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Segmental flexibility of the C1q subcomponent of human complement and its possible role in the immune response.

Fluorescence polarization techniques were used to study the rotational dynamics of the C1q subcomponent of human complement. C1q was covalently labeled with dansyl (DNS) chloride. Digestion of either C1q-DNS4.0 or C1q-DNS1.8 conjugates with pepsin showed that about 75% of the DNS probes were attached to the C1q globular heads and that the remainder were on the collagen-like stalk (peptic fragment). C1q-DNS conjugates readily agglutinated IgG-coated latex beads and combined with C1r2C1s2 to form hemolytically active 16 S C1-DNS. Both C1q-DNS and C1-DNS samples displayed steady-state rotational correlation time and fluorescence lifetime transitions near 48 degrees C. Hydrodynamic studies showed that C1q formed soluble aggregates near the transition temperature. In contrast, stalk samples with a DNS probe apparently attached to the large central fibril showed no thermal transitions or aggregation even when heated above 50 degrees C. Nanosecond fluorescence depolarization measurements detected restricted flexible motions of the C1q heads with an associated rotational correlation time, phi s, of about 25 ns. The C1q anisotropy decay was dominated, however, by a long component, phi L, of perhaps 1000 ns. Except for probe wiggle, the stalk-DNS anisotropy profile was essentially flat. The rapid rotations associated with phi s could represent restricted twisting motions of the arm-head segments or wobbling motions of the heads themselves. Such motions may facilitate binding of the C1q heads to immune complexes. Straightforward diffusion calculations indicated that phi L could represent either global tumbling of the entire C1q molecule or wagging motions of the individual arm-head segments, as suggested by electron micrographs. Upon binding of the C1q heads to an activator, some of the C1q segments may be held in a slightly more open or more closed conformation, which in turn may trigger activation of the C1 proenzymes. In conclusion, we suggest a plausible triggering mechanism for C1 activation that is compatible with the flexible properties of its subcomponents.

Complement Activating Enzymes↗

[Sensitivity to fosfomycin of bacteria isolated at the Pitié-Salpétrière Hospital in 1982 and 1983].

Susceptibility of 16 056 strains isolated in 1982 and 1983 to fosfomycin (FOS) was tested with Mueller-Hinton medium and discs 50 micrograms FOS + 25 micrograms glucose-6-phosphate (G6P) (cutoff diameter 14 mm). For 3 411 strains, inhibition zone diameters were recorded. Almost all E. coli, Citrobacter, Salmonella and S. aureus were susceptible to FOS as well as 70% of Serratia, E. cloacae, P. mirabilis and 50% of Klebsiella and Enterobacter sp. Only 27% of P. aeruginosa, 20% of indole positive Proteus and 8% of Acinetobacter were sensitive to FOS. E. coli, Citrobacter and S. aureus seem to exhibit the highest susceptibilities to FOS. Sensitivity rates of E. cloacae, Serratia, P. aeruginosa, and especially Klebsiella seemed slightly underestimated by the disk diffusion method, but differentiation between susceptible and resistant strains was facilitated by rating as susceptible those strains with inhibition zone diameters between 12 and 14 mm. This method also seemed to underrate the sensitivity of susceptible Enterobacteriaceae, probably because of inadequate G6P concentrations at this point of the disk.

Acinetobacter↗

[New trends in limb salvage. Endovascular techniques applied to leg arteries].

UNLABELLED: Femoro-distal bypasses are not always feasible in patients presenting with "critical" chronic ischemia. The results of endovascular therapies carried out over a 2-year period were analyzed. PATIENTS--METHODS: 23 patients with critical ischemia (rest pain 13%, gangrene 87%) had 25 procedures on 29 leg arteries; percutaneous transluminal angioplasty in 17 arteries, rotational atherectomy in 10 arteries, laser recanalization (1 artery), directional atherectomy (1 artery). RESULTS: The hospital mortality rate was 4.3%. The cumulative patency and limb salvage rates are respectively 51% and 77% at 6 months, 34% and 71% at 12 months. DISCUSSION: Candidates for tibial-peroneal endovascular techniques should have a threatened limb, as long as the consequences of failed procedures on patients presenting with claudication can be disastrous, and as long as mid-term patency rates reported in the literature are not fair enough. In localized stenoses or short occlusions with adequate runoff, endovascular techniques are a good alternative to femorotibial bypasses for limb salvage. In diffuse lesions with no possibility of bypass, endovascular techniques can facilitate limb salvage, even if the mid-term patency rate is poor. When conventional therapies cannot face critical ischemia, endovascular therapies can provide a fair limb salvage rate.

