MEASUREMENT OF THE PHOTO-PUMP STRENGTH OF THE 3D-5F TRANSITIONS IN THE AUTOMATICALLY LINE MATCHED NI-LIKE SM PHOTOPUMPED X-RAY LASER

The photo-pump strengths of both the ((3 (d) over bar(4))(0)(3d(6))(0))(0)-(((3 (d) over bar(3))(3/2)(3d(6))(0))(3/2)(5 (f) over bar)(5/2))(1) and the ((3 (d) over bar(4))(0)(3d(6))0)0-(((3 (d) over bar(4))(0)(3d(5))(5/2))(5/2)(5f)(7/2))(1) transitions in Ni-like Sm34+ have been measured to be 2.0 x 10(-4) and 2.4 x 10(-4) photons/mode respectively. The implications of the measurement are briefly discussed in a comparison of the merits of automatically line matched photo-pump scheme to those of the collisional excitation Ni-like Sm+34 scheme.

Temperature-regulated efflux pump/potassium antiporter system mediates resistance to cationic antimicrobial peptides in Yersinia

Most bacterial pathogens are resistant to cationic antimicrobial peptides (CAMPs) that are key components of the innate immunity of both vertebrates and invertebrates. In Gram-negative bacteria, the known CAMPs resistance mechanisms involve outer membrane (OM) modifications and specifically those in the lipopolysaccharide (LPS) molecule. Here we report, the characterization of a novel CAMPs resistance mechanism present in Yersinia that is dependent on an efflux pump/potassium antiporter system formed by the RosA and RosB proteins. The RosA/RosB system is activated by a temperature shift to 37 degrees C, but is also induced by the presence of the CAMPs, such as polymyxin B. This is the first report of a CAMPs resistance system that is induced by the presence of CAMPs. It is proposed that the RosA/RosB system protects the bacteria by both acidifying the cytoplasm to prevent the CAMPs action and pumping the CAMPs out of the cell.

The sarcolemmal calcium pump inhibits the calcineurin/nuclear factor of activated T-cell pathway via interaction with the calcineurin A catalytic subunit

The calcineurin/nuclear factor of activated T-cell (NFAT) pathway represents a crucial transducer of cellular function. There is increasing evidence placing the sarcolemmal calcium pump, or plasma membrane calcium/calmodulin ATPase pump (PMCA), as a potential modulator of signal transduction pathways. We demonstrate a novel interaction between PMCA and the calcium/calmodulin-dependent phosphatase, calcineurin, in mammalian cells. The interaction domains were located to the catalytic domain of PMCA4b and the catalytic domain of the calcineurin A subunit. Endogenous calcineurin activity, assessed by measuring the transcriptional activity of its best characterized substrate, NFAT, was significantly inhibited by 60% in the presence of ectopic PMCA4b. This inhibition was notably reversed by the co-expression of the PMCA4b interaction domain, demonstrating the functional significance of this interaction. PMCA4b was, however, unable to confer its inhibitory effect in the presence of a calcium/calmodulin-independent constitutively active mutant calcineurin A suggesting a calcium/calmodulin-dependent mechanism. The modulatory function of PMCA4b is further supported by the observation that endogenous calcineurin moves from the cytoplasm to the plasma membrane when PMCA4b is overexpressed. We suggest recruitment by PMCA4b of calcineurin to a low calcium environment as a possible explanation for these findings. In summary, our results offer strong evidence for a novel functional interaction between PMCA and calcineurin, suggesting a role for PMCA as a negative modulator of calcineurin-mediated signaling pathways in mammalian cells. This study reinforces the emerging role of PMCA as a molecular organizer and regulator of signaling transduction pathways.

