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Bis[2-(4-phosphonooxyphenoxy)carbonylethyl]phosphinc acid (BPPCEP) was prepared and evaluated as a site-directed affinity reagent for cross-linking human hemoglobin. It was synthesized in four steps starting from 4-benzyloxyphenol and was converted to its pentasodium salt so as to afford efficient cross-linking in an aqueous medium. The reagent was found to specifically cross-link human hemoglobin A0 in the β-cleft chains under oxygenated reaction conditions at neutral pH. The amino acid residues involved in the cross-linking were determined by mass spectral analyses of tryptic digest fragments of cross-linked hemoglobin, employing a MALDI-TOF mass spectrometer. The MS analyses suggested that the most likely amino acids involved in the cross-links are Val-1 or Lys-82 present on one of the β subunits and Lys-82 or Lys-144 on the other. Molecular modeling studies performed on the reagent−HbA0 complex corroborated the conclusions reached by MALDI-MS analyses. The oxygen equilibrium measurements of the three major BPPCEP-cross-linked Hb products, isolated and purified by preparative cation exchange chromatography, exhibited oxygen affinity (P50) values of 14.5, 12.1, and 15.5 Torr as compared with the P50 of 13.1 Torr for cell-free hemoglobin. The oxygen-binding cooperativity of the modified products, as determined by the Hill coefficient generated from the Hill plots of the respective P50 values, coupled with the absence of sigmoidal shape of the O2 equilibrium curves, was considerably lower than that of the native hemoglobin.

The murine natural resistance-associated macrophage protein, Nramp1, has multiple pleiotropic effects on macrophage activation and regulates survival of intracellular pathogens including Leishmania, Salmonella and Mycobacterium species. Nramp1 acts as an iron transporter, but precisely how this relates to macrophage activation and/or pathogen survival remains unclear. To gain insight into function, anti-Nramp1 monoclonal and polyclonal antibodies are used here to localise Nramp1 following activation and infection. Confocal microscope analysis in uninfected macrophages demonstrates that both the mutant (infection-susceptible) and wild-type (infection-resistant) forms of the protein localise to the membranes of intracellular vesicular compartments. Gold labelling and electron microscopy defines these compartments more precisely as electron-lucent late endosomal and electron-dense lysosomal compartments, with Nramp1 colocalizing with Lamp1 and cathepsins D and L in both compartments, with macrosialin in late endosomes, and with BSA-5 nm gold in pre-loaded lysosomes. Nramp1 is upregulated with interferon-(gamma) and lipopolysaccaride treatment, coinciding with an increase in labelling in lysosomes relative to late endosomes and apparent dispersion of Nramp1-positive vesicles from a perinuclear location towards the periphery of the cytoplasm along the microtubular network. In both control and activated macrophages, expression of the protein is 3- to 4-fold higher in wild-type compared to mutant macrophages. In Leishmania major-infected macrophages, Nramp1 is observed in the membrane of the pathogen-containing phagosomes, which retain a perinuclear localization in resting macrophages. In Mycobacterium avium-infected resting and activated macrophages, Nramp1-positive vesicles migrated to converge, but not always fuse, with pathogen-containing phagosomes. The Nramp1 protein is thus located where it can have a direct influence on phagosome fusion and the microenvironment of the pathogen, as well as in the more general regulation of endosomal/lysosomal function in macrophages.

Advanced aerospace programs have created a need for mechanical fastening which requires the development and application of new materials, processes and technologies. Some of these are already in place, but others will evolve as the needs are further defined by the structural designers. Some of these fastening systems are described herein.

