Giant thermal Hall conductivity in the pseudogap phase of cuprate superconductors.

The nature of the pseudogap phase of the copper oxides ('cuprates') remains a puzzle. Although there are indications that this phase breaks various symmetries, there is no consensus on its fundamental nature1. Fermi-surface, transport and thermodynamic signatures of the pseudogap phase are reminiscent of a transition into a phase with antiferromagnetic order, but evidence for an associated long-range magnetic order is still lacking2. Here we report measurements of the thermal Hall conductivity (in the x-y plane, κxy) in the normal state of four different cuprates-La1.6-xNd0.4SrxCuO4, La1.8-xEu0.2SrxCuO4, La2-xSrxCuO4 and Bi2Sr2-xLaxCuO6+δ. We show that a large negative κxy signal is a property of the pseudogap phase, appearing at its critical hole doping, p*. It is also a property of the Mott insulator at p ≈ 0, where κxy has the largest reported magnitude of any insulator so far3. Because this negative κxy signal grows as the system becomes increasingly insulating electrically, it cannot be attributed to conventional mobile charge carriers. Nor is it due to magnons, because it exists in the absence of magnetic order. Our observation is reminiscent of the thermal Hall conductivity of insulators with spin-liquid states4-6, pointing to neutral excitations with spin chirality7 in the pseudogap phase of cuprates.

Change of carrier density at the pseudogap critical point of a cuprate superconductor.

The pseudogap is a partial gap in the electronic density of states that opens in the normal (non-superconducting) state of cuprate superconductors and whose origin is a long-standing puzzle. Its connection to the Mott insulator phase at low doping (hole concentration, p) remains ambiguous and its relation to the charge order that reconstructs the Fermi surface at intermediate doping is still unclear. Here we use measurements of the Hall coefficient in magnetic fields up to 88 tesla to show that Fermi-surface reconstruction by charge order in the cuprate YBa2Cu3Oy ends sharply at a critical doping p = 0.16 that is distinctly lower than the pseudogap critical point p* = 0.19 (ref. 11). This shows that the pseudogap and charge order are separate phenomena. We find that the change in carrier density n from n = 1 + p in the conventional metal at high doping (ref. 12) to n = p at low doping (ref. 13) starts at the pseudogap critical point. This shows that the pseudogap and the antiferromagnetic Mott insulator are linked.

Prasugrel vs. clopidogrel in acute coronary syndrome patients treated with prasugrel.

WHAT IS KNOWN AND OBJECTIVE: Current guidelines recommend a combination of clopidogrel and aspirin for management of patients who have experienced an acute coronary syndrome (ACS). Additional antiplatelet agents have been recently approved. Few comparative effectiveness studies are available for these new agents. Accordingly, we evaluated effect on time to hospital admission and resource utilization (number of hospitalizations, ER visits and outpatient visits) of prasugrel vs. clopidogrel in prasugrel-treated patients as assessed in a matched cohort. METHODS: Based on the Truven Health Analytics MarketScan database from 01 January 2009 through 31 July 2012, a retrospective prasugrel-clopidogrel matched cohort was created. Inferences for average treatment effect over 1 and 12 months on time to hospitalization and resource utilization were performed by (i) frequentist Kaplan-Meier estimation with a Cox proportional hazard model and Lin's cost history method for censored resource utilization outcomes and (ii) Bayesian discrete-time hazard and negative binomial models. RESULTS AND DISCUSSION: The 10,963 matched pairs were well balanced on baseline characteristics. Frequentist analyses of time to hospital admission over 365 days and mean all-cause resource utilization over 30 and 365 days showed no statistical differences between prasugrel and clopidogrel (P-values > 0·05). Based on Bayesian analysis of time to admission over 12 months, there was positive evidence of equivalence (0·987 probability of equivalence at a 10% equivalence margin and a Bayes factor of 0·611). Although the frequentist analyses for number of all-cause hospitalizations showed a lack of a significant difference at Months 1 and 12, the Bayesian data analysis showed positive evidence of superiority of clopidogrel at Month 1 (Bayes factor: 5·369); however, at Month 12, there was little evidence of superiority of one treatment over the other (Bayes factor: 0·422). WHAT IS NEW AND CONCLUSION: Using frequentist and Bayesian data analyses, in prasugrel-treated patients, clopidogrel was equivalent to prasugrel for time to hospital admission over 12 months and there was positive evidence that it was superior to prasugrel for number of hospitalizations over the first month of treatment.

