Multiple signaling pathways of human interleukin-8 receptor A. Independent regulation by phosphorylation

Interleukin-8 (IL-8) receptor A (CXCR1) couples to a pertussis toxin-sensitive G protein to mediate phospholipase Cbeta (PLCbeta) activation and cellular responses. Responses to CXCR1 are attenuated by prior exposure of neutrophils to either IL-8, a cleavage product of the fifth component of complement (C5a) or n-formylated peptides (formylmethionylleucylphenylalanine, fMLP). To characterize the role of receptor phosphorylation in the regulation of the CXCR1, a phosphorylation-deficient mutant, M2CXCR1, was constructed. This receptor, stably expressed in RBL-2H3 cells, coupled more efficiently to G protein and stimulated enhanced phosphoinositide hydrolysis, cAMP production, exocytosis, and phospholipase D activation, and was resistant to IL-8-induced receptor internalization. The rate and total amount of ligand stimulated actin polymerization remained unchanged, but interestingly, chemotaxis was decreased by approximately 30% compared with the wild type receptor. To study the role of receptor phosphorylation in cross-desensitization of chemoattractant receptors, M2CXCR1 was coexpressed with cDNAs encoding receptors for either fMLP (FR), C5a (C5aR), or platelet-activating factor (PAFR). Both C5aR and PAFR were cross-phosphorylated upon M2CXCR1 activation, resulting in attenuated guanosine 5'-3'-O-(thio)triphosphate (GTPgammaS) binding in membranes. In contrast, FR and M2CXCR1 were resistant to cross-phosphorylation and cross-inhibition of GTPgammaS binding by other receptors. Despite the resistance of M2CXCR1 to cross-phosphorylation and receptor/G protein uncoupling, its susceptibility to cross-desensitization of its Ca2+ response by fMLP and C5a, was equivalent to CXCR1. Regardless of the enhancement in certain receptor functions in M2CXCR1 compared with the wild type CXCR1, the mutated receptors mediated equivalent PLCbeta3 phosphorylation and cross-desensitization of Ca2+ mobilization by FR, C5aR, and PAFR. The results herein indicate that phosphorylation of CXCR1 regulates some, but not all of the receptors functions. While receptor phosphorylation inhibits G protein turnover, PLC activation, Ca2+ mobilization and secretion, it is required for normal chemotaxis and receptor internalization. Since phosphorylation of CXCR1 had no effect on its ability to induce phosphorylation of PLCbeta3 or to mediate class-desensitization, these activities may be mediated by independently regulated pathways.     

Protein kinase C-mediated inhibition of transmembrane signalling through CCK(A) and CCK(B) receptors

