Altered molecular specificity of surfactant phosphatidycholine synthesis in patients with acute respiratory distress syndrome

Background

Acute respiratory distress syndrome (ARDS) is a life-threatening critical illness, characterised by qualitative and quantitative surfactant compositional changes associated with premature airway collapse, gas-exchange abnormalities and acute hypoxic respiratory failure. The underlying mechanisms for this dysregulation in surfactant metabolisms are not fully explored. Lack of therapeutic benefits from clinical trials, highlight the importance of detailed in-vivo analysis and characterisation of ARDS patients according to patterns of surfactant synthesis and metabolism.

Methods

Ten patients with moderate to severe ARDS were recruited. Most (90%) suffered from pneumonia. They had an infusion of methyl-D₉-choline chloride and small volume bronchoalveolar lavage fluid (BALF) was obtained at 0,6,12,24,48,72 and 96 hours. Controls were healthy volunteers, who had BALF at 24 and 48 hours after methyl-D₉-choline infusion. Compositional analysis and enrichment patterns of stable isotope labelling of surfactant phosphatidylcholine (PC) was determined by electrospray ionisation mass spectrometry.

Results

BALF of patients with ARDS consisted of diminished total PC and fractional PC16:0/16:0 concentrations compared to healthy controls. Compositional analysis revealed, reductions in fractional compositions of saturated PC species with elevated levels of longer acyl chain unsaturated PC species. Molecular specificity of newly synthesised PC fraction showed time course variation, with lower PC16:0/16:0 composition at earlier time points, but achieved near equilibrium with endogenous composition at 48 hours after methyl-D₉-choline infusion. The enrichment of methyl-D₉-choline into surfactant total PC is nearly doubled in patients, with considerable variation between individuals.

Conclusions

This study demonstrate significant alterations in composition and kinetics of surfactant PC extracted from ARDS patients. This novel approach may facilitate biochemical phenotyping of ARDS patients according to surfactant synthesis and metabolism, enabling individualised treatment approaches for the management of ARDS patients in the future.     

Sex-limited expression of gene Loci controlling flagellar membrane agglutination in the chlamydomonas mating reaction

Mutant strains of Chlamydomonas reinhardi that have lost their ability to undergo sexual agglutination via their flagellar tips have been induced to undergo zygotic cell fusion and meiosis, using a flagellar-directed antiserum. Genetic analysis of antiserum-mediated crosses involving five nonagglutinating mt⁺ mutant strains reveals the following: (1) None of the mutations is linked to the mt locus. (2) All of the mutations are "sex-limited," meaning that they can be carried and transmitted by, but not expressed in, mt^- cells. (3) Four of the mutations (imp-2, imp-5, imp-6, imp-7) are either allelic or closely linked to one another, with imp-8 defining a second locus.     

Sexual agglutinins from the Chlamydomonas flagellar membrane. Partial purification and characterization

Chlamydomonas sexual agglutinins have been quantitatively extracted from isolated flagella in vitro using the dialyzable nonionic detergent octyl-D-glucopyranoside and from cells in vivo with 12.5 mM EDTA. Both preparations elicit normal sexual responses from gametes of complementary, but not like, mating types. Extracts of vegetative cells and several agglutination-deficient (imp) mutants are totally inactive. Agglutinin activity is sensitive to trypsin, mild periodate oxidation, and heating at 60 degrees C for 1 min. These findings, coupled with the size of the molecule (it is excluded from Sepharose 6B and sediments as a 12 S particle in sucrose gradients) lead us to propose that the Chlamydomonas sexual agglutinins are large glycoproteins or glycoprotein aggregates which associate with the flagellar membrane in an extrinsic fashion. Partial purification of in vivo 125I-surface labeled EDTA extracts rules out several surface polypeptides, including the bulk of material migrating in the region of the major membrane glycoprotein (Mr 350,000), as agglutinin candidates and indicates that the active molecule is a minor component of the flagellar membrane. In addition, in vitro assays suggest a mechanism for in vivo sexual agglutination whereby stable adhesion is achieved by the active redistribution of agglutinins to the flagellar tips.     