Nerve growth factor localization in the nasal mucosa of patients with persistent allergic rhinitis.

BACKGROUND AND OBJECTIVES: Nerve growth factor (NGF) and NGF receptors have been shown to be expressed by structural and infiltrating inflammatory cells in the human allergic bronchial mucosa and conjunctiva. In the nose, a positive immunostaining for NGF was recently reported in biopsies of subjects undergoing surgery for refractory nasal obstruction. This study was aimed at studying by immunohistochemistry NGF expression and localization in the nasal mucosa from subjects with moderate/severe persistent allergic rhinitis and natural allergen exposure. METHODS: Immunostaining for NGF, tryptase and eosinophil cationic protein was performed in human nasal turbinate sections of 25 patients affected by persistent allergic rhinitis and sensitization to Dermatophagoides pteronyssinus. RESULTS: NGF was consistently expressed in the epithelium and in the submucosa of allergic rhinitic subjects, preferentially localized in eosinophils and mast cells. A strong NGF immunostaining was found in mucous cells of the epithelial lining and in the submucosal glands. CONCLUSIONS: As previously shown for allergic asthma and allergic conjunctivitis, NGF is also detectable in the nasal mucosa of patients with persistent allergic rhinitis. The preferential NGF localization in mucous cells of the epithelial lining and in the submucosal glands suggests a possible role for NGF in modulating secretion in allergic rhinitis and possibly other allergic diseases.

ARCTIC CHANGE AND POSSIBLE INFLUENCE ON MID-LATITUDE CLIMATE AND WEATHER: A US CLIVAR White Paper.