1. The rat CCK(A) and CCK(B) receptors were stably expressed in Chinese hamster ovary (CHO-09) cells in order to compare modes of signal transduction and effects of protein kinase C (PKC) thereupon. 2. Spectrofluorophotometry of Fura-2-loaded cells revealed that both receptors retained their pharmacological characteristics following expression in CHO cells. Sulphated cholecystokinin-(26-33)-peptide amide (CCK-8-S) increased the cytosolic Ca2+ concentration ([Ca2+]i) in CCK(A) cells, measured as an increase in Fura-2 fluorescence emission ratio, 1000 fold more potently than its non-sulphated form (CCK-8-NS) (EC50 values of 0.19 nM and 0.18 microM, respectively). By contrast, CCK-8-S and CCK-8-NS were equally potent in CCK(B) cells (EC50 values of 0.86 nM and 1.18 nM, respectively). The CCK(A) receptor agonist JMV-180 increased [Ca2+]i only in CCK(A) cells. Likewise, pentagastrin increased [Ca2+]i only in CCK(B) cells. Finally, CCK-8-S-induced Ca2+ signalling through the CCK(A) receptor was most potently inhibited by the CCK(A) receptor antagonist L364,718, whereas the CCK(B) receptor antagonist L365,260 was more potent in CCK(B) cells. 3. Receptor-mediated activation of adenylyl cyclase was measured in the presence of the inhibitor of cyclic nucleotide phosphodiesterase activity, 3-isobutyl-1-methylxanthine. CCK-8-S and, to a lesser extent, CCK-8-NS, but not JMV-180 or pentagastrin, stimulated the accumulation of cyclicAMP in CCK(A) cells. By contrast, none of these agonists increased cyclicAMP in CCK(B) cells. 4. Short-term (3 min) pretreatment with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) evoked a rightward shift of the dose-response curve for the Ca2+ mobilizing effect of CCK-8-S in both cell lines. In addition, short-term TPA pretreatment markedly reduced CCK-8-S-induced cyclicAMP accumulation in CCK(A) cells. In both cases, the inhibitory effect of TPA was abolished by the PKC inhibitors, GF-109203X and staurosporine, whereas no inhibition was observed with the inactive phorbol ester, 4-alpha-phorbol 12-myristate 13-acetate. 5. During prolonged TPA treatment, the cells gradually recovered from phorbol ester inhibition and in the case of CCK-8-S-induced Ca2+ mobilization complete recovery was achieved after 24 h of TPA treatment. Western blot analysis revealed that this recovery was paralleled by down-regulation of PKC-alpha, suggesting the involvement of this PKC isotype in the inhibitory action of TPA. 6. This study demonstrates that following expression in CHO cells (i) both CCK(A) and CCK(B) receptors are coupled to Ca2+ mobilization, (ii) only CCK(A) receptors are coupled to cyclicAMP formation and (iii) with both receptors signalling is inhibited by PKC.     

Spectral Properties and Sensitization of Ce3+ and Eu2+ Codoped Calcium Zinc Chlorosilicate

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Solvothermal fabrication and luminescent properties of Eu2＋/Ce3＋ doped barium lithium fluoride

Phosphors of BaLiF3 doped with Eu or/and Ce were solvothermally prepared at 200°C for 5d and characterized by means of X-ray powder diffraction (XRD) and environment scanning electron microscopy (ESEM). The excitation and emission spectra of the rare earth ions doped BaLiF3 were measured by fluorescence spectroscopy and the effects of Ce3+ ions on the luminescence of Eu2+ ions were investigated. In the codoped Eu2+ and Ce3+ system, the emission intensity of Eu2+ ion gradually increased with the Ce3+ concentration increasing, and the enhancement of Eu2+ fluorescence was due to efficient energy transfer from Ce3+ to Eu2+ in the host.     

Photoluminescence properties and effect of Bi~（3＋） dopant of Eu~（3＋） and Dy~（3＋） co-doped Sr_3V_2O_8

Photoluminescence properties of Sr 2.5 Dy 1/3-x Eu x V 2 O 8(x=0,0.06,0.12,0.18,0.24,0.33) were investigated.The excitation spectra included a broad band in the short wavelength region and several sharp lines in the longer wavelength region,and the spectral origin were discussed.The emission spectra were measured in two different exciting ways,i.e.,exciting the VO 4 group at 270 nm and the Eu 3+ ion at 398 nm,respectively,and the energy transferring process was reasonably suggested.Furthermore,multi-color emission could be achieved in Sr 2.5 Dy 1/3-x Eu x V 2 O 8,indicating that the studied samples had potential applications in the white light emitting diodes.Further investigation showed that reducing the concentration of Eu 3+ and Dy 3+ and introducing Bi 3+ as a sensitizer ion greatly enhanced the emission intensity.     

