Skin graft rejection elicited by beta 2-microglobulin as a minor transplantation antigen involves multiple effector pathways: role of Fas-Fas ligand interactions and Th2-dependent graft eosinophil infiltrates

Beta(2)-microglobulin (beta(2)m)-derived peptides are minor transplantation Ags in mice as beta(2)m-positive skin grafts (beta(2)m(+/+)) are rejected by genetically beta(2)m-deficient recipient mice (beta(2)m(-/-)). We studied the effector pathways responsible for the rejection induced by beta(2)-microglobulin-derived minor transplantation Ags. The rejection of beta(2)m(+/+) skin grafts by naive beta(2)m(-/-) mice was dependent on both CD4 and CD8 T cells as shown by administration of depleting mAbs. Experiments performed with beta(2)m(-/-)CD8(-/-) double knockout mice grafted with a beta(2)m(+/+) MHC class I-deficient skin showed that sensitized CD4 T cells directed at beta(2)m peptides-MHC class II complexes are sufficient to trigger rapid rejection. Rejection of beta(2)m(+/+) grafts was associated with the production of IL-5 in vitro, the expression of IL-4 and IL-5 mRNAs in the grafted tissue, and the presence within rejected grafts of a considerable eosinophil infiltrate. Blocking IL-4 and IL-5 in vivo and depleting eosinophils with an anti-CCR3 mAb prevented graft eosinophil infiltration and prolonged beta(2)m(+/+) skin graft survival. Lymphocytes from rejecting beta(2)m(-/-) mice also displayed an increased production of IFN-gamma after culture with beta(2)m(+/+) minor alloantigens. In vivo neutralization of IFN-gamma inhibited skin graft rejection. Finally, beta(2)m(+/+) skin grafts harvested from B6(lpr/lpr) donor mice, which lack a functional Fas molecule, survived longer than wild-type beta(2)m(+/+) skin grafts, showing that Fas-Fas ligand interactions are involved in the rejection process. We conclude that IL-4- and IL-5-dependent eosinophilic rejection, IFN-gamma-dependent mechanisms, and Fas-Fas ligand interactions are effector pathways in the acute rejection of minor transplantation Ags.     

Role of cholesterol in human immunodeficiency virus type 1 envelope protein-mediated fusion with host cells

In this study we examined the effects of target membrane cholesterol depletion and cytoskeletal changes on human immunodeficiency virus type 1 (HIV-1) Env-mediated membrane fusion by dye redistribution assays. We found that treatment of peripheral blood lymphocytes (PBL) with methyl-beta-cyclodextrin (MbetaCD) or cytochalasin reduced their susceptibility to membrane fusion with cells expressing HIV-1 Env that utilize CXCR4 or CCR5. However, treatment of human osteosarcoma (HOS) cells expressing high levels of CD4 and coreceptors with these agents did not affect their susceptibility to HIV-1 Env-mediated membrane fusion. Removal of cholesterol inhibited stromal cell-derived factor-1alpha- and macrophage inflammatory protein 1beta-induced chemotaxis of both PBL and HOS cells expressing CD4 and coreceptors. The fusion activity as well as the chemotactic activity of PBL was recovered by adding back cholesterol to these cells. Confocal laser scanning microscopy analysis indicated that treatment of lymphocytes with MbetaCD reduced the colocalization of CD4 or of CXCR4 with actin presumably in microvilli. These findings indicate that, although cholesterol is not required for HIV-1 Env-mediated membrane fusion per se, its depletion from cells with relatively low coreceptor densities reduces the capacity of HIV-1 Env to engage coreceptor clusters required to trigger fusion. Furthermore, our results suggest that coreceptor clustering may occur in microvilli that are supported by actin polymerization.     

Morphological studies on microfilaments and their organizing center in killifish (Fundulus heteroclitus L.) melanophores

Fish chromatophores serve as excellent study models for cytoskeleton-dependent organelle translocations because the distribution of pigmentary organelles can be observed against a time frame by microscopy. In this study the distribution of microfilaments along with microtubules in cultured melanophores of the killifish (Fundulus heteroclitus Linneaus) are examined using whole-cell transmission electron microscopy (WCTEM), fluorescence, and laser scanning confocal microscopy. Dispersing, dispersed, aggregating and aggregated states of pigment are induced by adding either caffeine (for dispersion) or epinephrine (for aggregation) to the cells in a standard culture medium. The cells that exhibited a random melanosome distribution in the standard culture media without these two reagents, served as the control. The results indicate that: (i) a structure considered to be the actin-filament organizing center (AFOC) is in close proximity to the microtubule-organizing center (MTOC); (ii) the radial layout of microfilaments remains similar over four physiological states of pigmentary response with the exception of epinephrine-aggregated pigment, in which the aggregate blocks the viewing of the AFOC and central microfilament rays, yet radial microfilaments, whether central and/or peripheral, are apparent in all physiological states of distribution; and (iii) microfilaments serve, together with microtubules, as scaffolding for melanosomes which migrate in bi-directional rows on cross-bridges, thus shedding light on the mechanisms for orderly melanosome translocations in a structural continuum.     