Novel functional interaction between the plasma membrane Ca2+ pump 4b and the proapoptotic tumor suppressor Ras-associated factor 1 (RASSF1)

Plasma membrane calmodulin-dependent calcium ATPases (PMCAs) are enzymatic systems implicated in the extrusion of calcium from the cell. We and others have previously identified molecular interactions between the cytoplasmic COOH-terminal end of PMCA and PDZ domain-containing proteins. These interactions suggested a new role for PMCA as a modulator of signal transduction pathways. The existence of other intracellular regions in the PMCA molecule prompted us to investigate the possible participation of other domains in interactions with different partner proteins. A two-hybrid screen of a human fetal heart cDNA library, using the region 652-840 of human PMCA4b (located in the catalytic, second intracellular loop) as bait, revealed a novel interaction between PMCA4b and the tumor suppressor RASSF1, a Ras effector protein involved in H-Ras-mediated apoptosis. Immunofluorescence co-localization, immunoprecipitation, and glutathione S-transferase pull-down experiments performed in mammalian cells provided further confirmation of the physical interaction between the two proteins. The interaction domain has been narrowed down to region 74-123 of RASSF1C (144-193 in RASSF1A) and 652-748 of human PMCA4b. The functionality of this interaction was demonstrated by the inhibition of the epidermal growth factor-dependent activation of the Erk pathway when PMCA4b and RASSF1 were co-expressed. This inhibition was abolished by blocking PMCA/RASSSF1 association with an excess of a green fluorescent protein fusion protein containing the region 50-123 of RASSF1C. This work describes a novel protein-protein interaction involving a domain of PMCA other than the COOH terminus. It suggests a function for PMCA4b as an organizer of macromolecular protein complexes, where PMCA4b could recruit diverse proteins through interaction with different domains. Furthermore, the functional association with RASSF1 indicates a role for PMCA4b in the modulation of Ras-mediated signaling.

The overexpression of the efflux pump Tpo1 leads to the bleomycin resistance in Saccharomyces cerevisiae.

La bléomycine est un antibiotique cytotoxique, son potentiel génotoxique est plus important quand elle est utilisée en combinaison avec des agents antinéoplasiques sur le cancer testiculaire, que sur les autres types qui développent souvent une résistance envers la drogue. Notre but consiste alors de mettre en évidence ce mécanisme de résistance en utilisant l’organisme modèle Saccharomyces cerevisiae. Nous avons démontré au sein de notre laboratoire, que les levures délétées au niveau de leur coactivateur transcriptionnel Imp2, présentent une hypersensibilité à la bléomycine, en raison de son accumulation toxique dans la cellule. Ceci suggère que Imp2 pourrait réguler l’expression d’une ou de plusieurs pompes à efflux, capables d’expulser la bléomycine à l’extérieur de la cellule. Pour tester notre hypothèse, nous avons recherché des suppresseurs multicopies capables de restaurer la résistance à la bléomycine chez le mutant imp2, et c’est ainsi que nous avons identifié l'activateur transcriptionnel Yap1. Ce dernier se lie à une région spécifique localisée au niveau du promoteur et permet d’activer l'expression d'un sous-ensemble de gènes, codant pour des pompes à efflux, impliquées dans la résistance aux drogues. Selon la littérature, au moins 27 pompes à efflux ont été identifiées chez la levure Saccharomyces cerevisiae, certaines d’entre elles disposent du site de liaison pour Yap1, tels que Qdr3, Tpo2 et Tpo1. Afin de déterminer si une de ces pompes expulse la bléomycine, nous avons créé des mutations simples et doubles en combinaison avec IMP2, aussi nous avons verifié si les mutants étaient sensibles à la drogue et enfin, nous avons testé si la surexpression de Yap1 pouvait restaurer le phénotype sauvage chez ces mutants, via l’activation de pompes à efflux.