Combined preoperative and postoperative immunotherapy for murine C1300 neuroblastoma

Preoperative treatment of murine C1300-neuroblastoma (C1300) with triple immunotherapy using low-dose cyclophosphamide (CY), retinyl palmitate (RP), and interleukin-2 (IL2), followed by tumor resection leads to significant initial tumor control and prolonged survival. However, because long-term tumor recurrence is 67%, the efficacy of continued postoperative immunotherapy is now evaluated. Thirty-two A/J mice with 1 cm subcutaneous C1300 tumors were treated for 13 days with CY-100 mg/kg, intraperitoneally (IP), on day 2 of treatment then 25 mg/kg on day 9, RP-2500 IU IP 2 x/week, and IL2 1.6 x 10(5) U IP BID on days 4 to 9 and 11 to 13. On day 14, mice were divided into five treatment groups: (1) OP (operated-tumor resection, n = 6); (2) OP+CY (resection and postoperative CY, n = 7); (3) OP+CY+RP (resection and postoperative CY+RP, n = 7); (4) OP+CY+RP+IL2 (resection and postoperative CY+RP+IL2, n = 7); and (5) CY+RP+IL2 (continued CY+RP+IL2 with no resection, n = 5). Survival and postoperative tumor recurrence were followed for 60 days. The cure rates were group 1 33% (2/6), group 2 43% (3/7), group 3 29% (2/7), group 4 71% (5/7), and group 5 20% (1/5). After surgery, tumors that recurred did so in 8 to 22 days, with no statistical difference noted between groups. MHC class I antigenic expression of tumors resected on day 14 and recurrent tumors was determined with monoclonal antibodies and flow cytometry. In tumors resected on day 14, class I expression measured by mean fluorescence, was 374.8 +/- 27.40.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of cytokine expression and leukotriene formation in human basophils by growth factors, chemokines and chemotactic agonists

Basophils stimulated with IL-3 plus C5a selectively express IL-4 and IL-13 and continuously produce leukotrienes (LT) for hours. C5a combined with IL-5 or granulocyte-macrophage colony-stimulated factor was, however, much less effective in promoting cytokine expression and a late continuous phase of LTC4 production, possibly due to lower expression levels of their receptor alpha chains. Basophils also express several chemoattractant receptors, including high levels of C5a receptors, macrophage chemotactic protein (MCP) receptors (CCR2) and eotaxin receptors (CCR3), intermediate levels of CXCR1, CXCR2 and platelet-activating factor receptors, and lower levels of N-formyl-Met-Leu-Phe (fMLP) receptors. However, among the corresponding agonists, only C5a, fMLP and much more weakly MCP1, were found to induce cytokine expression and continuous LTC4 release, and only when combined with IL-3. CCR3, which is highly expressed on basophils and has been shown to mediate strong migratory but weak release responses, does not regulate cytokine expression. The weakly expressed fMLP receptor is an efficient activator of several cell functions including LTC4 formation, while CXCR2 hardly affects basophil function despite considerable expression. Thus, chemoattractant-receptors mediate different cellular responses unrelated to their expression levels.     

Problems with the laryngeal mask airway: prevention and management

The aim of the present work was to define an FSH receptor (FSHR) peptide that can induce antibodies that will inhibit the bioactivity of FSH. Therefore, the hFSHR sequence was aligned with that of all other known G-protein coupled receptors. An area with increased sequence homology was identified between the FSH-, LH-, TSH receptors, the C5a receptor and the IL8 receptor. The similarity consists of a richness in acidic (D and E) and hydrophobic (Y and F) residues. In hFSHR the sequence is EDNESSYSRGFDMTYTEFDYDLCNEVVD (amino acid 299-326). Research on both the C5a- and IL8-receptor has indicated that this part is responsible for hormone binding but not for signal transduction. Protamine. an antagonist for both the C5a- and IL8 receptor also inhibited the bioactivities of FSH and LH when tested in a bioassay. This suggests that in the hFSHR this region might also be involved in hormone binding. Specificity of this region towards the diverse ligands all binding to the C5a or to the IL8 receptor might be attributed to differences in the profile of alternating basic and hydrophobic residues. Therefore, the hypothesis was tested as to whether antisera raised against peptides of this FSHR-domain would inhibit FSH-bioactivity but not LH-bioactivity. Indeed antisera were found (anti-hFSHR 309-322) that inhibited the biological activity of FSH in a bioassay. These antisera proved to be specific since they did not inhibit the bioactivity of LH. These data suggest that the core sequence (hFSHR 309-322) of the aligned domain of the hFSHR, in analogy to the IL8- and C5a receptors, is involved in hormone binding and ligand specificity. This domain therefore forms a valuable tool in FSH- and FSHR research for scientific and medical purposes.     