Aged↗

[Distal arteritis of the legs: endovascular treatment].

Femoro-tibial bypasses are not always feasible in patients presenting with "critical" chronic ischemia. The results of endovascular therapies carried out over a 2-year period are analyzed. Twenty-three patients with critical ischemia (rest pain 13%, gangrene 87%) had 25 procedures on 29 leg arteries: percutaneous transluminal angioplasty in 17 arteries, rotational atherectomy in 10 arteries, laser recanalization (1 artery), directional atherectomy (1 artery). The hospital mortality rate was 4.3%. The cumulative patency and limb salvage rates were respectively 51% and 77% at 6 months, 34% and 71% at 12 months. Candidates for tibial-peroneal endovascular techniques should have a threatened limb, as long as the consequences of failed procedures on patients presenting with claudication can be disastrous, and as long as mid-term patency rates reported in the literature are not fair enough. In localized stenosis or short occlusions with adequate runoff, endovascular techniques are a good alternative to femorotibial bypasses for limb salvage. In diffuse lesions with no possibility of bypass, endovascular techniques can facilitate limb salvage, even if the mid-term arterial patency rate is poor. When conventional therapies cannot face critical ischemia, endovascular therapies can provide a fair limb salvage rate.

Aged↗

Resistance of mice to experimental leishmaniasis is associated with more rapid appearance of mature macrophages in vitro and in vivo.

Resistance to murine leishmaniasis has been related to the propagation of specific Th cell subsets (Th1 and Th2). This study shows that there are differences between resistant and susceptible mice in the initial myelomonocytic infiltrate, which precede the specific T cell response. After subcutaneous injection of 2 x 10(7) Leishmania major into footpads of resistant C57Bl/6 and susceptible BALB/c mice we performed immunohistochemical studies on the infiltrate. Two days after infection the percentage of more mature, F4/80-positive macrophages in the lesion increased faster in C57Bl/6 mice (63%) than in BALB/c mice (29%). The same strain-specific differences were observed after infection of corresponding strains of athymic mice (57.2% in C57Bl/6 nu/nu; 33.6% in BALB/c nu/nu), thus excluding a T cell-controlled phenomenon. After 1 wk the infiltrate in susceptible mice began to reveal significantly more cells containing MRP14, which is expressed by granulocytes and less mature monocytes but not by mature macrophages. No corresponding differences were found between athymic strains, suggesting that at this point organization of the infiltrate falls under control of protective T cells. In bone marrow cultures of BALB/c and C57Bl/6 mice, the percentage of F4/80-positive macrophages was also increasing faster in C57Bl/6 mice than in BALB/c mice. Increased expression of the F4/80 Ag was associated with higher leishmanicidal activity of C57Bl/6 macrophages. MRP14-positive bone marrow cells on the other hand were rarely infected by parasites. We suggest 1) that the earlier appearance of leishmanicidal macrophages in lesions of C57Bl/6 mice could influence propagation of either Th1 or Th2 cells by reduction of parasite load or by differential secretion of decisive cytokines and 2) that the diffuse accumulation of granulocytes and inflammatory monocytes in susceptible mice facilitates spread of disease.

Animals↗

Apical membrane limits urea permeation across the rat inner medullary collecting duct.

