aminopeptidase M, dipeptidyl-peptidase IV and endopeptidase by primary cultures from rabbit kidney proximal tubule.

Techniques using microdissected tubules from rabbit kidney allow the isolation of well defined segments which can be cultured to obtain pure renal cell epithelia. From microdissected proximal tubules, we obtained epithelia the cells of which exhibit some of the antigenic expressions of the initial proximal cells. For this purpose, we used three monoclonal antibodies raised against apical brush border membranes of the proximal tubules. We determined with precision the identity and some of the molecular characteristics of the antigens bound by these three antibodies and found that they correspond to three hydrolases present in the brush borders of proximal renal cells (amino-peptidase, dipeptidyl-peptidase IV and endopeptidase). These apical markers are expressed by the growing cells of primary cultures from proximal tubules, suggesting strongly that they are effectively proximal cells and that no appreciable dedifferentiation occurred during the growth process. We have also shown that apical expression of these hydrolases on the plasma membrane of the epithelium occurred only after several days of culture and determined the complete polarization of the cells. Electron microscopy studies confirmed the degree of polarization of the cultured cells by the presence of numerous microvilli on their apical face.