A constitutively active mutant of the alpha 1B-adrenergic receptor can cause greater agonist-dependent down-regulation of the G-proteins G9 alpha and G11 alpha than the wild-type receptor.

Rat 1 fibroblasts transfected to express either the wild-type hamster alpha 1B-adrenergic receptor or a constitutively active mutant (CAM) form of this receptor resulting from the alteration of amino acid residues 288-294 to encode the equivalent region of the human beta 2-adrenergic receptor were examined. The basal level of inositol phosphate generation in cells expressing the CAM alpha 1B-adrenergic receptor was greater than for the wild-type receptor, The addition of maximally effective concentrations of phenylephrine or noradrenaline resulted in substantially greater levels of inositol phosphate generation by the CAM alpha 1B-adrenergic receptor, although this receptor was expressed at lower steady-state levels than the wild-type receptor. The potency of both phenylephrine and noradrenaline to stimulate inositol phosphate production was approx. 200-fold greater at the CAM alpha 1B-adrenergic receptor than at the wild-type receptor. In contrast, endothelin 1, acting at the endogenously expressed endothelin ETA, receptor, displayed similar potency and maximal effects in the two cell lines. The sustained presence of phenylephrine resulted in down-regulation of the alpha subunits of the phosphoinositidase C-linked, pertussis toxin-insensitive, G-proteins G9 and G11 in cells expressing either the wild-type or the CAM alpha 1B-adrenergic receptor. The degree of down-regulation achieved was substantially greater in cells expressing the CAM alpha 1B-adrenergic receptor at all concentrations of the agonist. However, in this assay phenylephrine displayed only a slightly greater potency at the CAM alpha 1B-adrenergic receptor than at the wild-type receptor. There were no detectable differences in the basal rate of G9 alpha/G11 alpha degradation between cells expressing the wild-type or the CAMalpha 1B-adrenergic receptor. In both cell lines the addition of phenylephrine substantially increased the rate of degradation of these G-proteins, with a greater effect at the CAM alpha 1B-adrenergic receptor. The enhanced capacity of agonist both to stimulate second-messenger production at the CAM alpha 1B-adrenergic receptor and to regulate cellular levels of its associated G-proteins by stimulating their rate of degradation is indicative of an enhanced stoichiometry of coupling of this form of the receptor to G9 and G11.

Functional and molecular evolutionary analysis of the "Brevix Radix" gene family in mono-and dicotyledonous plants