Quantification of the magnification and distortion effects of a pediatric flexible video-bronchoscope

Background

Flexible video bronchoscopes, in particular the Olympus BF Type 3C160, are commonly used in pediatric respiratory medicine. There is no data on the magnification and distortion effects of these bronchoscopes yet important clinical decisions are made from the images. The aim of this study was to systematically describe the magnification and distortion of flexible bronchoscope images taken at various distances from the object.

Methods

Using images of known objects and processing these by digital video and computer programs both magnification and distortion scales were derived.

Results

Magnification changes as a linear function between 100 mm (×1) and 10 mm (×9.55) and then as an exponential function between 10 mm and 3 mm (×40) from the object. Magnification depends on the axis of orientation of the object to the optic axis or geometrical axis of the bronchoscope. Magnification also varies across the field of view with the central magnification being 39% greater than at the periphery of the field of view at 15 mm from the object. However, in the paediatric situation the diameter of the orifices is usually less than 10 mm and thus this limits the exposure to these peripheral limits of magnification reduction. Intraclass correlations for measurements and repeatability studies between instruments are very high, r = 0.96. Distortion occurs as both barrel and geometric types but both types are heterogeneous across the field of view. Distortion of geometric type ranges up to 30% at 3 mm from the object but may be as low as 5% depending on the position of the object in relation to the optic axis.

Conclusion

We conclude that the optimal working distance range is between 40 and 10 mm from the object. However the clinician should be cognisant of both variations in magnification and distortion in clinical judgements.     

A neuroscientific approach to normative judgment in law and justice

Developments in cognitive neuroscience are providing new insights into the nature of normative judgment. Traditional views in such disciplines as philosophy, religion, law, psychology and economics have differed over the role and usefulness of intuition and emotion in judging blameworthiness. Cognitive psychology and neurobiology provide new tools and methods for studying questions of normative judgment. Recently, a consensus view has emerged, which recognizes important roles for emotion and intuition and which suggests that normative judgment is a distributed process in the brain. Testing this approach through lesion and scanning studies has linked a set of brain regions to such judgment, including the ventromedial prefrontal cortex, orbitofrontal cortex, posterior cingulate cortex and posterior superior temporal sulcus. Better models of emotion and intuition will help provide further clarification of the processes involved. The study of law and justice is less well developed. We advance a model of law in the brain which suggests that law can recruit a wider variety of sources of information and paths of processing than do the intuitive moral responses that have been studied so far. We propose specific hypotheses and lines of further research that could help test this approach.     

THE ISOLATION OF MOUSE HEPATOCYTE GAP JUNCTIONS : Preliminary Chemical Characterization and X-Ray Diffraction

A method is reported for isolating a preparation of hepatic gap junctions from the mouse. The method involves a collagenase digestion, treatment with the detergent Sarkosyl NL-97, and ultrasonication, followed by sucrose gradient ultracentrifugation. A run with 36 animals yields 0.1–0.5 mg protein. Electron microscopy with thin-sectioning and negative staining techniques reveals that the final pellet is a very pure preparation of gap junctions, accompanied by a small amount of amorphous contamination. Polyacrylamide-gel electrophoresis of sodium dodecyl sulfate (SDS)-solubilized material shows one major protein in the junction, with an apparent mol wt of 20,000, and two minor components. Thin-layer chromatography demonstrates one major and one minor phospholipid, and some neutral lipid. Low-angle X-ray diffraction of wet and dried specimens show reflections which index on an 86 A center-to-center hexagonal lattice, corresponding closely to electron microscope data. Dried specimens also show a lamellar diffraction, corresponding to the total profile thickness of the junction (150 A).     