The Arctic has warmed more than twice as fast as the global average since the mid 20th century, a phenomenon known as Arctic amplification (AA). These profound changes to the Arctic system have coincided with a period of ostensibly more frequent events of extreme weather across the Northern Hemisphere (NH) mid-latitudes, including extreme heat and rainfall events and recent severe winters. Though winter temperatures have generally warmed since 1960 over mid-to-high latitudes, the acceleration in the rate of warming at high-latitudes, relative to the rest of the NH, started approximately in 1990. Trends since 1990 show cooling over the NH continents, especially in Northern Eurasia. The possible link between Arctic change and mid-latitude climate and weather has spurred a rush of new observational and modeling studies. A number of workshops held during 2013-2014 have helped frame the problem and have called for continuing and enhancing efforts for improving our understanding of Arctic-mid-latitude linkages and its attribution to the occurrence of extreme climate and weather events. Although these workshops have outlined some of the major challenges and provided broad recommendations, further efforts are needed to synthesize the diversified research results to identify where community consensus and gaps exist. Building upon findings and recommendations of the previous workshops, the US CLIVAR Working Group on Arctic Change and Possible Influence on Mid-latitude Climate and Weather convened an international workshop at Georgetown University in Washington, DC, on February 1-3, 2017. Experts in the fields of atmosphere, ocean, and cryosphere sciences assembled to assess the rapidly evolving state of understanding, identify consensus on knowledge and gaps in research, and develop specific actions to accelerate progress within the research community. With more than 100 participants, the workshop was the largest and most comprehensive gathering of climate scientists to address the topic to date. In this white paper, we synthesize and discuss outcomes from this workshop and activities involving many of the working group members. WORKSHOP FINDINGS: Rapid Arctic change - Emergence of new forcing (external and internal) of atmospheric circulation: Rapid Arctic change is evident in the observations and is simulated and projected by global climate models. AA has been attributed to sea ice and snow decline (regionally and seasonally varying). However this cannot explain why AA is greatest in winter and weakest in summer. It was argued at the workshop that other factors can also greatly contribute to AA including: increased downwelling longwave radiation from greenhouse gases (including greater water vapor concentrations from local and remote sources); increasing ocean heat content, due to local and remote processes; regional and hemispheric atmospheric circulation changes; increased poleward heat transport in the atmosphere and ocean; and cloud radiative forcing. In particular, there is emerging observational evidence that an enhanced poleward transport of sensible and latent heat plays a very important role in the AA of the recent decades, and that this enhancement is mostly fueled by changes in the atmospheric circulation. We concluded that our understanding of AA is incomplete, especially the relative contributions from the different radiative, thermodynamic, and dynamic processes.Arctic mid-latitude linkages - Focusing on seasonal and regional linkages and addressing sources of inconsistency and uncertainty among studies: The topic of Arctic mid-latitude linkages is controversial and was vigorously debated at the workshop. However, we concluded that rapid Arctic change is contributing to changes in mid-latitude climate and weather, as well as the occurrence of extreme events. But how significant the contribution is and what mechanisms are responsible are less well understood. Based on the synthesis efforts of observational and modeling studies, we identified a list of proposed physical processes or mechanisms that may play important roles in linking Arctic change to mid-latitude climate and weather. The list, ordered from high to low confidence, includes: increasing geopotential thickness over the polar cap; weakening of the thermal wind; modulating stratosphere-troposphere coupling; exciting anomalous planetary waves or stationary Rossby wave trains in winter and modulating transient synoptic waves in summer; altering storm tracks and behavior of blockings; and increasing frequency of occurrence of summer wave resonance. The pathway considered most robust is the propagation of planetary/Rossby waves excited by the diminished Barents-Kara sea ice, contributing to a northwestward expansion and intensification of the Siberian high leading to cold Eurasian winters. OPPORTUNITIES AND RECOMMENDATIONS: An important goal of the workshop was achieved: to hasten progress towards consensus understanding and identification of knowledge gaps. Based on the workshop findings, we identify specific opportunities to utilize observations and models, particularly a combination of them, to enable and accelerate progress in determining the mechanisms of rapid Arctic change and its mid-latitude linkages.Observations: Due to the remoteness and harsh environmental conditions of the Arctic, in situ observational time series are highly limited spatially and temporally in the region.Six recommendations to expand approaches using observational datasets and analyses of Arctic change and mid-latitude linkages include: Synthesize new Arctic observations;Create physically-based sea ice-ocean surface forcing datasets;Systematically employ proven and new metrics;Analyze paleoclimate data and new longer observational datasets;Utilize new observational analysis methods that extend beyond correlative relationships; andConsider both established and new theories of atmospheric and oceanic dynamics to interpret and guide observational and modeling studies.Model experiments: We acknowledge that models provide the primary tool for gaining a mechanistic understanding of variability and change in the Arctic and at mid-latitudes. Coordinated modeling studies should include approaches using a hierarchy of models from conceptual, simple component, or coupled models to complex atmospheric climate models or fully coupled Earth system models. We further recommend to force dynamical models with consistent boundary forcings.Three recommendations to advance modeling and synthesis understanding of Arctic change and mid-latitude linkages include: Establish a Modeling Task Force to plan protocols, forcing, and output parameters for coordinated modeling experiments (Polar Amplification Model Intercomparison Project; PAMIP);Furnish experiment datasets to the community through open access (via Earth System Grid); andPromote analysis within the community of the coordinated modeling experiments to understand mechanisms for AA and to further understand pathways for Arctic mid-latitude linkages.

L-454,560, a potent and selective PDE4 inhibitor with in vivo efficacy in animal models of asthma and cognition.