Role of endothelin receptors, calcium and nitric oxide in the potentiation by endothelin-1 of the sympathetic contraction of rabbit ear artery during cooling

1. To examine further the potentiation by endothelin-1 on the vascular response to sympathetic stimulation, we studied the isometric response of isolated segments, 2 mm long, from the rabbit central ear artery to electrical field stimulation (1-8 Hz), under different conditions, at 37 degrees C and during cooling (30 degrees C). 2. Electrical stimulation produced frequency-dependent contraction, which was reduced (about 63% for 8 Hz) during cooling. At 30 degrees C, but not at 37 degrees C, endothelin-1 (1, 3 and 10 nM) potentiated the contraction to electrical stimulation in a dose-dependent way (from 43 +/- 7% to 190 +/- 25% for 8 Hz). 3. This potentiation by endothelin-1 was reduced by the antagonist for endothelin ETA receptors BQ-123 (10 microM) but not by the antagonist for endothelin ETB receptors BQ-788 (10 microM). The agonist for endothelin ETB receptors IRL-1620 (0.1 microM) did not modify the contraction to electrical stimulation. 4. The blocker of L-type Ca2+ channels verapamil (10 microM l-1) reduced (about 72% for 8 Hz) and the unspecific blocker of Ca(2+)-channels NiCl2 (1 mM) practically abolished (about 98%), the potentiating effects of endothelin-1 found at 30 degrees C. 5. Inhibition of nitric oxide synthesis with NG-nitro-L-arginine (L-NOARG, 0.1 mM) increased the contraction to electrical stimulation at 30 degrees C more than at 37 degrees C (for 8 Hz, this increment was 297 +/- 118% at 30 degrees C, and 66 +/- 15% at 37 degrees C). Endothelium removal increased the contraction to electrical stimulation at 30 degrees C (about 91% for 8 Hz) but not at 37 degrees C. Both L-NOARG and endothelium removal abolished the potentiating effects of endothelin-1 on the response to electrical stimulation found at 30 degrees C. 6. These results in the rabbit ear artery suggest that during cooling, endothelin-1 potentiates the contraction to sympathetic stimulation, which could be mediated at least in part by increasing Ca2+ entry after activation of endothelin ETA receptors. This potentiating effect of endothelin-1 may require the presence of an inhibitory tone due to endothelial nitric oxide.     

Luminescence properties of BaB8O13：Eu under UV and VUV excitation

The phosphor BaB8O13:Eu3+ were synthesized by solid-state reaction, and their luminescent properties were studied under 254 and 147 nm excitation. The excitation spectrum showed two broad bands in the range of 100-300 nm: one was the host lattice absorption with the maxima at 160 nm and the other was Ba-O absorption overlapped with the CT band of Eu3+, which indicated that the energy of the host lat-tice absorption could be efficiently transferred to the Eu3+. The overlapped bands were tended to separate when monitored by different wave-length, which indicated that at least two Ba2+ sites were available in BaB8O13. The emissions of Eu3+ (612 nm) and Eu2+ (405 nm) were both observed in the emission spectra of BaB8O13:Eu3+ under the excitation of either 254 or 147 nm. With the doping concentration of Eu3+ in-creasing, the 612 nm emission was enhanced while 405 nm emission was decreased under 254 nm excitation, which was due to the persistent energy transfer from Eu2+ to Eu3+. While under 147 nm excitation, the 612 nm emission was quenched and the 405 nm emission was en-hanced. It was concluded that the preferential excitation of Eu2+ under 147 nm excitation was one of the reasons for this facts.     