Ice premelting during differential scanning calorimetry

Premelting at the surface of ice crystals is caused by factors such as temperature, radius of curvature, and solute composition. When polycrystalline ice samples are warmed from well below the equilibrium melting point, surface melting may begin at temperatures as low as -15 degrees C. However, it has been reported (. Biophys. J. 65:1853-1865) that when polycrystalline ice was warmed in a differential scanning calorimetry (DSC) pan, melting began at about -50 degrees C, this extreme behavior being attributed to short-range forces. We show that there is no driving force for such premelting, and that for pure water samples in DSC pans curvature effects will cause premelting typically at just a few degrees below the equilibrium melting point. We also show that the rate of warming affects the slope of the DSC baseline and that this might be incorrectly interpreted as an endotherm. The work has consequences for DSC operators who use water as a standard in systems where subfreezing runs are important.     

Isolation of four major subunits from Clostridium thermocellum cellulosome and their synergism in the hydrolysis of crystalline cellulose

The cellulosome of Clostridium thermocellum, purified by affinity chromatography, was dissociated under mild conditions and separated by SDS-PAGE. Two major p-nitrophenylcellobiosidases (PNPCases I and II) corresponding to the S₅ (103 kDa) and S₈ (78 kDa) subunits and one major carboxymethylcellulase (CMCase) coinciding with the S₁₁ (60.5 kDa) subunit were isolated and characterized using carboxymethylcellulose (CMC), H₃PO₄-swollen cellulose and cello-oligosaccharides. Both PNPCases showed little effect on the viscosity of CMC and released twice as much total sugar as reducing sugar from H₃PO₄-swollen cellulose. The CMCase released ten times more total sugar than reducing sugar from H₃PO₄-swollen cellulose and reduced the viscosity of CMC rapidly. None of these enzymes was active on cellotriose. Both PNPCases released cellobiose from cellotetraose, and cellobiose and cellotriose from cellopentaose. In contrast, CMCase was active only on cellopentaose and released mainly glucose. Use of MeUmb(Glc)_n revealed that both PNPCases cleaved preferentially either the second or fourth linkage from the non-reducing end while the CMCase was specific for the internal glycosidic bonds. Thus, the PNPCases and CMCase behaved as typical exo- and endoglucanases, respectively. When tested individually, all three enzymes degraded Avicel only to a small extent. A 1.5–2.0-fold increase in sugar release was observed when CMCase was combined with either PNPCase I, II or both. Combining S₁ with either PNPCase II or CMCase resulted in fourfold synergism in the hydrolysis of Avicel. Synergism was sevenfold when all three enzymes were combined with S₁.     

Factors affecting the behavior of captive white rhinoceros (Ceratotherium simum) and the accuracy of ad-hoc keeper data

Although white rhinoceros (Ceratotherium simum) are common in captivity, few behavioral studies have been conducted and there is seemingly no research for immersive exhibits where potential for visitor effects is high. Moreover, little information exists on possible effects of weather and temperature on rhino outside their native range. Here we analyze 14,501 observations of rhino in a drive-through enclosure. Data were collected by researchers (n = 12,160 datapoints) and keepers (n = 2341 datapoints) over a 4-month period. We aimed to: (1) quantify behavior using detailed researcher-collected data and contemporaneous but ad hoc keeper-collected data; (2) compare datasets statistically; (3) establish effects of visitors, temperature, and weather on behavior; and (4) assess the influence of visitors on similarity of researcher/keeper datasets. Activity budgets were similar to the wild and the single previous study from a traditional (nondrive-through) enclosure. There was some discrepancy in activity budgets between researcher and keeper data due to significant differences in recorded frequency of two rare behaviors (horn rub; social interaction) and two behaviors that could be easily confused (grazing vs. standing with head-down): recording of other behaviors matched well. Weather and temperature affected behavior, with rhino becoming more sedentary (−locomotion, grazing; +resting, standing, and sedentary eating of hay) on hot/sunny days compared to cool/wet days. The number of visitor vehicles had a fairly negligible effect but resting was lower on busy days, possibly as vigilance increased. The match between researcher/keeper datasets was lowest when visitor numbers were high, suggesting visitors might affect keeper ability to accurately record behavior.