UNLABELLED: Urea diffuses across the terminal inner medullary collecting duct (IMCD) via a facilitated transport pathway. To examine the mechanism of transcellular urea transport, membrane-apparent urea (Purea) and osmotic water (Pf) permeabilities of IMCD cells were measured by quantitative light microscopy in isolated IMCD-2 tubules perfused in the absence of vasopressin. Basolateral membrane Pf, determined by addition of raffinose to the bath, was 69 microns/s. Basolateral membrane Purea, determined by substituting urea for raffinose without change in osmolality, was 14 X 10(-5) cm/s. Bath phloretin inhibited basolateral Purea by 85% without a significant effect on Pf. The basolateral reflection coefficient for urea, determined by addition of urea in the presence of phloretin, was 1.0. These results indicate that urea crosses the basolateral membrane by diffusion, and not by solvent drag. In perfused tubules, the rate of cell swelling following substitution of urea for mannitol was significantly greater with bath than lumen changes. After correcting for membrane surface area, the basolateral membrane was twofold more permeable than the apical membrane. CONCLUSIONS: (a) in the absence of vasopressin, urea permeation across the IMCD cell is limited by the apical membrane; (b) the basolateral membrane contains a phloretin-sensitive urea transporter; (c) transepithelial urea transport occurs by movement of urea through the IMCD cell.

Animals↗

Continuous measurement of porcine renal cortex microcirculation with enhanced thermal diffusion technology.

Continuous monitoring of renal cortical blood flow (RCBF) in the perioperative setting of aortic or renal vascular surgery could facilitate the early detection of vascular complications, possibly resulting in a reduction of postoperative renal failure. A new prototype system for measurement of parenchymous organ perfusion based on the principle of thermal diffusion ("TD"-Thermal Diffusion Electrode, Thermal Technologies Inc., Cambridge, MA, USA) was used for RCBF measurements in the outer cortex of the porcine kidney. We validated the sensitivity of the device to detect renal blood flow impairment, comparing TD flow data with renal artery blood flow values (RABF), measured by ultrasonic flow probes. The hypothesis was tested that acute disturbances of RCBF, induced by a variable degree of renal artery stenosis, can be immediately detected and continuously monitored by TD measurements in the porcine renal cortex. Mean baseline RCBF measured by TD electrodes was 68.1 +/- 25.0 ml/100 g/min. Mean baseline RABF was 102.1 +/- 26.6 ml/min. Controlled induction of a variable degree of renal arterial occlusion by implanted vascular balloon occluders was always followed by an immediate and proportional decline of RCBF, as measured by TD. Flow data obtained with both methods were significantly correlated by linear regression (r=.82, r2=.68; P < 0.0001). Dynamic changes of RABF in the time course of renal artery partial/total occlusion and arterial flow release could be continuously followed by detection of corresponding flow changes of RCBF. We conclude that the TD system investigated in the current study allows a continuous and sensitive determination of porcine renal cortex perfusion. A clinical evaluation of the method, e.g., in the perioperative setting of aortic or renal transplantation surgery, now appears to be justified.

Animals↗

[Economic analysis of the dissemination of a medical innovation: prenatal diagnosis by early amniocentesis as an example. Part 2. Facilitating decision making in public health for optimal dissemination of an innovation].

Only a specific public health policy can resolve the technical, ethical, and financial problems posed by the diffusion of prenatal diagnosis by amniocentesis for the prevention of Down's syndrome. The purpose of this article is to facilitate decision making in this field by evaluating the costs and benefits associated with the different prevention strategies. Beginning with projections of the number of Down's syndrome births to be expected among women of 35 and older to the year 2000 (assuming that no prenatal diagnosis are made), various objectives combining the age of the target population and usage rates are presented and discussed. An evaluation of the overall costs of prenatal diagnosis and caring for Down's syndrome patients shows that a policy reaching a 50% usage rate for women 38 and older would save up to 66 millions French francs (using the 1981 franc value). On the other hand, it is shown that lowering the incidence of Down's syndrome in a target population of women over 35 to the level of the younger population would imply a usage rate of at least 80%. This goal is unrealistic unless amniocentesis were to become a compulsory examination. In such case, women would be deprived of an essential freedom, in view of the related ethical issues. The authors lastly discuss the different means of increasing usage rates without inhibiting women's freedom of choice.

Adult↗

Getting across the nuclear pore complex: new insights into nucleocytoplasmic transport.

Small ions and molecules can traverse the nuclear pore complex (NPC) simply by diffusion, whereas larger proteins and RNAs require specific signals and factors that facilitate their passage through the NPC. Our understanding of the factors that participate and regulate nucleocytoplasmic transport has increased tremendously over the past years, whereas the actual translocation step through the NPC has remained largely unclear. Here, we present and discuss recent findings on the interaction between the NPC and transport receptors and provide new evidence that the NPC acts as a constrained diffusion pore for molecules and particles without retention signal and as an affinity gate for signal-bearing cargos.

