Burn Injury Reduces Neutrophil Directional Migration Speed in Microfluidic Devices

Thermal injury triggers a fulminant inflammatory cascade that heralds shock, end-organ failure, and ultimately sepsis and death. Emerging evidence points to a critical role for the innate immune system, and several studies had documented concurrent impairment in neutrophil chemotaxis with these post-burn inflammatory changes. While a few studies suggest that a link between neutrophil motility and patient mortality might exist, so far, cumbersome assays have prohibited exploration of the prognostic and diagnostic significance of chemotaxis after burn injury. To address this need, we developed a microfluidic device that is simple to operate and allows for precise and robust measurements of chemotaxis speed and persistence characteristics at single-cell resolution. Using this assay, we established a reference set of migration speed values for neutrophils from healthy subjects. Comparisons with samples from burn patients revealed impaired directional migration speed starting as early as 24 hours after burn injury, reaching a minimum at 72–120 hours, correlated to the size of the burn injury and potentially serving as an early indicator for concurrent infections. Further characterization of neutrophil chemotaxis using this new assay may have important diagnostic implications not only for burn patients but also for patients afflicted by other diseases that compromise neutrophil functions.     

Age-related variability in occlusal wear planes

Investigation of two populations of 136 individuals shows several patterns of occlusal wear plane change which are positively correlated with age. For individuals up to the age of 18, there is a characteristic pattern in which the occlusal wear planes of the mandibular teeth are lingually sloped and those of the maxillary dentition buccally sloped, with the exception of the maxillary premolars, which are also lingually sloped. The long axes of the mandibular teeth give them a lingual orientation relative to the maxillary teeth, and the long axes of the maxillary molars, by contrast, are buccally oriented. In the 18-30 age range for all sexes, the mandibular M1 becomes buccally sloped on its occlusal surface while the occlusal wear plane on the maxillary M1 becomes lingually sloped. Later age changes indicate a trend for the mandibular premolars to become buccally sloped, while the wear planes of the maxillary premolars remain lingually sloped. There is a corresponding tendency for the maxillary and mandibular second molars to undergo changes in the initial orientation of the occlusal wear planes.     

Candida utilis NAD+ kinase: purification, properties and affinity gel studies

1. NAD+ kinase (ATP:NAD+ 2' phosphotransferase, EC 2.7.1.23) has been purified to apparent enzymic homogeneity on Blue Sepharose CL-6B. 2. The molecular weight of the active species is about 260,000 as determined by PAGE and gel chromatography. Protein staining (PAGE) revealed minor bands with molecular weight values of 40,000, 140,000 and 550,000. Subunit studies (SDS-PAGE) gave evidence of a single band of molecular weight approximately 32,000. 3. On the basis of the release patterns of this enzyme from several affinity gels, an elution diagram is proposed as a device to assess the contribution of any of the several displacing agents that can be used to manipulate the desorption of a (enzyme) ligate from an immobilized ligand.     

Kinetics and mechanism of the reduction of ferricytochrome c by the superoxide anion

The temperature and pH dependence of the reaction of the superoxide radical anion with ferricytochrome c have been measured using the pulse-radiolysis technique. The temperature dependence of the reaction at low ionic strength yields an activation energy of 31 +/- 5 kJ/mol as compared to 14 +/- 3 kJ/mol for the reaction of CO2.(-) under the same conditions. The pH dependence fits the single pK'a of ferricytochrome c of 9.1. The bimolecular rate constant for the reaction of the superoxide anion with ferricytochrome c at pH 7.8, 21 +/- 2 degrees C, in the presence of 50 mM phosphate and 0.1 mM EDTA is (2.6 +/- 0.1) X 10(5) M-1 s-1. Using this value, 1 unit of superoxide dismutase activity (McCord, J. M., and Fridovich, I. (1969) J. Biol. Chem. 244, 6049-6055) is calculated to be 3.6 +/- 0.3 pmol of enzyme if the assay is performed in a total volume of 3.0 ml. Copper ions reduce the yield of the reaction of ferricytochrome c with CO2.(-). The reactivities of native and singly modified 4-carboxy-2,4-dinitrophenyllysine cytochromes c towards the superoxide anion radical are in the order native greater than 4-carboxy-2,4-dinitrophenyllysine 60 greater than lysine 13 greater than lysine 87 greater than lysine 27 greater than lysine 86 greater than lysine 72, indicating that electron transfer takes place at or close to the solvent accessible heme edge. The mechanism of the reaction is discussed in terms of the approach of superoxide anion radicals to the heme edge and the available molecular orbitals of both heme and free radicals.     

Use of phospholipase A to compare phospholipid organization in synaptic membranes,myelin, and liposomes

Summary The pattern of fatty acid release from rat synaptic membranes in the presence of phospholipase A₂ (Vipera russelli) was compared to that from liposomes comprised of phospholipids. Phospholipase A₂ more readily attacked myelin and synaptic membranes than liposomes prepared from total phospholipids derived from myelin. Although hydrolysis of liposomal phospholipids occurred in the absence of added calcium, the presence of 2mm CaCl₂ or 2% bovine serum albumin significantly enhanced the phospholipase attack of liposomes, but not synaptic membranes or myelin. Phospholipase exhibited a marked preference for phospholipids containing docosahexaenoic acid (226) in the synaptic membranes, while with liposomes the pattern of released fatty acid reflected the fatty acid composition in the two-position of the phospholipids. Although either calcium or albumin markedly increased the phospholipase hydrolysis of liposomes, neither affected the hydrolysis of synaptic membranes or the pattern of fatty acid release from liposomes. It was concluded that the nonlipid constituents, particularly the proteins, of biomembranes were responsible for the organization of the phospholipids and accounted for the observed differences between liposomes and synaptic membranes with respect to enzymic accessibility.