Tadpole erythrocytes: luminescent properties with dark field microscopy

To characterize and classify erythrocytes of ranid tadpoles, alcohol-fixed blood smears were studied with dark field illumination. All preclimax stage (limb bud-foot) Rana castesbeiana tadpoles from ponds in Massachusetts had red blood cells that were polymorphic. The majority of cells (88%) showed a bright, granular luminescence varying from white to blue-grey, whereas, cytoplasm of the other cells was smooth, black, and nonluminescent. On the other hand, tadpoles in similar stages from other species (Rana clamitans, Mass. and Rana pipens, Vermont) and from R. catesbeiana tadpoles from other locations (Wisconsin and North Carolina) had no observable cytoplasmic luminescence in any of their red blood cells. Moreover, as Mass. R. catesbeiana underwent metamorphic climax their luminescent cells disappeared and were replaced by small, round, dark, nonluminescent cells, precursors of the oval, nonluminescent erythrocytes characteristic of adult frogs. Cells with black nonluminescent cytoplasm generally contained nuclei which were luminescent. In conclusion, two main types of red blood cells-those with and those without cytoplasmic luminescence-are distinguishable with dark field microscopy. Luminescence of the cells varies with species, geographic location, and developmental stage of the tadpoles.     

An Investigation of the Ability of the Glutaraldehyde Test to Distinguish between Acute and Chronic Inflammatory Disease in Horses

The Glutaraldehyde test (GT), a rapid and inexpensive test, has been utilized empirically for many years in bovine practice for diagnosing inflammatory diseases. GT is used primarily to demonstrate increased serum concentrations of fibrinogen and globulin. Glutaraldehyde binds with free amino groups in fibrinogen and immunoglobulin to create a clot in a first degree chemical reaction. The clotting time of the GT estimates the content of proteins produced in response to inflammation. The applicability of GT for diagnosing inflammation in the horse has never been investigated. The objective of this study was to determine the ability of GT to distinguish between acute and chronic inflammatory disease in horses. Thirty-seven horses with suspected inflammatory diseases were evaluated using the GT, history, complete clinical examination and routine blood analysis. GT-times, laboratory results and clinical outcome were compared statistically. Horses that were determined to be acutely affected (based on history, clinical examination and routine blood analysis) tended to have a negative GT (75%). Results of the GT did not correlate with blood fibrinogen concentration. Positive GT also predicted a fatal outcome in 69% of the clinical cases. The results of this trial indicate that GT can be a useful screening test to distinguish between acute and chronic inflammatory disease in horses.     

Experimental evaluation of the relationship between lethal or non-lethal virulence and transmission success in malaria parasite infections

Background  

Evolutionary theory suggests that the selection pressure on parasites to maximize their transmission determines their optimal host exploitation strategies and thus their virulence. Establishing the adaptive basis to parasite life history traits has important consequences for predicting parasite responses to public health interventions. In this study we examine the extent to which malaria parasites conform to the predicted adaptive trade-off between transmission and virulence, as defined by mortality. The majority of natural infections, however, result in sub-lethal virulent effects (e.g. anaemia) and are often composed of many strains. Both sub-lethal effects and pathogen population structure have been theoretically shown to have important consequences for virulence evolution. Thus, we additionally examine the relationship between anaemia and transmission in single and mixed clone infections.     

Synthesis,antiviral activity and pharmacokinetics of P1/P1' substituted 3-aminoindazole cyclic urea HIV protease inhibitors

A series of P1/P1' substituted cyclic urea analogues were prepared in an attempt to increase the intra-cellular antiviral potency of the nonsymmetrical 3-aminoindazoles DMP 850 and DMP 851. The effect of alkyl substitution of the P1/P1' residues on cellular antiviral potency, protein binding, resistance profile and pharmacokinetics are described.     

Cyclosporin A suspends transplantation reactions in the marine sponge Microciona prolifera

Sponges are the simplest extant animals but nevertheless possess self-nonself recognition that rivals the specificity of the vertebrate MHC. We have used dissociated cell assays and grafting techniques to study tissue acceptance and rejection in the marine sponge Microciona prolifera. Our data show that allogeneic, but not isogeneic, cell contacts trigger cell death and an increased expression of cell adhesion and apoptosis markers in cells that accumulate in graft interfaces. Experiments investigating the possible existence of immune memory in sponges indicate that faster second set reactions are nonspecific. Among the different cellular types, gray cells have been proposed to be the sponge immunocytes. Fluorescence confocal microscopy results from intact live grafts show the migration of autofluorescent gray cells toward graft contact zones and the inhibition of gray cell movements in the presence of nontoxic concentrations of cyclosporin A. These results suggest that cell motility is an important factor involved in sponge self/nonself recognition. Communication between gray cells in grafted tissues does not require cell contact and is carried by an extracellular diffusible marker. The finding that a commonly used immunosuppressor in human transplantation such as cyclosporin A blocks tissue rejection in marine sponges indicates that the cellular mechanisms for regulating this process in vertebrates might have appeared at the very start of metazoan evolution.     