Femtosecond time-resolved wave packet motion in molecular multiphoton ionization and fragmentation

The dynamics of molecular multiphoton ionization and fragmentation of a diatomic molecule (Na_2) have been studied in molecular beam experiments. Femtosecond laser pulses from an amplified colliding-pulse mode-locked (CPM) ring dye laser are employed to induce and probe the molecular transitions. The final continuum states are analyzed by photoelectron spectroscopy, by ion mass spectrometry and by measuring the kinetic energy of the formed ionic fragments. Pump-probe spectra employing 70-fs laser pulses have been measured to study the time dependence of molecular multiphoton ionization and fragmentation. The oscillatory structure of the transient spectra showing the dynamics on the femtosecond time scale can best be understood in terms of the motion of wave packets in bound molecular potentials. The transient Na_2^+ ionization and the transient Na^+ fragmentation spectra show that contributions from direct photoionization of a singly excited electronic state and from excitation and autoionization of a bound doubly excited molecular state determine the time evolution of molecular multiphoton ionization.

Femtosecond time-resolved molecular multiphoton ionization: the Na_2 system

We report here the first experimental study of femtosecond time-resolved molecular multiphoton ionization. Femtosecond pump-probe techniques are combined with time-of-flight spectroscopy to measure transient ionization spectra of Na_2 in a molecular-beam experiment. The wave-packet motions in different molecular potentials show that incoherent contributions from direct photoionization of a singly excited state and from excitation and autoionization of a bound doubly excited molecular state determine the observed transient ionization signal.

Femtosecond dynamics of molecular and cluster ionization and fragmentation

The real-time dynamics of molecular (Na_2 . Na_3) and cluster Na_n (n=4-2l) multiphoton ionization and -fragmentation has been studied in beam experiments applying femtosecond pump-probe techniques in combination with ion and electron spectroscopy. Wave packet motion in the dimer Na_2 reveals two independent multiphoton ionization processes while the higher dimensional motion in the trimer Na_3 reflects the chaotic vibrational motion in this floppy system. The first studies of cluster properties (energy, bandwidth and lifetime of intermediate resonances Na^*_n) ) with femtosecond laser pulses give a striking illustration of the transition from "molecule-like" excitations to "surfaceplasma"-like resonances for increasing cluster sizes. Time-resolved fragmentation of cluster ions Na_n^* indicate that direct photo-induced fragmentation processes are more important at short times than the statistical unimolecular decay.

Femtosecond pump-probe study of the spreading and recurrence of a vibrational wave packet in Na_2

The motion of a vibrational wave packet in the bound A(^1 \summe^+_u) electronic state of the sodium dimer is detected in a femtosecond pump/probe molecular beam experiment. For short times harmonic motion is seen in the total ion yield of Na^+_2 as a function of delay time between the two laser pulses. The spreading of the wave packet results in the loss of the periodic variation of the ion signal. For longer delay times (47 ps) the wave packet regains its initial form which is reflected in the revival structure of the Na^+_2 signal. Time-dependent quantum calculations reproduce the measured effects.

Femtosecond time-resolved molecular multiphoton ionization and fragmentation of Na_2

We present a comparison between experimental and theoretical results for pump/probe multiphoton ionizing transitions of the sodium dimer, initiated by femtosecond laser pulses. It is shown that the motion of vibrational wavepackets in two electronic states is probed simultaneously and their dynamics is reflected in the total Na^+_2 ion signal which is recorded as a function of the time delay between pump and probe pulse. The time dependent quantum calculations demonstrate that two ionization pathways leading to the same final states of the molecularion exist: one gives an oscillating contribution to the ion signal, the other yields a constant background. From additional measurements of the Na^+ -transient photofragmentation spectrum it is deduced that another ionization process leading to different final ionic states exists. The process includes the excitation of a doubly excitedbound Rydberg state. This conclusion is supported by the theoretical simulation.

Multimilligram enantioresolution of sulfoxide proton pump inhibitors by liquid chromatography on polysaccharide-based chiral stationary phase

The enantiomers of sulfoxide proton pump inhibitors - omeprazole, lansoprazole, rabeprazole and Ro 18-5364 - were enantiomerically separated by liquid chromatography at multimilligram scale on a poly saccharide-based chiral stationary phase using normal and polar organic conditions as mobile phase. The values of the recovery and production rate were significant for each enantiomer; better results were achieved using a solid-phase injection system. However, this system was applied just for the enantionteric separation of omeprazole to demonstrate the applicability of this injection mode at milligram scale. The chiroptical characterization of the compounds was performed using a polarimeter and a circular dichroism detector. The higher enantiomeric purity obtained for the isolated enantiomers suggests that the methods here described should be considered as a simple and rapid way to obtain enantiomeric pure standards for analytical purpose. (C) 2007 Elsevier B.V. All rights reserved.