ABSTRACTIn contrast to animals, plants cannot move from their place of birth and, therefore, need to adapt to their particular habitat in order to survive. Thus, plant development is remarkably plastic, making plants an ideal system for the isolation of genes that account for intraspecific natural variation and possibly environmental adaptation. However, to date, this approach mostly identified null alleles and missed mutations with subtle effects. For instance, BREVIS RADIX (BRX) has been isolated as a key regulator of root growth through a naturally occurring loss-of-function allele in the Arabidopsis thaliana accession Uk-1 and is the founding member of a highly-conserved plant-specific gene family.In this work, we show that a strong selective pressure is acting on the BRX gene family and dates back before the monocot-dicot divergence. However, functional diversification is observed mainly in dicotyledon BRX family genes and is correlated with acceleration in the evolutionary rates in the N-terminal regions. Population genetic data revealed that BRX is highly conserved across Arabidopsis accessions and presents signatures of adaptation. Interestingly, a seven amino acid deletion polymorphism in BRX sequence was found in a few accessions, which seems to be responsible for their enhanced primary root growth. Nevertheless, BRX might not only be active in the root, as suggested by its expression in the shoot. Indeed, leaves and cotyledons of brx mutants are significantly smaller than wild- type. This phenotype is a direct consequence of the absence of BRX function in the shoot rather than an indirect effect of an altered root system growth. Interestingly, cotyledons of brx plants reflect the same physiological defects as the root. Moreover, phenotypes in BRX gain-of-function plants, such as epinastic leaves and increased epidermal cell size, could be associated with an increase in leaf brassinosteroid content.Collectively, these results indicate that BRX contributes to local adaptation by ubiquitously regulating plant growth, probably through the modulation of brassinosteroid biosynthesis.RÉSUMÉContrairement à la plupart des animaux, les plantes ne peuvent se mouvoir et doivent ainsi s'adapter à leur environnement pour survivre. Pour cette raison, elles représentent un système idéal pour l'identification de gènes contribuant à la variation naturelle intra- spécifique, ainsi qu'à l'adaptation. Cependant, cette approche a, jusqu'à présent, surtout permis d'isoler des allèles nuls et non des mutations conférant des effets plus subtiles. C'est le cas du gène Β REVIS RADIX (BRX), un régulateur clé de la croissance racinaire, qui a été identifié grâce à un allèle non-fonctionnel présent dans l'accession naturelle d'Arabidopsis thaliana Uk-1. BRX et ses homologues des plantes mono- et dicotylédones forment une famille très conservée et spécifique aux plantes.Dans ce travail, nous démontrons que la famille de gènes BRX est soumise à une forte pression de sélection qui remonte avant la divergence entre mono- et dicotylédones. Cependant, une diversification fonctionnelle a été observée chez les gènes des dicotylédones et corrèle avec une accélération de la vitesse d'évolution dans leur région N- terminale. Une analyse génétique de différentes accessions naturelles d'Arabidopsis a révélé que BRX est hautement conservé et présente des signatures d'adaptation. Remarquablement, un polymorphisme de délétion de sept acides aminés a été détecté dans quelques accessions et a pour conséquence une plus forte croissance de la racine primaire. Néanmoins, il semble que le rôle de BRX ne se limite pas qu'à la racine, comme indiqué par son expression dans les parties aériennes de la plante. En effet, les mutants brx présentent des cotylédons et des feuilles significativement plus petits que le type sauvage, une conséquence directe de l'absence d'activité de BRX dans ces organes. Nous avons aussi noté que les cotylédons des mutants brx, à l'instar des racines, ont une perception altérée de l'auxine et peuvent être complémentés par l'application exogène de brassinostéroïdes. De plus, dans des plantes présentant un gain de fonction BRX, les feuilles sont épinastiques et les cellules de leur épiderme plus grandes. Ces phénotypes sont accompagnés d'une augmentation de la concentration de brassinostéroïdes dans les feuilles. Conjointement, ces résultats démontrent que BRX contribue à une adaptation locale de la plante par la régulation générale de sa croissance, probablement en modulant la biosynthèse des brassinostéroïdes.

Enhanced heat shock protein 70 expression alters proteasomal degradation of IkappaB kinase in experimental acute respiratory distress syndrome.