Perforin oligomers form arcs in cellular membranes: a locus for intracellular delivery of granzymes

Perforin-mediated cytotoxicity is an essential host defense, in which defects contribute to tumor development and pathogenic disorders including autoimmunity and autoinflammation. How perforin (PFN) facilitates intracellular delivery of pro-apoptotic and inflammatory granzymes across the bilayer of targets remains unresolved. Here we show that cellular susceptibility to granzyme B (GzmB) correlates with rapid PFN-induced phosphatidylserine externalization, suggesting that pores are formed at a protein-lipid interface by incomplete membrane oligomers (or arcs). Supporting a role for these oligomers in protease delivery, an anti-PFN antibody (pf-80) suppresses necrosis but increases phosphatidylserine flip-flop and GzmB-induced apoptosis. As shown by atomic force microscopy on planar bilayers and deep-etch electron microscopy on mammalian cells, pf-80 increases the proportion of arcs which correlates with the presence of smaller electrical conductances, while large cylindrical pores decline. PFN appears to form arc structures on target membranes that serve as minimally disrupting conduits for GzmB translocation. The role of these arcs in PFN-mediated pathology warrants evaluation where they may serve as novel therapeutic targets.The cytotoxic cell granule-secretory pathway depends on perforin (PFN) to deliver granzyme (Gzm) proteases to the cytosol of target cells where they induce apoptosis and other biological effects, such as inflammation.¹ Ring-shaped transmembrane PFN pores hereafter called ‘cylindrical pores'', are presumed to act as the gateway for cytosolic entry, either at the plasma membrane or after endocytosis.^{2, 3, 4} In either case the highly cationic Gzms are thought to diffuse through these cylindrical pores formed by poly-PFN. Nevertheless, a mechanistic understanding of the phenomenon (how the cationic globular protein exchanges from its carrier proteoglycan, serglycin, to the pore and crosses the plasma and/or vesicular membranes) has been lacking due to limitations in imaging technology and in our detailed understanding of the molecular forms that PFN may adopt following interaction with a target cell plasma membrane.Here we show under conditions where cylindrical pore formation is minimal,⁵ that granzyme B (GzmB) translocation readily occurs. We previously demonstrated that a prelude to granzyme translocation is PFN-mediated, Ca-independent phosphatidylserine (PS) externalization (flip-flop) measured by annexin-V and lactadherin binding.⁶ This rapid PS flip-flop also occurs when mouse CD8 cells contact antigen-pulsed target cells. Inasmuch as the proteinaceous cylinders offer a formidable barrier to lipid flow, we have speculated that the observed movement of anionic phospholipids to the external leaflet is due to the formation of proteo-lipidic structures, which consists of oligomerized PFN monomers bearing an arc morphology and plasma membrane lipids.^{6, 7, 8}In the work reported here, the topology of PFN embedded into homogeneous planar bilayers and tumor cell plasma membranes was imaged by atomic force microscopy (AFM) and deep etch electron microscopy (DEEM), respectively. Further, the influence of an anti-human PFN mAb (pf-80) that rescues target cells from necrosis,⁹ was examined. The AFM data show that PFN forms arcs as well as rings in planar bilayers, while conductance measurements across equivalent membranes in parallel experiments measured functional pore sizes consistent with these varied structures. The pf-80 mAb increased the frequency of arc formation and reduced conductance values. Interestingly, PS flip-flop and granzyme delivery were both increased in target cells after PFN oligomerization was interrupted by the pf-80 mAb. A similar effect was seen in T24 bladder carcinoma cells imaged by DEEM. Treatment with PFN leads to deposition of rings (barrel stave pores) and arcs and the pf-80 mAb increased the ratio of arcs to rings on the surface of these cells. We suggest that the observed protein arcs function as toroidal pores in whole cells, explaining PS flip-flop, and act as focal points for granzyme translocation across lipid bilayer.     

SAMPI: Protein Identification with Mass Spectra Alignments

Background  

Mass spectrometry based peptide mass fingerprints (PMFs) offer a fast, efficient, and robust method for protein identification. A protein is digested (usually by trypsin) and its mass spectrum is compared to simulated spectra for protein sequences in a database. However, existing tools for analyzing PMFs often suffer from missing or heuristic analysis of the significance of search results and insufficient handling of missing and additional peaks.