FITC-albumin as a marker for assessment of endothelial permeability in mice: comparison with 125I-albumin.

Transvascular transport of labeled-albumin is used to study endothelial permeability in experimental murine models of pulmonary infections. But radio-tagged albumin necessitates heavy safety procedures in terms of storage, manipulation and evacuation. The authors tested fluorescein isothiocyanate-tagged albumin (FITC-albumin) as a new marker for determination of endothelial permeability in a murine model of lung infection by Pseudomonas aeruginosa PAO1, in comparison with a standard method with (125)I-albumin. The mean permeability +/- SEM measured with (125)I-albumin was 2.45%/2 h +/- 0.37 for the control mice and 6.65%/2 h +/- 0.77 for the infected ones (P < .0001). With FITC-albumin, results obtained for both groups were respectively 4.96%/2 h +/- 0.64 and 11.5%/2 h +/- 1.2 (P < .0001). Spearman's rank coefficient was equal to .88 (P < .0001), showing a very strong correlation between both methods of measurement. The Bland-Altman analysis of bias revealed that there was no significant bias between FITC-albumin-derived and (125)I-albumin-derived values. The correction of the values obtained in plasma and lung homogenate supernatants by the subtraction of natural spontaneous fluorescence measured in these samples was crucial for the calculation of endothelial permeability in this new method. We believe that FITC-albumin can be useful for assessment of endothelial permeability in murine models of pulmonary diseases.

Antithrombin reduces mesenteric venular leukocyte interactions and small intestine injury in endotoxemic rats.

We examined the hypothesis that recombinant human antithrombin would reduce mesenteric venule leukocyte adhesion and small intestine injury in endotoxemic rats. Endotoxemic (endotoxin 10 mg/kg, intravenously) rats were treated either with saline or recombinant human antithrombin (250 and 500 U/kg). In some rats, indomethacin (100 mg/kg, intraperitoneally) was injected 60 min prior to endotoxin and recominant human antithrombin (500 U/kg) treatment. Compared to controls, intravital videomicroscopy of the mesentric venule showed an increase of leukocyte rolling (55+/-17 versus 70+/-19 leukocytes/min; P < 0.05) and firm adhesion (1.1+/-0.3 versus 5.8+/-0.8 leukocytes/100 microm; P < 0.05) in endotoxemic rats. Recombinant human antithrombin attenuated endotoxin-induced venular endothelium leukocyte adhesive cascade. The beneficial effects of recombinant human antithrombin on leukocyte adhesion were inhibited by indomethacin (100 mg/kg, intraperitoneally) in endotoxemic rats. Endotoxin treatment increased fluorescein isothiocyanate (FITC)-labeled dextran 4,000 (FD4) gut lumen to plasma ratio and wet weight/dry weight ratio. Recombinant human antithrombin (500 U/kg) attenuated endotoxin-induced gut injury. These observations suggest that recombinant human antithrombin reduces endothelium-leukocyte interactions in endotoxemic rats by interacting with local prostacyclin production.

In vivo application of intestinal pH measurement using 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) fluorescence imaging.

Measurement of gastrointestinal intramucosal pH (pHim) has been recognized as an important factor in the detection of hypoxia-induced dysfunctions. However, current pH measurement techniques are limited in terms of time and spatial resolutions. A major advance in accurate pH measurement was the development of the ratiometric fluorescent indicator dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). This study aimed to set up and validate a fluorescence imaging technique to measure in vivo the intramucosal pH (pHim) of the intestine. The intestine was inserted into an optical chamber placed under a microscope. Animals were injected intravenously with the pH-sensitive fluorescent dye BCECF. Fluorescence was visualized by illuminating the intestine alternately at 490 and 470 nm. The emitted fluorescence was directed to an intensified camera. The ratio of emitted fluorescence at excitation wavelengths of 490 and 470 nm was measured, corrected and converted to pHim by constructing a calibration curve. The pHim controls were performed with a pH microelectrode and were correlated with venous blood gas sampling. Results show that pHim is determined with an accuracy of +/- 0.07 pH units and a response time of 1 min. In conclusion pHim mapping of rat intestine can be obtained by fluorescence imaging using BCECF. This technology could be easily adapted for endoscopic pH measurements.