OBJECTIVES: Acute respiratory distress syndrome is a common and highly lethal inflammatory lung syndrome. We previously have shown that an adenoviral vector expressing the heat shock protein (Hsp)70 (AdHSP) protects against experimental sepsis-induced acute respiratory distress syndrome in part by limiting neutrophil accumulation in the lung. Neutrophil accumulation and activation is modulated, in part, by the nuclear factor-kappaB (NF-kappaB) signal transduction pathway. NF-kappaB activation requires dissociation/degradation of a bound inhibitor, IkappaBalpha. IkappaBalpha degradation requires phosphorylation by IkappaB kinase, ubiquitination by the SCFbeta-TrCP (Skp1/Cullin1/Fbox beta-transducing repeat-containing protein) ubiquitin ligase, and degradation by the 26S proteasome. We tested the hypothesis that Hsp70 attenuates NF-kappaB activation at multiple points in the IkappaBalpha degradative pathway. DESIGN: Laboratory investigation. SETTING: University medical center research laboratory. SUBJECTS: Adolescent (200 g) Sprague-Dawley rats and murine lung epithelial-12 cells in culture. INTERVENTIONS: Lung injury was induced in rats via cecal ligation and double puncture. Thereafter, animals were treated with intratracheal injection of 1) phosphate buffer saline, 2) AdHSP, or 3) an adenovirus expressing green fluorescent protein. Murine lung epithelial-12 cells were stimulated with tumor necrosis factor-alpha and transfected. NF-kappaB was examined using molecular biological tools. MEASUREMENTS AND MAIN RESULTS: Intratracheal administration of AdHSP to rats with cecal ligation and double puncture limited nuclear translocation of NF-kappaB and attenuated phosphorylation of IkappaBalpha. AdHSP treatment reduced, but did not eliminate, phosphorylation of the beta-subunit of IkappaB kinase. In vitro kinase activity assays and gel filtration chromatography revealed that treatment of sepsis-induced lung injury with AdHSP induced fragmentation of the IkappaB kinase signalosome. This stabilized intermediary complexes containing IkappaB kinase components, IkappaBalpha, and NF-kappaB. Cellular studies indicate that although ubiquitination of IkappaBalpha was maintained, proteasomal degradation was impaired by an indirect mechanism. CONCLUSIONS: Treatment of sepsis-induced lung injury with AdHSP limits NF-kappaB activation. This results from stabilization of intermediary NF-kappaB/IkappaBalpha/IkappaB kinase complexes in a way that impairs proteasomal degradation of IkappaBalpha. This novel mechanism by which Hsp70 attenuates an intracellular process may be of therapeutic value.

Controlled contrast transcranial Doppler and arterial blood gas analysis to quantify shunt through patent foramen ovale.

BACKGROUND AND PURPOSE: A right-to-left shunt can be identified by contrast transcranial Doppler ultrasonography (c-TCD) at rest and/or after a Valsalva maneuver (VM) or by arterial blood gas (ABG) measurement. We assessed the influence of controlled strain pressures and durations during VM on the right-to-left passage of microbubbles, on which depends the shunt classification by c-TCD, and correlated it with the right-to-left shunt evaluation by ABG measurements in stroke patients with patent foramen ovale (PFO). METHODS: We evaluated 40 stroke patients with transesophageal echocardiography-documented PFO. The microbubbles were recorded with TCD at rest and after 4 different VM conditions with controlled duration and target strain pressures (duration in seconds and pressure in cm H2O, respectively): V5-20, V10-20, V5-40, and V10-40. The ABG analysis was performed after pure oxygen breathing in 34 patients, and the shunt was calculated as percentage of cardiac output. RESULTS: Among all VM conditions, V5-40 and V10-40 yielded the greatest median number of microbubbles (84 and 95, respectively; P<0.01). A significantly larger number of microbubbles were detected in V5-40 than in V5-20 (P<0.001) and in V10-40 than in V10-20 (P<0.01). ABG was not sensitive enough to detect a shunt in 31 patients. CONCLUSIONS: The increase of VM expiratory pressure magnifies the number of microbubbles irrespective of the strain duration. Because the right-to-left shunt classification in PFO is based on the number of microbubbles, a controlled VM pressure is advised for a reproducible shunt assessment. The ABG measurement is not sensitive enough for shunt assessment in stroke patients with PFO.

Enhanced Model Selection for motion segmentation

In this paper a novel rank estimation technique for trajectories motion segmentation within the Local Subspace Affinity (LSA) framework is presented. This technique, called Enhanced Model Selection (EMS), is based on the relationship between the estimated rank of the trajectory matrix and the affinity matrix built by LSA. The results on synthetic and real data show that without any a priori knowledge, EMS automatically provides an accurate and robust rank estimation, improving the accuracy of the final motion segmentation

The mc2-CMX vaccine induces an enhanced immune response against Mycobacterium tuberculosis compared to Bacillus Calmette-Guérin but with similar lung inflammatory effects