Active Transport, Cell Nucleus↗

Identifying optimal lipid raft characteristics required to promote nanoscale protein-protein interactions on the plasma membrane.

The dynamic lateral segregation of signaling proteins into microdomains is proposed to facilitate signal transduction, but the constraints on microdomain size, mobility, and diffusion that might realize this function are undefined. Here we interrogate a stochastic spatial model of the plasma membrane to determine how microdomains affect protein dynamics. Taking lipid rafts as representative microdomains, we show that reduced protein mobility in rafts segregates dynamically partitioning proteins, but the equilibrium concentration is largely independent of raft size and mobility. Rafts weakly impede small-scale protein diffusion but more strongly impede long-range protein mobility. The long-range mobility of raft-partitioning and raft-excluded proteins, however, is reduced to a similar extent. Dynamic partitioning into rafts increases specific interprotein collision rates, but to maximize this critical, biologically relevant function, rafts must be small (diameter, 6 to 14 nm) and mobile. Intermolecular collisions can also be favored by the selective capture and exclusion of proteins by rafts, although this mechanism is generally less efficient than simple dynamic partitioning. Generalizing these results, we conclude that microdomains can readily operate as protein concentrators or isolators but there appear to be significant constraints on size and mobility if microdomains are also required to function as reaction chambers that facilitate nanoscale protein-protein interactions. These results may have significant implications for the many signaling cascades that are scaffolded or assembled in plasma membrane microdomains.

Animals↗

Ultrarapid desalting of protein solutions for electrospray mass spectrometry in a microchannel laminar flow device.

The adverse effects of nonvolatile salts on the electrospray (ESI) mass spectra of proteins and other biological analytes are a major obstacle for a wide range of applications. Numerous sample cleanup approaches have been devised to facilitate ESI-MS analyses. Recently developed microdialysis techniques can shorten desalting times down to several minutes, the bottleneck being diffusion of the contaminant through a semipermeable membrane. This work introduces an approach that allows the on-line desalting of macromolecule solutions within tens of milliseconds. The device does not employ a membrane; instead, it uses a two-layered laminar flow geometry that exploits the differential diffusion of macromolecular analytes and low molecular weight contaminants. To maximize desalting efficiency, diffusive exchange between the flow layers is permitted only for such a time as to allow full exchange of salt, while incurring minimal macromolecule exchange. Computer simulations and optical studies show that the device can reduce the salt concentration by roughly 1 order of magnitude, while retaining approximately 70% of the original protein concentration. Application of this approach to the on-line purification of salt-contaminated protein solutions in ESI-MS results in dramatic improvements of both the signal-to-noise ratio and the absolute signal intensity. However, efficient desalting requires the diffusion coefficients of salt and analyte to differ by roughly 1 order of magnitude or more. This technique has potential to facilitate high-throughput analyses of biological macromolecules directly from complex matrixes. In addition, it may become a valuable tool for process monitoring and for on-line kinetic studies on biological systems.

Bradykinin↗

Lac repressor-operator interaction: DNA length dependence.

The interaction of the E. coli lac operon repressor with its operator DNA has been directly examined as a function of the length of operator-containing DNA. The apparent bimolecular association rate constants were calculated as ka = (kd/KD), where the dissociation equilibrium constant, KD and the dissociation rate constant, kd, were measured by nitrocellulose filter adsorption assays. The values obtained for the overall association rate constants are compared with theoretical association rate curves for specific mechanisms. Association of the repressor with short operator containing DNA fragments (less than 70 base pairs) occurs at rates expected of three-dimensional diffusion. Our data also imply that at longer DNA lengths a combination of three-dimensional diffusion with one-dimensional sliding along with hopping and/or intersegment transfer must be involved to facilitate the repressor operator association.

Base Composition↗

W/O/W double emulsion technique using ethyl acetate as organic solvent: effects of its diffusion rate on the characteristics of microparticles.