1,2,4-Oxadiazoles identified by virtual screening and their non-covalent inhibition of the human 20S proteasome.

Although several constitutive proteasome inhibitors have been reported these recent years, potent organic, noncovalent and readily available inhibitors are still poorly documented. Here we used a structure- and ligand-based in silico approach to identify commercially available 1,2,4-oxadiazole derivatives as non-covalent human 20S proteasome inhibitors. Their optimization led to the newly synthesized compound 4h that is a mixed proteasomal inhibitor of the chymotrypsin- like activity (K(i) of 26,1 nM and K'(i) of 7.5 nM) which is in addition selective versus the challenging cathepsin B and calpain proteases. Molecular modelling studies corroborated the mechanism of inhibition and suggest an unusual binding of the inhibitor within the S5 binding pocket (ß6 subunit). The cellular effects of our compounds validate their utility as potential pharmacological agents for anti-cancer pre-clinical studies.

Performance of a fixed-taper in-vacuum undulator at SPring-8.

With an in-vacuum undulator, the smallest gaps can be used to achieve high-brilliance radiation within a small spectral width around the harmonics of the fundamental. However, some experiments require a scan over a much wider range of energy within timescales which are impossible to reach via gap tuning. For standard undulators a flat spectrum is usually obtained by using a variable tapered gap. Unfortunately, the mechanical design of the in-vacuum undulator used at SPring-8 is hardly compatible with the extra degree of freedom necessary to adjust the taper mechanically. New magnetic designs are investigated to overcome this problem; their performances are compared with the performances of a fixed-taper in-vacuum undulator for a source of photons in the 5-15 keV range (energy of the fundamental) with an energy width of 1.5 keV.

In-vacuum undulators of SPring-8.

Most of the SPring-8 insertion devices are in-vacuum undulators except for the soft X-ray devices and one elliptical wiggler. The standard-type SPring-8 in-vacuum undulator has a period of 32 mm and a minimum gap of 8 mm. The fundamental radiation energy ranges from 5.2 to 18.5 keV. Three standard in-vacuum undulators are already installed in the ring and are operating without any problems. The magnetic field correction, the vacuum system and the commissioning of the in-vacuum undulators are described in this paper.

Development of an in-vacuum minipole undulator.

An in-vacuum minipole (short-period) insertion device has been developed in a collaboration between SPring-8 and the National Synchrotron Light Source (NSLS). The magnetic arrays were constructed by SPring-8 and were installed in a chamber with mechanical parts in the X-ray ring (E = 2.584 GeV) at the NSLS in May 1997. The device is made of permanent magnets with 30.5 periods and a period length of 11 mm. It is designed to produce fundamental radiation at 4.6 keV, and, with a modest value of deflection parameter (K = 0.7 at 3.3 mm gap), enables higher harmonics to be used for a variety of experiments.

In-vacuum figure-8 undulator for hard X-rays with both horizontal and vertical polarization.

A figure-8 undulator of the in-vacuum type has been adopted as an insertion device for BL24XU, the Hyogo Beamline at SPring-8, to provide hard X-rays with both horizontal and vertical polarization instead of a tandem undulator consisting of horizontal and vertical undulators. The undulator will be operated with the gap almost fixed at 11.6 mm to provide the fundamental radiation with horizontal polarization at 9.5 keV and the 1.5th harmonic with vertical polarization at 14 keV.

Construction of a vertical undulator at SPring-8.

A new-type insertion device (ID) has been constructed for the structural-biology beamline (BL45XU) at SPring-8. The ID consists of two undulators which can provide vertical polarized radiation (vertical undulators). By changing the individual gap of each undulator independently, photons with two different energies can be obtained. Magnetic field measurements show that the maximum horizontal field is 0.49 T and the magnetic performance is as good as expected.

SPring-8 twin helical undulator.