Although the attenuated Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccine has been used since 1921, tuberculosis (TB) control still proceeds at a slow pace. The main reason is the variable efficacy of BCG protection against TB among adults, which ranges from 0-80%. Subsequently, the mc2-CMX vaccine was developed with promising results. Nonetheless, this recombinant vaccine needs to be compared to the standard BCG vaccine. The objective of this study was to evaluate the immune response induced by mc2-CMX and compare it to the response generated by BCG. BALB/c mice were immunised with both vaccines and challenged withMycobacterium tuberculosis (Mtb). The immune and inflammatory responses were evaluated by ELISA, flow cytometry, and histopathology. Mice vaccinated with mc2-CMX and challenged with Mtb induced an increase in the IgG1 and IgG2 levels against CMX as well as recalled specific CD4+ T-cells that produced T-helper 1 cytokines in the lungs and spleen compared with BCG vaccinated and challenged mice. Both vaccines reduced the lung inflammatory pathology induced by the Mtb infection. The mc2-CMX vaccine induces a humoral and cellular response that is superior to BCG and is efficiently recalled after challenge with Mtb, although both vaccines induced similar inflammatory reductions.

Enhanced antitumor activity of doxorubicin in breast cancer through the use of poly(butylcyanoacrylate) nanoparticles.

The use of doxorubicin (DOX), one of the most effective antitumor molecules in the treatment of metastatic breast cancer, is limited by its low tumor selectivity and its severe side effects. Colloidal carriers based on biodegradable poly(butylcyanoacrylate) nanoparticles (PBCA NPs) may enhance DOX antitumor activity against breast cancer cells, thus allowing a reduction of the effective dose required for antitumor activity and consequently the level of associated toxicity. DOX loading onto PBCA NPs was investigated in this work via both drug entrapment and surface adsorption. Cytotoxicity assays with DOX-loaded NPs were performed in vitro using breast tumor cell lines (MCF-7 human and E0771 mouse cancer cells), and in vivo evaluating antitumor activity in immunocompetent C57BL/6 mice. The entrapment method yielded greater drug loading values and a controlled drug release profile. Neither in vitro nor in vivo cytotoxicity was observed for blank NPs. The 50% inhibitory concentration (IC50) of DOX-loaded PBCA NPs was significantly lower for MCF-7 and E0771 cancer cells (4 and 15 times, respectively) compared with free DOX. Furthermore, DOX-loaded PBCA NPs produced a tumor growth inhibition that was 40% greater than that observed with free DOX, thus reducing DOX toxicity during treatment. These results suggest that DOX-loaded PBCA NPs have great potential for improving the efficacy of DOX therapy against advanced breast cancers.

Consensus guidelines for enhanced recovery after gastrectomy: Enhanced Recovery After Surgery (ERAS®) Society recommendations.

BACKGROUND: Application of evidence-based perioperative care protocols reduces complication rates, accelerates recovery and shortens hospital stay. Presently, there are no comprehensive guidelines for perioperative care for gastrectomy. METHODS: An international working group within the Enhanced Recovery After Surgery (ERAS®) Society assembled an evidence-based comprehensive framework for optimal perioperative care for patients undergoing gastrectomy. Data were retrieved from standard databases and personal archives. Evidence and recommendations were classified according to the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) system and were discussed until consensus was reached within the group. The quality of evidence was rated 'high', 'moderate', 'low' or 'very low'. Recommendations were graded as 'strong' or 'weak'. RESULTS: The available evidence has been summarized and recommendations are given for 25 items, eight of which contain procedure-specific evidence. The quality of evidence varies substantially and further research is needed for many issues to improve the strength of evidence and grade of recommendations. CONCLUSION: The present evidence-based framework provides comprehensive advice on optimal perioperative care for the patient undergoing gastrectomy and facilitates multi-institutional prospective cohort registries and adequately powered randomized trials for further research.

Stimulation of hematopoiesis in vivo by recombinant bacterial murine interleukin 3.