Type 4 phosphodiesterases (PDE4) inhibitors are emerging therapeutics in the treatment of a number of chronic disorders including asthma, chronic obstructive pulmonary disease (COPD) and cognitive disorders. This study delineates the preclinical profile of L-454,560, which is a potent, competitive and preferential inhibitor of PDE4A, 4B, and 4D with IC50 values of 1.6, 0.5 and 1.2 nM, respectively. In contrast to the exclusive binding of cilomilast and the preferential binding of roflumilast to the PDE4 holoenzyme state (Mg2+-bound form), L-454,560 binds to both the apo-(Mg2+-free) and holoenzyme states of PDE4. The intrinsic enzyme potency for PDE4 inhibition by L-454,560 also results in an effective blockade of LPS-induced TNFalpha formation in whole blood (IC50 = 161 nM) and is comparable to the human whole blood potency of roflumilast. The cytokine profile of inhibition of L-454,560 is mainly a Th1 profile with significant inhibition of IFNgamma and no detectable inhibition of IL-13 formation up to 1 microM. L-454,560 was also found to be efficacious in two models of airway hyper-reactivity, the ovalbumin (OVA) sensitized and challenged guinea pig and the ascaris sensitized sheep model. Furthermore, L-454560 was also effective in improving performance in the delayed matching to position (DMTP) version of the Morris watermaze, at a dose removed from that associated with potential emesis. Therefore, L-454,560 is a novel PDE4 inhibitor with an overall in vivo efficacy profile at least comparable to roflumilast and clearly superior to cilomilast.

Pseudogap phase of cuprate superconductors confined by Fermi surface topology.

The properties of cuprate high-temperature superconductors are largely shaped by competing phases whose nature is often a mystery. Chiefly among them is the pseudogap phase, which sets in at a doping p* that is material-dependent. What determines p* is currently an open question. Here we show that the pseudogap cannot open on an electron-like Fermi surface, and can only exist below the doping p FS at which the large Fermi surface goes from hole-like to electron-like, so that p* ≤ p FS. We derive this result from high-magnetic-field transport measurements in La1.6-x Nd0.4Sr x CuO4 under pressure, which reveal a large and unexpected shift of p* with pressure, driven by a corresponding shift in p FS. This necessary condition for pseudogap formation, imposed by details of the Fermi surface, is a strong constraint for theories of the pseudogap phase. Our finding that p* can be tuned with a modest pressure opens a new route for experimental studies of the pseudogap.

Neuroanatomical and pharmacological assessment of Fos expression induced in the rat brain by the phosphodiesterase-4 inhibitor 6-(4-pyridylmethyl)-8-(3-nitrophenyl) quinoline.

A major obstacle in the therapeutic development of phosphodiesterase-4 (PDE4) inhibitors is the production of adverse side effects such as nausea and vomiting. Immunohistochemical detection of Fos-like immunoreactivity (FLI) was used to address the neuroanatomical basis for the pharmacological actions of PDE4 inhibitors. The potent and selective PDE4 inhibitors 6-(4-pyridylmethyl)-8-(3-nitrophenyl) quinoline (PMNPQ) and rolipram elevated FLI in brain regions potentially relevant to the anti-depressant and emetic effects of PDE4 inhibition. PMNPQ and rolipram elevated FLI in the locus coeruleus, habenula, paraventricular nucleus of the thalamus, amygdala and nucleus accumbens, all structures with strong limbic connectivity implicated in arousal, memory and affective aspects of behaviour. Consistent with the emetic effects of PDE4 inhibitors such as PMNPQ and rolipram, these compounds elevated FLI in caudal brainstem nuclei such as the area postrema and nucleus of the solitary tract. Administration of the NK(1) antagonist RP 67580 prior to PMNPQ reversed increases in FLI produced by PMNPQ in these regions. RP 67580 did not, however, reduce PMNPQ-induced FLI in limbic structures. These findings suggest that PDE4 inhibitors produce emesis by increasing NK(1) receptor activation in the AP/NTS and implicate brain regions associated with reward and mood such as the amygdala, paraventricular nucleus of the thalamus, habenula and nucleus accumbens in the anti-depressant activity of such compounds.