Substâncias húmicas: sistema de fracionamento sequencial por ultrafiltração com base no tamanho molecular

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Caracterização da estrutura molecular e propriedades viscográficas de amidos de mandioca (Manihot esculenta Crantz) para aplicações industriais

Pós-graduação em Agronomia (Energia na Agricultura) - FCA

Acinetobacter baumannii isolates from pets and horses in Switzerland: molecular characterization and clinical data

OBJECTIVES: We investigated whether Acinetobacter baumannii isolates of veterinary origin shared common molecular characteristics with those described in humans. METHODS: Nineteen A. baumannii isolates collected in pets and horses were analysed. Clonality was studied using repetitive extragenic palindromic PCR (rep-PCR) and multilocus sequence typing (MLST). PCR and DNA sequencing for various beta-lactamase, aminoglycoside-modifying enzyme, gyrA and parC, ISAba1 and IS1133, adeR and adeS of the AdeABC efflux pump, carO porin and class 1/2/3 integron genes were performed. RESULTS: Two main clones [A (n = 8) and B (n = 9)] were observed by rep-PCR. MLST indicated that clone A contained isolates of sequence type (ST) ST12 (international clone II) and clone B contained isolates of ST15 (international clone I). Two isolates of ST10 and ST20 were also noted. Seventeen isolates were resistant to gentamicin, 12 to ciprofloxacin and 3 to carbapenems. Isolates of ST12 carried bla(OXA-66), bla(ADC-25), bla(TEM-1), aacC2 and IS1133. Strains of ST15 possessed bla(OXA-69), bla(ADC-11), bla(TEM-1) and a class 1 integron carrying aacC1 and aadA1. ISAba1 was found upstream of bla(ADC) (one ST10 and one ST12) and/or bla(OXA-66) (seven ST12). Twelve isolates of different STs contained the substitutions Ser83Leu in GyrA and Ser80Leu or Glu84Lys in ParC. Significant disruptions of CarO porin and overexpressed efflux pumps were not observed. The majority of infections were hospital acquired and in animals with predisposing conditions for infection. CONCLUSIONS: STs and the molecular background of resistance observed in our collection have been frequently described in A. baumannii detected in human patients. Animals should be considered as a potential reservoir of multidrug-resistant A. baumannii.

Different anticoagulation strategies in off-pump coronary artery bypass operations: a European survey

In order to determine anticoagulation strategies in OPCAB a questionnaire survey among 750 European cardio-thoracic surgeons was performed. Questions addressed volume of OPCAB procedures performed, intra- and perioperative heparinization and antiplatelet therapy, as well as perioperative management. A total of 325 (43.7%) questionnaires were returned and validated. Perioperative protocols for administration of antiplatelets differed among the respondent surgeons. Perioperative prophylaxis of thrombosis (low or high molecular weight heparin) is performed by 78%. Intraoperative heparin dosage range between 70 U/kg to 500 U/kg, 60% of respondents prefer a low-dose regimen (< or = 150 U/kg). Correspondingly, the lowest activated clotting time (ACT) during surgery is accepted to be 200 s by 24%, 250 s by 18% and 300 s by 26% of surgeons. Protamine is used by 91% of respondents, while 52% perform a 1:1 reversal. A cell-saver and antifibrinolytics are used by 70% and 40%, respectively. Interestingly, 56% of respondents think bleeding in OPCAB patients is not reduced when compared to on-pump CABG. In addition, 34% of respondents believe there is an increased risk of early graft occlusion following OPCAB. This survey demonstrates widely different intra- and perioperative anticoagulation strategies for OPCAB procedures among European surgeons.