Mouse interleukin 3 (IL-3) cDNA was cloned into a plasmid construction, allowing the synthesis of very high quantities of IL-3 in Escherichia coli. The recombinant (r) IL-3, purified to homogeneity, was active in vitro on the proliferation and differentiation of various hematopoietic progenitor cells at 1 pM. To maintain detectable blood levels of IL-3, osmotic pumps containing rIL-3 or control solutions were placed under the skin of normal and irradiated C3H/HeJ and (BALB X B10) F1 mice. The effect of IL-3 on hematopoietic progenitor cell numbers in spleen and bone marrow was evaluated 3 and 7 days later by using an in vitro clonal assay. The results demonstrated the following: (i) Doses of IL-3 infused at the rate of 2.5-5 ng per g of body weight per hr were sufficient to increase the numbers of hematopoietic progenitors in normal mice by at least 2-fold within 3 days. (ii) In mice with progenitor cell levels depressed by sublethal irradiation, 7-day treatment with IL-3 resulted in a 10-fold increase to near normal levels. (iii) The erythroid and myeloid lineages appeared to be enhanced to the same extent. (iv) Enhancement of hematopoiesis occurred primarily in spleen, but hematopoietic foci were also evident in the liver; in contrast, total cell and progenitor cell numbers were decreased in the bone marrow.

Identification of serum and urine proteins responsible for enhanced pigment production by group B streptococci as amylases.

The serum and urine proteins responsible for enhanced pigment production in Streptococcus agalactiae in culture media were purified by chromatography and were identified as amylases by comparison of their amino acid composition with that calculated for proteins with known sequences. Similar pigment-enhancing activity was displayed by other amylases of nonanimal origin and by maltooligosaccharides.

Diffusion spectrum magnetic resonance imaging (DSI) tractography of crossing fibers.

MRI tractography is the mapping of neural fiber pathways based on diffusion MRI of tissue diffusion anisotropy. Tractography based on diffusion tensor imaging (DTI) cannot directly image multiple fiber orientations within a single voxel. To address this limitation, diffusion spectrum MRI (DSI) and related methods were developed to image complex distributions of intravoxel fiber orientation. Here we demonstrate that tractography based on DSI has the capacity to image crossing fibers in neural tissue. DSI was performed in formalin-fixed brains of adult macaque and in the brains of healthy human subjects. Fiber tract solutions were constructed by a streamline procedure, following directions of maximum diffusion at every point, and analyzed in an interactive visualization environment (TrackVis). We report that DSI tractography accurately shows the known anatomic fiber crossings in optic chiasm, centrum semiovale, and brainstem; fiber intersections in gray matter, including cerebellar folia and the caudate nucleus; and radial fiber architecture in cerebral cortex. In contrast, none of these examples of fiber crossing and complex structure was identified by DTI analysis of the same data sets. These findings indicate that DSI tractography is able to image crossing fibers in neural tissue, an essential step toward non-invasive imaging of connectional neuroanatomy.

