CAP 37, a 37 kD human neutrophil granule cationic protein shares homology with inflammatory proteinases

We have previously shown that a major granule-associated cationic protein CAP 37 (Mr = 37 kD) derived from human PMN is a monocyte-specific chemoattractant. The N-terminal amino acid sequence of this novel chemotactic protein shares significant homology with a number of inflammatory molecules with protease activity including elastase and cathepsin G. However, a critical substitution of a serine for a histidine at position 41, results in its lack of serine protease activity.     

Subcellular localization of NAD(P)H oxidase(s) in human neutrophilic polymorphonuclear leucocytes.

NADH and NADPH oxidase activities in a homogenate of human neutrophils co-sediment in a linear sucrose density gradient under either velocity or isopycnic conditions of centrifugation. The position of these activities in the gradient does not correspond to any known subcellular granule or to the cell-membrane fraction. These data suggest that the oxidase activities may reside in a unique granule that has previously not been recognized.     

Characterization of recombinant amino-terminal NC4 domain of human collagen IX: interaction with glycosaminoglycans and cartilage oligomeric matrix protein

The N-terminal NC4 domain of collagen IX is a globular structure projecting away from the surface of the cartilage collagen fibril. Several interactions have been suggested for this domain, reflecting its location and its characteristic high isoelectric point. In an attempt to characterize the NC4 domain in more detail, we set up a prokaryotic expression system to produce the domain. The purified 27.5-kDa product was analyzed for its glycosaminoglycan-binding potential by surface plasmon resonance and solid-state assays. The results show that the NC4 domain of collagen IX specifically binds heparin with a K(d) of 0.6 microm, and the full-length recombinant collagen IX has an even stronger interaction with heparin, with an apparent K(d) of 3.6 nm. The heparin-binding site of the NC4 domain was located in the extreme N terminus, containing a heparin-binding consensus sequence, whereas electron microscopy suggested the presence of at least three additional heparin-binding sites on full-length collagen IX. The NC4 domain was also shown to bind cartilage oligomeric matrix protein. This interaction and the association of cartilage oligomeric matrix protein with other regions of collagen IX were found to be heparin-competitive. Circular dichroism analyses of the NC4 domain indicated the presence of stabilizing disulfide bonds and a thermal denaturation point of about 80 degrees C. The pattern of disulfide bond formation within the NC4 domain was identified by tryptic peptide mass mapping of the NC4 in native and reduced states. A similar pattern was demonstrated for the NC4 domain of full-length recombinant collagen IX.     

Photochemistry of dyes and fluorochromes used in biology and medicine: some physicochemical background and current applications

An overview of the basic principles of photochemistry is presented to facilitate discussion of fluorescence, quenching and quantum yields. These topics in turn provide the foundation for an account of fluorescence spectroscopy and its application to microscopy. A brief overview of light microscopy and the application of fluorescence microscopy is given. The influences of molecular features, such as aromatic character and substitution patterns, on color and fluorescence are described. The concept of color fading is considered with particular reference to its effect on microscopic preparations. A survey of representative fluorescent probes is provided, and their sensitivity, application, and limitations are described. The phototoxicity of fluorescent molecules is discussed using biomembranes and DNA as examples of targets of toxicity. Photodynamic therapy, a relatively new clinical application of phototoxicity, is described. Both anticancer and antimicrobial applications are noted, and an assessment is given of the current ideas on the ideal physicochemical properties of the sensitizing agents for such applications.     

Scanning electron microscopic observations of early stages of phagocytosis of E. coli by human neutrophils

Summary Changes in surface morphology, as observed by scanning electron microscopy, appear rapidly when human polymorphonuclear neutrophils (PMN) are challenged with bacteria. Monolayers of PMN adhering to glass were incubated with opsonized E. coli from 5 sec to 10 min, and then fixed and prepared for SEM. As early as 5 sec after phagocytic challenge, E. coli are found in contact with PMN and in the process of engulfment into open cavities formed by lamellipodia. The shape of the mouth of the forming phagocytic vacuole is related to the orientation of bacteria during entry. Bacteria engulfed into early forming phagosomes are surrounded by a large open space between the bacteria and the phagosome wall. As phagocytosis proceeds, the space is reduced and the loose fit around the entering bacteria becomes tight. By 30 sec, bacteria may be completely internalized and by 1 min phagocytized E. coli are packed into bulging PMN. The observations reveal the variability and rapidity of the phagocytic response and confirm the presence of sensitive mechanisms for host defense by PMN.This work was supported by research grants from the University of North Carolina Research Council and the National Institutes of Health (A1 02430)     

Cortisol stress response and histopathological alteration index in Clarias gariepinus exposed to sublethal concentrations of Qua Iboe crude oil and rig wash

The histological effect on and stress response of post juvenile Clarias gariepinus exposed to Qua Iboe crude oil and rig wash were investigated. Fish weighing 60–90 g and measuring 16–18 cm were exposed for 7–28 days to 8.00 ml^?1 Qua Iboe crude oil and 0.0018 ml^–1 rig wash, both being 0.1 of the 96 hr LC₅₀. Blood samples of C. gariepinus were collected every seven days and evaluated for stress by measuring cortisol concentration. The gills and liver were studied and scored for Gill Alteration Index (GAI) and Hepatic Alteration Index (HAI), respectively. There was an increase in cortisol level up to the 7th and 14th day among the group exposed to Qua Iboe crude oil, with a decrease on the 21st and 28th day. The rig wash group increased in cortisol level up to the 7th day and decreased slightly on the 14th day, after which the trend became irregular. The toxic effects of the Qua Iboe crude oil and rig wash were time dependent, as shown by the histopathological alteration index (HAI) of gill and liver. After 28 days of exposure, the gills had irreparable damage due to high frequency of cellular necrosis and degeneration, whereas the liver had from moderate to severe damage due to the high frequency of cellular degeneration and inflammation. Qua Iboe crude oil and rig wash are both toxic to C. gariepinus, therefore their indiscriminate discharge to the environment must be discouraged.