Structure of the histone mRNA hairpin required for cell cycle regulation of histone gene expression

Expression of replication-dependent histone genes requires a conserved hairpin RNA element in the 3' untranslated regions of poly(A)-less histone mRNAs. The 3' hairpin element is recognized by the hairpin-binding protein or stem-loop-binding protein (HBP/SLBP). This protein-RNA interaction is important for the endonucleolytic cleavage generating the mature mRNA 3' end. The 3' hairpin and presumably HBP/SLBP are also required for nucleocytoplasmic transport, translation, and stability of histone mRNAs. RNA 3' processing and mRNA stability are both regulated during the cell cycle. Here, we have determined the three-dimensional structure of a 24-mer RNA comprising a mammalian histone RNA hairpin using heteronuclear multidimensional NMR spectroscopy. The hairpin adopts a novel UUUC tetraloop conformation that is stabilized by base stacking involving the first and third loop uridines and a closing U-A base pair, and by hydrogen bonding between the first and third uridines in the tetraloop. The HBP interaction of hairpin RNA variants was analyzed in band shift experiments. Particularly important interactions for HBP recognition are mediated by the closing U-A base pair and the first and third loop uridines, whose Watson-Crick functional groups are exposed towards the major groove of the RNA hairpin. The results obtained provide novel structural insight into the interaction of the histone 3' hairpin with HBP, and thus the regulation of histone mRNA metabolism.     

Mice lacking the metalloprotease-disintegrin MDC9 (ADAM9) have no evident major abnormalities during development or adult life

MDC9 (ADAM9/meltrin gamma) is a widely expressed and catalytically active metalloprotease-disintegrin protein that has been implicated in the ectodomain cleavage of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and as an alpha secretase for the amyloid precursor protein. In this study, we evaluated the expression of MDC9 during development and generated mice lacking MDC9 (mdc9(-/-) mice) to learn more about the function of this protein during development and in adults. During mouse development, MDC9 mRNA is ubiquitously expressed, with particularly high expression levels in the developing mesenchyme, heart and brain. Despite the ubiquitous expression of MDC9, mdc9(-/-) mice appear to develop normally, are viable and fertile, and do not have any major pathological phenotypes compared to wild-type mice. Constitutive and stimulated ectodomain shedding of HB-EGF is comparable in embryonic fibroblasts isolated from mdc9(-/-) and wild-type mice, arguing against an essential role of MDC9 in HB-EGF shedding in these cells. Furthermore, there were no differences in the production of the APP alpha and gamma secretase cleavage product (p3) and of beta- and gamma-secretase cleavage product (A beta) in cultured hippocampal neurons from mdc9(-/-) or wild-type mice, arguing against an essential major role of MDC9 as an alpha-secretase in mice. Further studies, including functional challenges and an evaluation of potential compensation by, or redundancy with, other members of the ADAM family or perhaps even with other molecules will be necessary to uncover physiologically relevant functions for MDC9 in mice.     

Structural analysis of the oligosaccharide alditols released from the jelly coat of Rana dalmatina eggs by reductive beta-elimination

A combination of ion-exchange chromatography and high performance liquid chromatography (HPLC) has been used to separate the reduced oligosaccharides produced by alkaline borohydride degradation of oviducal mucins obtained from the jelly coat of Rana dalmatina. The primary structures of 26 O-glycans were determined by one-dimensional and two-dimensional 1H and 1H13C NMR spectroscopy. As observed for 20 other amphibian species, these carbohydrate chains are highly species-specific. The main typical feature of the species R. dalmatina consists in the presence of the backbone Gal(beta1-3)[Gal(beta1-4)]Gal(beta1-3)GalNAc-ol, previously observed among Ranidae, such as R. temporaria and R. ridibunda. Nevertheless, the nature of carbohydrates present at the periphery of the glycans perfectly differentiates the three species.     

Pharmaco-redox regulation of cytokine-related pathways: from receptor signaling to pharmacogenomics

Cytokines represent a multi-diverse family of polypeptide regulators; they are relatively low molecular weight (< 30 kDa), pharmacologically active proteins that are secreted by one cell for the purpose of altering either its own functions (autocrine effect) or those of adjacent cells (paracrine effect). Cytokines are small, nonenzymatic glycoproteins whose actions are both diverse and overlapping (specificity/redundancy) and may affect diverse and overlapping target cell populations. In many instances, individual cytokines have multiple biological activities. Different cytokines can also have the same activity, which provides for functional redundancy (network) within the inflammatory and immune systems. As biological cofactors that are released by specific cells, cytokines have specific effects on cell-cell interaction, communication, and behavior of other cells. As a result, it is infrequent that loss or neutralization of one cytokine will markedly interfere with either of these systems. The biological effect of one cytokine is often modified or augmented by another. Because an interdigitating, redundant network of cytokines is involved in the production of most biological effects, both under physiologic and pathologic conditions, it usually requires more than a single defect in the network to alter drastically the outcome of the process. This fact, therefore, may have crucial significance in the development of therapeutic strategies for biopharmacologic intervention in cytokine-mediated inflammatory processes and infections.     