Human cytosolic phospholipase A2 expressed in insect cells is extensively phosphorylated on Ser-505.

Cytosolic PLA2 (cPLA2) has been implicated in the release of the arachidonic acid utilized in the inflammatory cascade. Phosphorylation of cPLA2 on Ser-505 by MAP kinase in response to agonist treatment, is thought to be one of the mechanisms required for activation of the enzyme in the cell. In order to obtain enough material for enzymological studies as well as to investigate the role of phosphorylation in the activation of cPLA2, the human enzyme was overexpressed in insect cells using a recombinant baculovirus. We report here on the characterization of the phosphorylation state of cPLA2 overexpressed in Sf9 cells. The level of overexpressed cPLA2 was shown to peak between 48 and 60 h post-infection, by this time the phosphorylated enzyme could easily be detected because of its reduced mobility on polyacrylamide gels. The reduced mobility or gel-shift has been shown to be due to phosphorylation of Ser-505. To determine whether this was also the case for insect cell overexpressed cPLA2, Ser-505 was replaced by Ala, and this mutant (cPLA2S505A) was expressed in Sf9 cells. Analysis of the overexpressed cPLA2S505A showed that it migrated only as the lower unshifted cPLA2 band confirming that the baculovirus overexpressed cPLA2 is extensively phosphorylated on Ser-505. Furthermore, treatment of infected Sf9 cells expressing the wild-type cPLA2 with phorbol 12-tetradecanoate 13-acetate (TPA) shifted all of the overexpressed cPLA2 to the phosphorylated Ser-505 form. When infected Sf9 cells were labelled with [32P], in addition to labelling of Ser-505 other sites were also labelled. Both cPLA2 and cPLA2S505A were purified from infected Sf9 cells and the specific activity for each of the enzymes was measured in a phosphatidylcholine vesicle fluorescence assay using 1-(10-pyrenedecanyl)arachidonyl-sn-glycero-3-phosphocholine as substrate. Under these conditions the specific activity of cPLA2 was, 2 mumol/min per mg, whereas cPLA2S505A was 7-fold less active. These findings suggest that Sf9 cells have a mechanism for phosphorylating cPLA2 similar to that found in mammalian cells which probably proceeds through a MAP kinase. Thus, insect cell overexpressed cPLA2 is a very good source for the Ser-505 phosphorylated enzyme.

Atmospheric dynamics. Constrained work output of the moist atmospheric heat engine in a warming climate.

Incoming and outgoing solar radiation couple with heat exchange at Earth's surface to drive weather patterns that redistribute heat and moisture around the globe, creating an atmospheric heat engine. Here, we investigate the engine's work output using thermodynamic diagrams computed from reanalyzed observations and from a climate model simulation with anthropogenic forcing. We show that the work output is always less than that of an equivalent Carnot cycle and that it is constrained by the power necessary to maintain the hydrological cycle. In the climate simulation, the hydrological cycle increases more rapidly than the equivalent Carnot cycle. We conclude that the intensification of the hydrological cycle in warmer climates might limit the heat engine's ability to generate work.

Evidence for a small hole pocket in the Fermi surface of underdoped YBa2Cu3Oy.

In underdoped cuprate superconductors, the Fermi surface undergoes a reconstruction that produces a small electron pocket, but whether there is another, as yet, undetected portion to the Fermi surface is unknown. Establishing the complete topology of the Fermi surface is key to identifying the mechanism responsible for its reconstruction. Here we report evidence for a second Fermi pocket in underdoped YBa2Cu3Oy, detected as a small quantum oscillation frequency in the thermoelectric response and in the c-axis resistance. The field-angle dependence of the frequency shows that it is a distinct Fermi surface, and the normal-state thermopower requires it to be a hole pocket. A Fermi surface consisting of one electron pocket and two hole pockets with the measured areas and masses is consistent with a Fermi-surface reconstruction by the charge-density-wave order observed in YBa2Cu3Oy, provided other parts of the reconstructed Fermi surface are removed by a separate mechanism, possibly the pseudogap.