The murine chromosome 1 gene Lsh/Ity/Bcg (candidate Nramp) regulates macrophage activation for antimicrobial activity against Salmonella typhimurium, Leishmania donovani, and Mycobacterium spp. To determine early events in the activation pathway, the ability of mycobacterial lipoarabinomannan (LAM) to induce early gene (KC and JE) expression in macrophages from susceptible (S) C57BL/10ScSn (Lshs) and congenic resistant (R) B10.L-Lshr mice was investigated. Stimulation with 1.8 microgram of arabinofuranosyl-terminated LAM (AraLAM) per ml resulted in similar kinetics for KC or JE expression in S and R macrophages. However, whereas JE/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA ratios remained equivalent, R macrophages consistently showed enhanced KC/GAPDH ratios within 30 to 40 min of stimulation compared with S macrophages. Significant differences in KC/GAPDH ratios were observed throughout the peak period (0.5 to 6 h) of the KC response and with doses of AraLAM ranging from 0.01 to 2.5 micrograms/ml. Heavily mannosylated LAM from virulent Mycobacterium tuberculosis Erdman, in doses of up to 2.5 micrograms/ml, failed to stimulate KC or JE in S or R macrophages. Gamma interferon alone (25 U/ml) stimulated equivalent JE expression in S and R macrophages and synergized with AraLAM to enhance JE in both. In contrast, AraLAM-induced KC expression was inhibited in the presence of gamma interferon. Agonist/inhibitor studies were undertaken to determine the signal transduction pathways mediating KC expression. The protein kinase C (PKC) inhibitor Calphostin C (200 nM) inhibited AraLAM-induced KC by 34% +/- 4% in S macrophages and 43% +/- 5% in R macrophages; the cyclic AMP-dependent PKA inhibitor KT5720 (2 microM) inhibited AraLAM-induced KC by 33% +/- 4% (S) and 25% +/- 5% (R). A role for Ca2+ was indicated because ionophore alone stimulated KC expression and synergized with AraLAM to give a dramatically enhanced response. Induction of KC was also inhibited by (i) blocking constitutive nitric oxide (NO) production by preincubation of macrophages with NG-monomethyl-L-arginine (400 microM) (48% +/- 8% [S] and 40% +/- 11% [R]) and (ii) incubation of macrophages with the cyclic GMP-dependent kinase inhibitor KT5823 (4 microM) (65% +/- 4% [S] and 72% +/- 6% [R]). The manner in which these PKC-, PKA-, and Ca(2+)-dependent, NO-mediated cyclic GMP-dependent kinase signal transduction pathways may relate to function of the candidate Lsh/Ity/Bcg gene Nramp is discussed.

The capacity of mature bone-marrow-derived macrophages and resident peritoneal macrophages from Lshr versus Lshs congenic mice to kill intracellular Leishmania donovani amastigotes when activated by recombinant gamma interferon-lipopolysaccharide (rIFN-gamma-LPS) was examined. IFN-gamma alone in doses up to 100 U/ml was unable to activate macrophages to kill L. donovani amastigotes in vitro; LPS was a necessary secondary stimulus. Similarly, LPS alone in doses up to 100 ng/ml produced no leishmanicidal activity. In bone marrow macrophages, a dose-dependent increase in leishmanicidal activity was observed as increasing rIFN-gamma-LPS dose combinations were introduced, with Lshr macrophages maintaining a significant but not dramatic advantage within any particular dose combination. For peritoneal macrophages, the reverse was true, with macrophages from Lshs mice being more efficient at killing for doses of LPS up to 10 ng/ml with doses of rIFN-gamma in the range of 11 to 33 U/ml. The degree of killing in both bone marrow and peritoneal macrophages correlated well with the levels of nitrites measured in the supernatants at 72 h, and a highly significant correlation was observed between 4-, 24-, or 72-h tumor necrosis factor alpha (TNF-alpha) release and nitrite production measured at 72 h. Inclusion of 200 microM NG-monomethyl-L-arginine, a competitive inhibitor of the L-arginine-dependent pathway for the synthesis of inorganic nitrogen oxides, inhibited the killing, as did the addition of neutralizing anti-TNF-alpha antibody. These results are consistent with previous data showing an important autocrine role for TNF-alpha in enhancing production of inorganic nitrogen oxides by primed or activated macrophages. In addition, our results suggest that production of TNF-alpha and nitrites after priming or activation signals may be under a different regulatory control in mature bone marrow macrophages than in the resident peritoneal macrophage population.

The Lsh/Ity/Bcg locus on mouse chromosome 1 regulates macrophage (m phi) priming/activation for antimicrobial activity against intracellular pathogens. A candidate Bcg gene, designated natural resistance-associated m phi protein (Nramp), recently isolated from a pre-B cell cDNA library encodes a polytopic integral membrane protein with structural features common to prokaryotic and eukaryotic transporters. In the present study, an activated m phi cDNA library yielded new Nramp clones that differ in the 5' region from the published pre-B cell-derived clone sequence, resulting in addition of 64 amino acids at the NH2 terminus of the predicted protein. This new domain is rich in proline, serine, and basic amino acids, and includes three protein kinase C phosphorylation sites and a putative Src homology 3 binding domain. RNAs containing this domain are the only form found in the m phi. Hence, the protein encoded by this RNA is the candidate molecule mediating natural resistance to intra-m phi pathogens.