Monomethoxypoly(ethylene glycol)-b-poly(DL-lactide) copolymer (PELA) microparticles loading lysozyme were prepared through a modified W/O/W double emulsion-solvent diffusion method using ethyl acetate (EA) as organic solvent. The modified process was divided into five steps: (1) primary emulsification (W1/O), (2) re-emulsification (W1/O/W2), (3) pre-solidification, (4) solidification and (5) purification. The pre-solidification step was carried out in the modified process to control the diffusion rate of EA from oil phase into outer aqueous phase, in order to prevent the wall polymer from precipitation, which usually occurred when the diffusion rate was too fast. The adequately rapid solidification of microparticle caused by controlled fast diffusion of EA and the use of amphiphilic copolymer PELA as wall material, facilitated a high protein entrapment (always above 94%) and full preservation of bioactivity of entrapped lysozyme. It was found that the volume of the outer aqueous phase in the re-emulsification step and the shear stress in the pre-solidification step had a significant effect on the diffusion rate of EA from the droplets into outer aqueous solution, and thereby on the characteristics of the resultant microparticles. With the volume or the shear stress increasing, the removal rate of EA increased, resulting in rapid solidification of the microparticles. This result led to a lower burst effect and a slower lysozyme release from the microparticles. This study suggests that the modified W/O/W double emulsion-solvent diffusion method with EA as organic solvent is a prospective technique to prepare biodegradable microparticles containing water-soluble sensitive agents.

Acetates↗

A dimensionless number analysis of the hybridization process in diffusion- and convection-driven DNA microarray systems.

The present theoretical analysis aims at providing a general understanding of the combined effect the many different process variables have on the hybridization rate in diffusion- and convection-driven DNA microarray systems. It is shown that all process variables can be grouped into only four different dimensionless numbers (the Damkohler number Da, the dimensionless association constant kappa(A), the dimensionless initial concentration C'(0) and a geometrical ratio alpha). These four numbers have a straightforward physical meaning and only contain easily measurable parameters. Reducing the solution space from 7D to 4D, the dimensionless number representation greatly facilitates the insight in the conditions leading to the occurrence of diffusion-limited hybridization rates in both diffusion- and convection-driven DNA microarray systems. This in turn simplifies their design and the interpretation of the experimental results that are obtained with these systems.

Diffusion↗

Cytoplasmic codiffusion of fatty acids is not specific for fatty acid binding protein.

The intracellular movement of fatty acids is thought to be facilitated through codiffusion with fatty acid binding protein (FABP). Previous work suggested that FABP decreases fatty acid binding to immobile membranes, causing faster cytoplasmic diffusion. However, the specificity for binding to FABP has not been addressed. The aim of the current study was to determine whether specific FABP binding is required or whether binding to other proteins will produce the same effect. A model cytoplasm consisted of a fatty acid, proteins, and liposomes to simulate intracellular membranes. Laser photobleaching (fluorescence recovery after photobleaching) was used to measure the movement of the fluorescent fatty acid 12-N-methyl-7-nitrobenzo-2-oxa-1,3-diazoaminostearate (NBD-stearate) in model cytoplasm, in normal and permeabilized Hep G2 cells, and after incubation of permeabilized cells with bovine serum albumin (BSA) or FABP. Increasing protein in the model cytoplasm increased the diffusion rate in proportion to the extent of protein binding. Cell permeabilization reduced diffusion of NBD-stearate to < 5% of controls. Incubation of permeabilized cells with FABP or BSA resulted in a concentration-dependent increase in the NBD-stearate diffusion rate. BSA was more effective than FABP in binding NBD-stearate and increasing its diffusion rate after permeabilization. Proteins like FABP promote the diffusion of fatty acids. Removal of these proteins drastically reduces cytoplasmic diffusion. Substitution with BSA reestablishes the diffusive flux, suggesting that specific binding to FABP is not required. These data support a role for intracellular binding proteins in facilitating the cytoplasmic movement of fatty acids.

4-Chloro-7-nitrobenzofurazan↗

pH compartmented w/o/w multiple emulsion: a diffusion study.