The expression in Escherichia coli of a synthetic gene coding for the precursor of papain is prevented by its own putative signal sequence.

A 1048-bp gene coding for prepropapain was assembled from chemically synthesized oligodeoxyribonucleotides and cloned into a variety of Escherichia coli expression plasmids. We observed loss of plasmid when the preproP gene was expressed in E. coli either as the native precursor or fused at the C terminus of the first 592 amino acids (aa) of beta-galactosidase (beta Gal). Deletion of the putative 26-aa signal peptide (pre-region) increased plasmid stability. The level of maintenance for the different plasmid constructs correlated with the level of expression detected by immunoblotting. Constitutive expression of the beta Gal-propapain fusion generated insoluble granules in a protease-deficient E. coli host. The fusion protein was easily purified to near homogeneity by differential solubilization of the granules.

Enhanced secretion from insect cells of a foreign protein fused to the honeybee melittin signal peptide.

The baculovirus/insect cell system has been remarkably successful in yielding high levels of synthesis of many proteins which have been difficult to synthesize in other host/vector systems. The system is also capable of secreting heterologous proteins, but with generally low efficiency. We have increased the efficiency of secretion of the system by using signal peptides of insect origin to direct the secretion of a foreign protein. The precursor of the plant cysteine protease papain (propapain) has been used as a report enzyme to compare secretion efficiency. Insect cells infected with a baculovirus recombined with the gene encoding propapain fused to the sequence encoding the honeybee melittin signal peptide secreted over five times more papain precursor than the wild-type prepropapain which used the plant signal peptide. Based on these results, we have assembled pVT-Bac, an Autographa californica nuclear polyhedrosis virus transfer vector that may enhance secretion of other foreign proteins from insect cells. The vector incorporates a number of features: phage f1 ori to facilitate site-directed mutagenesis, the strong polyhedrin promoter upstream from the melittin signal peptide-encoding sequence, and eight unique restriction sites to facilitate fusion of heterologous genes.

Angiotensin I converting enzyme in gastric mucosa of the rabbit: localization by autoradiography, immunofluorescence, and immunoelectron microscopy.

To localize angiotensin converting enzyme (ACE) in the fundic mucosa of the rabbit, we used autoradiography with the ACE inhibitor [3H]-trandolaprilate and post-embedding immunocytochemical techniques with a goat anti-rabbit ACE, using fluorescence and electron microscopy. Autoradiographic localization of [3H]-trandolaprilate in rabbit fundus sections shows that ACE is present in the fundic mucosa and mainly in the gland area. Fundic mucosa was fixed with 4% formaldehyde and embedded in Lowicryl K4M. Semi-thin (1 micron) or thin sections (800-900 A) were stained with anti-rabbit ACE followed by fluorescein isothyocyanate-labeled rabbit anti-goat IgG or protein A-gold reagent, respectively. Label was present on endothelium of all blood vessels running through the mucosa. Label was prominently localized in the granules of mucous surface and neck cells and on the granules of chief cells. The intracellular localization of ACE, and particularly its intragranular presence within chief and mucous cells, suggests that the enzyme, at the fundic level, is involved in the intragranular processing of a peptide, the nature of which remains to be determined.

Localization of angiotensin-converting enzyme (ACE) mRNA in rabbit gastric mucosa by in situ hybridization.

