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.     

The Invisibility of Disability: Using Dance to Shake from Bioethics the Idea of ‘Broken Bodies’

Complex social and ethical problems are often most effectively solved by engaging them at the messy and uncomfortable intersections of disciplines and practices, a notion that grounds the InVisible Difference project, which seeks to extend thinking and alter practice around the making, status, ownership, and value of work by contemporary dance choreographers by examining choreographic work through the lenses of law, bioethics, dance scholarship, and the practice of dance by differently‐abled dancers. This article offers a critical thesis on how bioethics has come to occupy a marginal and marginalizing role in questions about the differently‐abled body. In doing so, it has rendered the disabled community largely invisible to and in bioethics. It then defends the claim that bioethics – as a social undertaking pursued collaboratively by individuals from different disciplines – must take much better notice of the body and the embodied individual if it is to better achieve its ends, which include constructing a moral and just society. Finally, this article considers how the arts, and specifically dance (and here dance by differently‐abled dancers), provides us with rich evidence about the body and our ability to respond positively to normally ‘othered’ bodies. It concludes that greater attention to empirical evidence like that being generated in InVisible Difference will help to expand the reach and significance of bioethics, and thereby its relevance to (and consciousness of) important questions about the status of bodies and bodily differences, which must be considered as central to its ambitions.     

Specific associations of fluorescent beta-2-microglobulin with cell surfaces. The affinity of different H-2 and HLA antigens for beta-2-microglobulin

We prepared single-labeled FITC derivatives of beta-2-microglobulin (b2m) and examined their interactions with class I MHC Ag H chains on living cells. Human b2m was reacted with FITC under mild conditions and separated by hydroxylapatite chromatography into three peaks containing single labeled derivatives of b2m peaks A, B, and C, and a peak containing the unmodified protein. The three fluorescent derivatives labeled the surfaces of cells bearing class I MHC Ag. The labeling was specific for class I MHC Ag as indicated by failure to label cells in the presence of excess unlabeled b2m and failure to label the HLA-negative cell lines Daudi and 721.221. Mouse cells labeled with fluorescent human b2m were recognized by mAb to the class I MHC Ag and by virus-restricted cytotoxic T lymphocytes, suggesting that labeling with the fluorescent b2m does not significantly alter the structure of class I MHC Ag or impair their ability to present viral antigens to cytotoxic T lymphocytes. We determined the kinetic and equilibrium binding parameters for the fluorescent b2m derivatives associating with the class I H chains of mouse and human cells. Peaks B and C exhibited biphasic binding to the mouse lymphoma cells EL-4(G-CSA-) (Kd1 = 1 x 10(-9) M; K2 = 1.5 to 3.0 x 10(-8) M whereas peak A bound to a small number of low affinity binding sites. In contrast to the biphasic binding observed with EL-4(G-CSA-), only monophasic binding was observed for peak C binding to RDM4 cells. Biphasic binding was also observed with the human B cell line LCL 721. Analysis of a series of LCL 721 class I MHC loss mutants and gene transferents revealed that the heterogeneity in binding is due to differences in the affinity of different class I encoded H chains for b2m.     

Aggregation of macrophages and fibroblasts is inhibited by a monoclonal antibody to the hyaluronate receptor

To examine the role of the hyaluronate receptor in cell to cell adhesion, we have employed the K-3 monoclonal antibody (MAb) which specifically binds to the hyaluronate receptor and blocks its ability to interact with hyaluronate. In the first set of experiments, we investigated the spontaneous aggregation of SV-3T3 cells, which involves two distinct mechanisms, one of which is dependent upon the presence of divalent cation and the other is independent. The divalent cation-independent aggregation was found to be completely inhibited by both intact and Fab fragments of the K-3 MAb. In contrast, the K-3 MAb had no effect on the divalent cation-dependent aggregation of cells. In a second set of experiments, we examined alveolar macrophages. The presence of hyaluronate receptors on alveolar macrophages was demonstrated by the fact that detergent extracts of these cells could bind [3H]hyaluronate, and this binding was blocked by the K-3 MAb. Immunoblot analysis of alveolar macrophages showed that the hyaluronate receptor had a Mr of 99,500, which is considerably larger than the 85,000 Mr for that on BHK cells. When hyaluronate was added to suspensions of alveolar macrophages, the cells were induced to aggregate. This effect was inhibited by the K-3 MAb, suggesting that the hyaluronate-induced aggregation was mediated by the receptor.     

The complete amino acid sequences of cytosolic and mitochondrial aspartate aminotransferases from horse heart,and inferences on evolution of the isoenzymes

Summary We report here the complete amino acid sequences of the cytosolic and mitochondrial aspartate aminotransferases from horse heart. The two sequences can be aligned so that 48.1% of the amino acid residues are identical. The sequences have been compared with those of the cytosolic isoenzymes from pig and chicken, the mitochondrial isoenzymes from pig, chicken, rat, and human, and the enzyme fromEscherichia coli. The results suggest that the mammalian cytosolic and mitochondrial isoenzymes have evolved at equal and constant rates whereas the isoenzymes from chicken may have evolved somewhat more slowly. Based on the rate of evolution of the mammalian isoenzymes, the geneduplication event that gave rise to cytosolic and mitochondrial aspartate aminotransferases is estimated to have occurred at least 10⁹ years ago. The cytosolic and mitochondrial isoenzymes are equally related to the enzyme fromE. coli; the prokaryotic and eukaryotic enzymes diverged from one another at least 1.3×10⁹ years ago.     

Mass characteristics of DNA obtained from chromosomes of a human carcinoma cell line

Sedimentation studies of DNA from chromosomes extracted from human mitotic cells showed that highmolecular-weight DNA can be obtained if cell hypotonic treatments and prolonged metaphase blocks are avoided. Two types of large double-stranded DNA were observed. One of these (M _r = 2.5×10⁸) appeared as a size class with characteristics reminiscent of the chromosomal DNA subunit hypothesis. However, this DNA is the decay product of larger molecules, whose minimum molecular weight is 6×10⁸.     

Isolation and properties of the soluble c-type cytochromes of the dinoflagellate Peridinium cinctum

Four soluble cytochromes of the c type were isolated from the freshwater dinoflagellate Peridinium cinctum collected from Lake Kinneret, Israel. Cytochrome c with alpha-band maximum at 550 nm in the reduced state had a molecular mass of 10,200 Da, pI 7.4, and Em of 278 m V. This cytochrome was active in the respiratory chain of beef heart Keilin-Hartree particles. Cytochrome c-553 had a molecular mass of 13,200 Da, pI 4.9, and Em of 384 m V, and was active in light induced electron transport of Euglena gracilis chloroplast fragments. Cytochrome c-554 had a molecular mass of 13,500 Da, pI 4.4, and Em of 326 m V. This cytochrome was inactive in light induced electron transport but competed with cytochrome c-552 of Euglena in the assay. The acidic cytochrome c-557 was present in very small quantities. The properties of the soluble c-type cytochromes of P. cinctum are compatible with the classification of dinoflagellates as primitive eucaryotes.     

Nitrate reductase: an improved assay method for phytoplankton

A new assay for measuring the activity of nitrate reductasein phytoplankton, based upon the permeability of cells treatedwith toluene to substrates and products, is described. The methodis simple and, since the reaction is carried out directly ona glass fiber filter, can be easily performed in the field oron shipboard. In comparison with previous methods, this techniquegave higher absolute amounts of NO^–₂ formed per unit tuneand higher enzymatic activities per sample volume when testedwith axenic algal cultures and with natural phytoplankton populationsfrom Lake Kinneret, the River Jordan and the Eastern Mediterranean.