There are several ways of producing circularly polarized light, such as using asymmetric devices, crossed undulators etc. The SPring-8 helical undulator introduces a simple way of producing both horizontal and vertical fields in one undulator. All the magnet arrays are arranged above and below the plane of the electron orbit, so there is no limitation of access from the sides of the undulator. For the SPring-8 BL25SU, two helical undulators will be installed in tandem, and the helicity of the polarization can be switched at up to 10 Hz using five kicker magnets.

Development of an elliptical multipole wiggler at SPring-8.

An elliptical multipole wiggler (EMPW) based on a new concept has been installed on the SPring-8 storage ring. The EMPW, with a 120 mm period, has a critical energy of 50 keV at a gap of 20 mm. It will provide high-brilliance hard circularly polarized X-rays in the 100-300 keV range to the pilot beamline dedicated to materials science: the Compton scattering beamline. Field measurements, field integrals, the expected fluxes, polarization rates, power and power densities are presented for two operating gaps: 30 mm during commissioning, 20 mm later.

An insertion device for the soft X-ray photochemistry beamline at SPring-8.

A figure-8 undulator is an insertion device (ID) proposed at SPring-8 to reduce the heat-load problems encountered with ordinary linear undulators. This device is useful when the K value is high in order to obtain low-energy photons as the fundamental of the undulator radiation. It has therefore been adopted as the ID for the soft X-ray photochemistry beamline (BL27SU) at SPring-8 because linearly polarized photons between 100 and 2000 eV are necessary for the experiments on this beamline. The figure-8 undulator for BL27SU is now under construction and, in the first phase of operation, is expected to cover the energy range down to 500 eV. In the second and third phases, the lowest energy may be reduced to 170 and 100 eV, respectively.

Gastric mucosal pH and blood flow during weaning from mechanical ventilation in patients with chronic obstructive pulmonary disease.

To determine if gastric intramucosal pH changes during weaning from mechanical ventilation are related to gastric mucosal blood flow modifications, we studied 16 ventilator-supported patients with chronic obstructive pulmonary disease (COPD) who tolerated a 2-h trial of spontaneous breathing with pressure support ventilation and were successfully extubated and 11 patients with COPD who failed such a trial. Gastric mucosal perfusion was assessed using gastric intramucosal pH (pH(i)) by tonometry and laser-Doppler flowmetry. During the weaning attempt, the failure weaning group developed a rapid, shallow breathing pattern with acute respiratory acidosis. The pH(i) was lower and gastric intramucosal PCO(2) (PCO(2)im) was higher in the failure weaning group than in the successful weaning group (p < 0.05). No change in gastric intramucosal-arterial PCO(2) difference was observed and a linear correlation was found between arterial PCO(2) and PCO(2)im (r(2) = 0.70; p < 0.001). Cardiac index increased in the failure group (p < 0.05) and remained stable in the success group whereas gastric mucosal blood flow decreased in the failure group (H(120) (min): -22 +/- 11% from baseline; p < 0.05) and increased in the success group (H(120) (min): 85 +/- 27% from baseline; p < 0.05). We conclude that gastric intramucosal pH changes during a 2-h weaning trial are mainly due to arterial PCO(2) variations. Nevertheless, gastric mucosal blood flow changes do occur and differ according to the weaning success or failure.

Inhaled nitric oxide modulates leukocyte kinetics in the mesenteric venules of endotoxemic rats.