In a previous study we showed, by immunohistochemical analysis on rabbit fundic mucosa, that in addition to its usual presence on the luminal plasma membrane of endothelial cells, angiotensin converting-enzyme (ACE) was localized inside granules of surface and neck mucous cells and within granules of chief cells. The aim of the present study was to localize ACE mRNA in cells of the rabbit fundic mucosa by in situ hybridization with a 35S-labeled probe. This probe was a cDNA fragment (406 BP) encoding a portion of the rabbit ACE mRNA obtained by reverse transcription followed by polymerase chain reaction on total RNA extracted from fundic mucosa. ACE mRNAs were detected in mucous and chief cells and in endothelial cells of the mucosal vasculature. These results are in complete agreement with our prior studies which showed by immunohistochemical analysis that ACE is present in these cells. Our findings therefore suggest that ACE previously detected in epithelial cells of the rabbit gastric mucosa is actually synthesized within these cells.

Inhibition of enzymatic activity of phospholipases A2 by minocycline and doxycycline.

Extracellular phospholipases A2 play an important role in articular and extra-articular inflammatory processes. Secretory non-pancreatic phospholipase A2 (PLA2) has been implicated in the pathogenesis of articular inflammation in rheumatoid arthritis, whereas pancreatic PLA2 contributes to the tissue damage associated with acute pancreatitis. Since in experimental models lipophilic tetracyclines such as minocycline and doxycycline are antiinflammatory, we examined their effects on PLA2 activity using two assay systems in vitro. We found that minocycline and to a lesser degree doxycycline were markedly inhibitory to both pancreatic and non-pancreatic PLA2. Using [14C]oleic acid labeled Escherichia coli membrane phospholipids as substrate, the IC50 values for minocycline and doxycycline were 3.6 x 10(-5) M (18 micrograms/mL) and 0.98 x 10(-4) M (47 micrograms/mL), respectively. In a scooting mode assay using the synthetic phospholipid 1-palmitoyl-2-(10-pyrenedecanoyl)-3-L-phosphatidylmethanol as substrate, IC50 values for minocycline were 5 microM (2.47 micrograms/mL) for non-pancreatic PLA2 and 8 microM (3.95 micrograms/mL) for pancreatic PLA2. Addition of excess calcium up to 50 mM did not reverse the inhibitory activity of tetracyclines. We conclude that lipophilic tetracyclines inhibit PLA2, probably by interaction with the substrate, and may be a useful adjunct in the therapy of inflammatory conditions in which PLA2 is implicated pathogenetically.

A new experimental method for the dynamic study of the antibody transfer mechanism from mother to fetus in the rat.

The yolk-sac is known to be a route for the transport of passive immunity from mother to fetus in the rat. The main purpose of the present paper is to describe an experimental system for ultrastructurally studying the kinetics of the uptake and transport of immunoglobulin by rat yolk-sac. This system has the advantage of enabling the membrane to be externalized and then exposed to protein under controlled environmental conditions whilst at the same time maintaining the conceptus in connection with the in situ placenta. Preliminary investigations have utilized homologous anti-horseradish peroxidase (HRP) IgG (detected as antibody by application of HRP) or HRP alone. Comparison has been made with the localization of endogenous IgG transmitted in vivo after immunization of the female rat with HRP. The results show the rapid binding of IgG to membrane since only 30 sec were sufficient for this attachment to occur. Moreover, the endocytic process also appears to be very fast as localization of IgG in clusters or patches, caveolae or pits and even rare microvesicles is observed within 8 min. On the other hand, no binding of HRP to microvilli was observed and, unlike IgG, HRP became located in the apico-tubulocanalicular system.

CDP840: a novel inhibitor of PDE-4.