In order to develop w/o/w emulsions characterized by two separate aqueous phases of different pH, a preliminary study was carried out to obtain a better insight into the possible diffusion processes taking place between an inner acidic aqueous phase and an external phase of higher pH (pH approximately 6). In fact, such systems could be of great interest for pharmaceutical use. For this purpose, a model emulsion was formulated. The study of pH and conductivity showed that acidic species transport take place between the two aqueous compartments. The three main release mechanisms that might be responsible for this passage across the oil phase were investigated: breakdown of oil globules, facilitated transport by surfactant micelles across the oil phase or by Fickian diffusion. It appears that this last mechanism was involved. In order to control this diffusion process, an alkaline species, octadecylamine was introduced in the oil phase. This compound could form an ion pair with the lactate ion at the interface of the external aqueous phase and the oil phase, thus, limiting the acidification of the external aqueous phase.

Algorithms↗

Numerical and experimental analysis of an axial flow left ventricular assist device: the influence of the diffuser on overall pump performance.

Thousands of adult cardiac failure patients may benefit from the availability of an effective, long-term ventricular assist device (VAD). We have developed a fully implantable, axial flow VAD (LEV-VAD) with a magnetically levitated impeller as a viable option for these patients. This pump's streamlined and unobstructed blood flow path provides its unique design and facilitates continuous washing of all surfaces contacting blood. One internal fluid contacting region, the diffuser, is extremely important to the pump's ability to produce adequate pressure but is challenging to manufacture, depending on the complex blade geometries. This study examines the influence of the diffuser on the overall LEV-VAD performance. A combination of theoretical analyses, computational fluid (CFD) simulations, and experimental testing was performed for three different diffuser models: six-bladed, three-bladed, and no-blade configuration. The diffuser configurations were computationally and experimentally investigated for flow rates of 2-10 L/min at rotational speeds of 5000-8000 rpm. For these operating conditions, CFD simulations predicted the LEV-VAD to deliver physiologic pressures with hydraulic efficiencies of 15-32%. These numerical performance results generally agreed within 10% of the experimental measurements over the entire range of rotational speeds tested. Maximum scalar stress levels were estimated to be 450 Pa for 6 L/min at 8000 rpm along the blade tip surface of the impeller. Streakline analysis demonstrated maximum fluid residence times of 200 ms with a majority of particles exiting the pump in 80 ms. Axial fluid forces remained well within counter force generation capabilities of the magnetic suspension design. The no-bladed configuration generated an unacceptable hydraulic performance. The six-diffuser-blade model produced a flow rate of 6 L/min against 100 mm Hg for 6000 rpm rotational speed, while the three-diffuser-blade model produced the same flow rate and pressure rise for a rotational speed of 6500 rpm. The three-bladed diffuser configuration was selected over the six-bladed, requiring only an incremental adjustment in revolution per minute to compensate for and ease manufacturing constraints. The acceptable results of the computational simulations and experimental testing encourage final prototype manufacturing for acute and chronic animal studies.

Blood Circulation↗

Intravascular lymphoma: magnetic resonance imaging correlates of disease dynamics within the central nervous system.

BACKGROUND: Intravascular lymphoma (IVL) is a rare non-Hodgkin's lymphoma with relative predilection for the central nervous system. In the absence of extraneural manifestations, the disease is not recognised until autopsy in the majority of cases underlining the need for new clinical markers. METHODS: This is a retrospective series of five patients with IVL seen at a single institution over three years. An advanced magnetic resonance imaging (MRI) protocol was performed at various time points prior to diagnosis and during treatment. RESULTS: MRI revealed multiple lesions scattered throughout the cerebral hemispheres; the brainstem, cerebellum, and spinal cord were less frequently involved. On initial presentation, hyperintense lesions were seen on diffusion weighted images suggestive of ischaemia in three of four patients in whom the images were obtained at that time point. In four patients lesions were also identifiable as hyperintense areas on fluid attenuated inversion recovery (FLAIR) sequences. Initial contrast enhancement was encountered in three cases. Diffusion weighted imaging lesions either vanished or followed the typical pattern of an ischaemic small vessel stroke with evolution of abnormal FLAIR signal followed by enhancement with gadolinium in the subacute stage and tissue loss in the chronic stage. Diffusion weighted imaging and FLAIR abnormalities proved to be partially reversible, correlating with the response to chemotherapy. CONCLUSION: We provide the first detailed description of the dynamic pattern of diffusion weighted MRI in IVL. These patterns in combination with systemic findings may facilitate early diagnosis and serve as a new tool to monitor treatment response.

Adult↗