OBJECTIVE: to determine whether inhaled nitric oxide (NO) would alter leukocyte kinetics in the septic microvasculature. DESIGN: Randomized, controlled trial. SETTING: Experimental laboratory. SUBJECTS: Male Sprague Dawley rats. INTERVENTIONS: Rats were treated with either saline or endotoxin (10 mg/kg, iv) and then allowed to breathe either air or air plus NO (10 ppm). MEASUREMENTS AND MAIN RESULTS: After a 4-hr period, rolling, firm adhesion, and emigration of leukocytes and endothelial dysfunction were monitored in mesenteric venules by using intravital videomicroscopy. Compared with controls, endotoxemic rats exhibited a profound influx in mesenteric venule rolling leukocytes (55+/-17 vs. 70+/-19 leukocytes/min; p < .05), associated with a reduction of leukocyte rolling velocity (83+/-14 vs. 34+/-3 microm/sec; p < .05). In endotoxemic rats, venular endothelium leukocyte firm adhesion (1.15+/-0.32 vs. 4.08+/-0.96 leukocytes/ 100 microm; p < .05) and emigration (0.84+/-0.47 vs. 4.23+/-1.2 leukocytes/100 microm; p < .05) increased compared with controls. Inhaled NO had no effect on leukocyte kinetics in control rats. Inhaled NO significantly attenuated endotoxin-induced venular endothelium leukocyte adhesion (4.08+/-0.96 vs. 1.86+/-0.76 leukocytes/100 microm; p < .05) and emigration (4.23+/-1.2 vs. 1.68+/-0.72 leukocytes/100 microm; p < .05). Compared with control rats, macromolecular (FITC-dextran) vascular leakage, expressed as the perivenular/intravenular fluorescence intensity ratio, increased in endotoxemic rats (0.56+/-0.02 vs. 0.81+/-0.05; p < .01). Endotoxin-induced macromolecular vascular leakage increases were partially prevented by inhaled NO (0.66+/-0.01 vs. 0.56+/-0.02; p < .05). CONCLUSION: These observations suggest that inhaled NO reduces leukocyte adhesion and the degree of vascular permeability dysfunction in mesenteric venule of endotoxemic rats.

Dobutamine improves gastrointestinal mucosal blood flow in a porcine model of endotoxic shock.

OBJECTIVE: To test the hypothesis that saline solution plus dobutamine increases gastrointestinal mucosal perfusion better than saline solution alone in a model of endotoxic shock. DESIGN: Prospective, randomized, unblinded study. SETTING: Animal research laboratory affiliated with a university teaching hospital. SUBJECTS: Twelve female pigs, weighing 30 to 32 kg. INTERVENTIONS: Animals were anesthetized, and their lungs were mechanically ventilated. Catheters were inserted into the right atrium, pulmonary artery, and carotid artery for blood sampling and blood pressure and cardiac output measurements. A tonometer and a laser Doppler probe were placed in the lumen of the stomach and the ileum for determination of mucosal acid-base status and measurement of mucosal blood flow. Group 1 animals (n = 6) received an infusion (T = 0 min) of 150 mcirog/kg Escherichia coli endotoxin and normal saline solution (0.3 mL/kg/min). Group 2 animals (n = 6) received an infusion of endotoxin and were resuscitated with the same method as used in group 1, but an infusion of dobutamine (5 microg/kg/min) was begun at T = 60 mins, and continued for the duration of the experiment. MEASUREMENTS AND MAIN RESULTS: Both experimental regimens produced shock, with decreased mean arterial pressure and systemic vascular resistance, without change in cardiac output and oxygen delivery. Endotoxin plus saline infusion decreased gastrointestinal mucosal blood flow to <60% of baseline and decreased gastrointestinal pH. In contrast, gastrointestinal mucosal blood flow returned to baseline values, and intramucosal pH tended to normalize by the end of the saline solution plus dobutamine resuscitative protocol. CONCLUSION: Compared with saline solution alone, saline solution plus dobutamine increased blood flow to the gastrointestinal mucosa, and may have partially improved oxygenation.

SPring-8 in-vacuum undulator beam test at the ESRF.

Before the commissioning of SPring-8, the in-vacuum hybrid undulator developed at SPring-8 had been brought to the ESRF for the first beam test in the summer of 1996. The purpose of this test was to investigate the influence of the in-vacuum undulator on the beam and check its vacuum system. However, heating by the resistive wall impedance turned out to be a critical issue for the in-vacuum undulators.

Performance of a helical undulator of the UVSOR.

A helical undulator was installed in the 0.75 GeV storage ring of the UVSOR facility of the Institute for Molecular Science. The undulator was designed to produce the fundamental of the circularly polarized undulator radiation in the energy range 2-43 eV, and the higher harmonics with elliptical polarization in the energy range up to 300 eV. Recently, the first spectrum from the undulator was observed. The performance of the undulator and the obtained spectrum are reported.