We present the in vitro characterization of a novel phosphodiesterase type 4 inhibitor, CDP840 (R-[+]-4-[2-¿3-cyclopentyloxy-4-methoxyphenyl¿-2-phenylethyl]pyridine), which has shown efficacy in a phase II allergen challenge study in asthmatics without adverse effects. CDP840 potently inhibits PDE-4 isoenzymes (IC50 2-30 nM) without any effect on PDE-1, 2, 3, 5, and 7 (IC50 > 100 microM). It exhibited no significant selectivity in inhibiting human recombinant isoenzymes PDE-4A, B, C or D and was equally active against the isoenzymes lacking UCR1 (PDE-4B2 and PDE-4D2). In contrast to rolipram, CDP840 acted as a simple competitive inhibitor of all PDE-4 isoenzymes. Studies with rolipram indicated a heterogeneity within all the preparations of PDE-4 isoenzymes, indicative of rolipram inhibiting the catalytic activity of PDE-4 with both a low or high affinity. These observations were confirmed by the use of a PDE-4A variant, PDE-4A330-886, which rolipram inhibited with low affinity (IC50 = 1022 nM). CDP840 in contrast inhibited this PDE-4A variant with similar potency (IC50 = 3.9 nM), which was in good agreement with the Kd of 4.8 nM obtained from [3H]-CDP840 binding studies. Both CDP840 and rolipram inhibited the high-affinity binding of [3H]-rolipram binding to PDE-4A, B, C, and D with similar Kd app (7-19 nM and 3-5 nM, respectively). Thus, the activity of CDP840 at the [3H]-rolipram binding site was in agreement with the inhibitor's activity at the catalytic site. However, rolipram was approximately 100-fold more potent than CDP840 at inhibiting the binding of [3H]-rolipram to mouse brain in vivo. These data clearly demonstrate that CDP840 is a potent selective inhibitor of all PDE-4 isoenzymes. In contrast to rolipram, CDP840 was well-tolerated in humans. This difference, however, cannot at present be attributed to either isoenzyme selectivity or lack of activity in vitro at the high-affinity rolipram binding site (Sr).

Expression of angiotensin I converting enzyme mRNA in rabbit gastric epithelial cells.

Angiotensin I converting enzyme (ACE) is a dipeptidyl carboxypeptidase synthesized by endothelial cells from many vascular beds as well as by extravascular tissues. Two forms of ACE have been characterized, a pulmonary form and a testicular form. Previously, in the gastrointestinal tract, we localized ACE in the rabbit gastric fundic tissue. In the present study, Northern blot analysis demonstrated the expression of a 5 kb ACE mRNA in fundic mucosa, identical in size to pulmonary ACE mRNA. In order to confirm the epithelial origin of this ACE, we have purified fundic epithelial cells by a flow cytometry technique by which endothelial cells were excluded and the population was enriched in intermediate and chief cells. Using reverse transcription and polymerase chain reaction with specific oligonucleotides, we have amplified from the enriched fundic epithelial cell RNA a 874 bp fragment, the restriction map of which is identical to that of rabbit lung. These findings demonstrate that in gastric mucosa ACE is expressed in fundic epithelial cells and seems to be similar to the pulmonary form.

Expression of angiotensin I-converting enzyme in the human gastric HGT-1 cell line.

We have previously shown that angiotensin I-converting enzyme (ACE) was expressed by epithelial cells of the rabbit gastric mucosa. In a search to obtain a cell model to study the regulation of ACE expression of gastric origin and its relationship with gastrin-cholecystokinin peptides, which have been proposed as ACE substrates, we investigated whether the HGT-1 human gastric cell line, which expresses gastrin, could also express ACE, using enzymatic and immunodetection methods as well as Northern-blot analysis and polymerase chain reaction. Results show that HGT-1 cells expressed a protein with a molecular weight of 130-140 kDa whose enzymatic and immunological properties were identical to those of ACE. More than 80% of ACE activity was found to be ectoenzymatic. However, immunocytochemical localization has mainly shown an intracellular localization, suggesting that most of intracytoplasmic ACE was not enzymatically active. In addition, Northern-blot analysis and polymerase chain reaction showed that the mRNA encoding that protein displayed a size and a sequence identical to those of somatic ACE. It therefore appears that the HGT-1 cell line could be a useful model to study both the regulation of gastric ACE and its interactions with gastrin-cholecystokinin peptides.