A Factor analysis of hidrochemical composition of Llobregat river basin

Hydrogeological research usually includes some statistical studies devised to elucidate mean background state, characterise relationships among different hydrochemical parameters, and show the influence of human activities. These goals are achieved either by means of a statistical approach or by mixing modelsbetween end-members. Compositional data analysis has proved to be effective with the first approach, but there is no commonly accepted solution to the end-member problem in a compositional framework.We present here a possible solution based on factor analysis of compositions illustrated with a case study.We find two factors on the compositional bi-plot fitting two non-centered orthogonal axes to the most representative variables. Each one of these axes defines a subcomposition, grouping those variables thatlay nearest to it. With each subcomposition a log-contrast is computed and rewritten as an equilibrium equation. These two factors can be interpreted as the isometric log-ratio coordinates (ilr) of three hiddencomponents, that can be plotted in a ternary diagram. These hidden components might be interpreted as end-members.We have analysed 14 molarities in 31 sampling stations all along the Llobregat River and its tributaries, with a monthly measure during two years. We have obtained a bi-plot with a 57% of explained totalvariance, from which we have extracted two factors: factor G, reflecting geological background enhanced by potash mining; and factor A, essentially controlled by urban and/or farming wastewater. Graphicalrepresentation of these two factors allows us to identify three extreme samples, corresponding to pristine waters, potash mining influence and urban sewage influence. To confirm this, we have available analysisof diffused and widespread point sources identified in the area: springs, potash mining lixiviates, sewage, and fertilisers. Each one of these sources shows a clear link with one of the extreme samples, exceptfertilisers due to the heterogeneity of their composition.This approach is a useful tool to distinguish end-members, and characterise them, an issue generally difficult to solve. It is worth note that the end-member composition cannot be fully estimated but only characterised through log-ratio relationships among components. Moreover, the influence of each endmember in a given sample must be evaluated in relative terms of the other samples. These limitations areintrinsic to the relative nature of compositional data

Imagerie cardiaque non invasive: apport spécifique en clinique des nouvelles modalités (I). Evaluation morphologique [Cardiac imaging: specific clinical role of newly developed non invasive techniques. Part 1: Morphological evaluation].

Echocardiography is the preferred initial test to assess cardiac morphology and ventricular function. Cardiac MRI enables an optimal visualisation of heart muscle without contrast injection, and precise measurement of the ventricular volumes and systolic function. It is therefore an ideal test for patients with poor echocardiographic windows or for the specific evaluation of right heart chambers. Heart CT also remarkably images heart muscle and precisely measures ventricular systolic function after intravenous injection of iodinated contrast. Coronary CT may also, in selected cases, avoid the need for diagnostic coronary angiography. Although very accurate, these imaging modalities are expensive and may be contra-indicated for a particular patient. Their use in clinical practice has to follow the accepted guidelines.

Postmortem angiography: review of former and current methods.

OBJECTIVE: Postmortem investigations are becoming more and more sophisticated. CT and MRI are already being used in pathology and forensic medicine. In this context, the impact of postmortem angiography increases because of the rapid evaluation of organ-specific vascular patterns, vascular alteration under pathologic and physiologic conditions, and tissue changes induced by artificial and unnatural causes. CONCLUSION: In this article, the advantages and disadvantages of former and current techniques and contrast agents are reviewed.

Reactive hyperreninemia is a major determinant of plasma angiotensin II during ACE inhibition.

The new ACE inhibitor trandolapril was administered to normal volunteers at daily doses of 0.5, 2, and 8 mg for 10 days. Twenty-one volunteers, aged 21-30 years, were included in the study. To randomly selected groups of seven subjects, each dose was administered in a single-blind fashion. None of the doses induced a consistent fall in blood pressure. Angiotensin-converting enzyme activity (ACE) was measured in vitro using three different synthetic substrates (i.e., Hip-Gly-Gly, Z-Phe-His-Leu, or angiotensin I). Although the degree of ACE inhibition assessed with the three methods varied widely, all methods clearly indicated dose-dependent ACE inhibition. These in vitro results were confirmed by measuring ACE inhibition in vivo using the ratio of plasma angiotensin II (ANG II) to blood angiotensin I (ANG I). The dose-dependent ACE inhibition was paralleled by a dose-dependent rise in active renin and blood angiotensin I levels, most evident on day 10. In contrast, plasma ANG II levels on day 10 were not different whether the volunteers received 0.5 or 8 mg trandolapril. Thus, whereas increasing doses of this new ACE inhibitor progressively enhanced the blockade of ACE activity, this was not reflected by additional reductions of plasma ANG II levels. The progressive enhancement of ACE inhibition seemed to be offset by the accentuation of the compensatory rise in renin and ANG I, which was still partially converted to ANG II.(ABSTRACT TRUNCATED AT 250 WORDS)