Use of a biomimetic chromatographic stationary phase for study of the interactions occurring between inorganic anions and phosphatidylcholine membranes

A liquid chromatographic method for the study of ion-membrane interactions is reported. A phosphatidylcholine biomimetic stationary phase was established by loading dimyristoylphosphatidylcholine (DMPC) onto a reversed-phase octadecylsilica packed column. This column was then used to study the interaction of some inorganic anions with the stationary phase by UV and conductivity detection. Ten inorganic anions were selected as model ions and were analyzed with the proposed chromatographic system. Anion-DMPC interactions of differing magnitudes were observed for all of the model anions. Perchlorate-DMPC interactions were strongest, followed by thiocyanate-DMPC, iodide-DMPC, chlorate-DMPC, nitrate-DMPC, bromide-DMPC, chloride-DMPC, fluoride-DMPC, and then sulfate-DMPC. Cations in the eluent, especially H(+) ions and divalent cations such as Ca(2+), showed strong effects on anion-DMPC interactions. The chromatographic data suggest that DMPC interacts with both the anions and the cations. Anion-DMPC interactions were dependent on the surface potential of the stationary phase: at low surface potentials anion-DMPC interactions were predominantly solvation dependent in nature whereas at more positive surface potentials anion-DMPC interactions were predominantly electrostatic in nature. Cation-DMPC interactions served to raise the surface potential, causing the anion-DMPC interactions to vary from solvation dependent to electrostatic. The chromatographic data were used to provide quantitative estimates of the enthalpies of the anion-DMPC interactions.     

Intracellular pH regulation in U-2 OS human osteosarcoma cells transfected with P-glycoprotein

The molecular mechanisms responsible for intracellular pH regulation in the U2-OS osteosarcoma cell line were investigated by loading with 2',7'-bis(2-carboxyethyl)-5(6) carboxyfluorescein ester and manipulation of Cl(-) and Na(+) gradients, both in HEPES- and HCO(3)(-)/CO(2)-buffered media. Both acidification and alkalinisation were poorly sensitive to 4,4'-diisothiocyanate dihydrostilbene-2,2'-disulfonic acid, inhibitor of the anion exchanger, but sensitive to amiloride, inhibitor of the Na(+)/H(+) exchanger. In addition to the amiloride-sensitive Na(+)/H(+) exchanger, another H(+) extruding mechanism was detected in U-2 OS cells, the Na(+)-dependent HCO(3)(-)/Cl(-) exchanger. No significant difference in resting pH(i) and in the rate of acidification or alkalinisation was observed in clones obtained from U-2 OS cells by transfection with the MDR1 gene and overexpressing P-glycoprotein. However, both V(max) and K' values for intracellular [H(+)] of the Na(+)/H(+) exchanger were significantly reduced in MDR1-transfected clones, in the absence and/or presence of drug selection, in comparison to vector-transfected or parental cell line. NHE1, NHE5 and at a lower extent NHE2 mRNA were detected in similar amount in all U2-OS clones. It is concluded that, although overexpression of P-glycoprotein did not impair pH(i) regulation in U-2 OS cells, the kinetic parameters of the Na(+)/H(+) exchanger were altered, suggesting a functional relationship between the two membrane proteins.     

Nuclear factor-kappa B-independent regulation of lipopolysaccharide-mediated interleukin-6 biosynthesis

The possible involvement of nuclear factor (NF)-kappa B in mediating the regulation of interleukin (IL)-6 biosynthesis in response to E. coli-derived lipopolysaccharide-endotoxin (LPS) was investigated in vitro. In alveolar epithelial cells, irreversible inhibition of the proteasome complex by carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132; 1-50 muM) did not affect LPS-mediated IL-6 secretion. Whereas the selective inhibition of the NF-kappa B pathway by the action of caffeic acid phenyl ethyl ester (CAPE; 1-100 microM) attenuated LPS-dependent IL-6 production at 100 muM, sulfasalazine (SSA; 0.1--10 mM), a potent and irreversible inhibitor of NF-kappa B, did not inhibit LPS-dependent IL-6 secretion. Incorporation of a selectively permeant inhibitor of NF-kappa B, SN-50 (1-20 microM), a peptide which contains the nuclear localization sequence (NLS) for the p50 NF-kappa B subunit and the amino-terminal sequence of Kaposi fibroblast growth factor to promote cell permeability, did not reduce LPS-mediated release of IL-6. These data indicate a NF-kappa B-independent pathway mediating LPS-dependent regulation of IL-6 biosynthesis in the airway epithelium.