Expression and functional activity of the IL-8 receptor type CXCR1 and CXCR2 on human mast cells

To further elucidate mechanisms involved in mast cell accumulation at sites of cutaneous inflammation, we have studied the ability of human leukemic mast cells (HMC-1 cells) to express functionally active IL-8 receptors. Expression of mRNA for both types of IL-8 receptors (CXCR1 and CXCR2) was demonstrated by PCR and of both proteins by flow cytometry. Binding and competition studies with 125I-labeled IL-8 and its homologue melanoma growth stimulating activity (125I-labeled MGSA) revealed two specific binding sites for IL-8, K1 = 1.1 x 10(11) M(-1) and K2 = 5 x 10(7) M(-1); and for MGSA, K1 = 2.8 x 10(10) M(-1) and K2 = 5 x 10(7) M(-1). This finding was supported by a dose-dependent rise of cytosolic free calcium concentration ([Ca2+]i) induced by both chemokines and to a lesser extent by the homologue neutrophil-activating peptide-2 (NAP-2). A significant migratory response of human leukemic mast cells (HMC-1) was observed with all three chemokines at a range from 10(-8) M to 10(-9) M. Moreover, the formation of cellular F-actin was induced in a rapid, dose-dependent fashion, with a maximally 1.7-fold increase at 10(-7) M. Using postembedding immunoelectron microscopy, we could show the expression of CXCRI on the cytoplasmatic membrane of isolated human skin mast cells whereas CXCR2 was located in mast cell-specific granules. These findings demonstrate for the first time the functional expression of both types of IL-8 receptors on human mast cells, suggesting a role for their ligands during mast cell activation and recruitment.     

Study on Luminescence Properties and Crystal-Lattice Environment of Eu~(2 ) in Sr_(4-x)Mg_x Si_3O_8Cl_4∶Eu~(2 ) Phosphor

ChlorosilicatecrystalmaterialM4 Si3O8Cl4 (M =Ba ,Sr ,Ca)isasuitablehostlatticeforluminescencematerials .Adivalenteuropiumactivatedstrontiumchlorosilicatephosphorisakindofgoodblue greenemissionphotoluminescencematerialunderUVexcita tion .Itsluminescencepropertiesandcrystalstructurehavebeenintensivelystudied[1～4 ] .Inthechlorosili catehost ,theluminescenceofEu2 consistsofa4f6 5d1- 4f7(8S7/2 )broad bandemission ,whichbe longstoelectric dipoleallowedtransitionandhasthepropertiesoflargeabso…     

Synthesis, characterization and properties of some rare earth complexes with 2,6-pyridine dicarboxylic acid and α-Picolinic acid

Five novel ternary complexes were synthesized by the rare earth with 2,6-pyridine dicarboxylic acid andα-picolinic acid.These complexes were characterized by elemental analysis,molar conductivity,FT-IR,UV-Vis,Raman,TG-DTA and XRD.The general formula of the complexes was[RE(DPA)(L α )(H2O)]·2H2O(RE=Pr 3+ ,Nd 3+ ,Sm 3+ ,Eu,Er 3+ ;DPA=2,6-pyridine dicarboxylic acid;L α =α-picolinic acid). The investigation of fluorescence properties of the Eu-complex showed that the Eu(III)ion could be sensitized efficiently by the ligand to some extent.The antibacterial activity test indicated that all the five complexes exhibited antibacterial ability against Escherichia coli and Staphylococcus aureus with broad antimicrobial spectrums.     

Effect of gadolinium incorporation on optical characteristics of YNbO_4：Eu~（3＋） phosphors for lamp applications

Eu3+-doped (Y,Gd)NbO4 phosphor was synthesized by solid-state reaction for possible application in cold cathode fluorescent lamps. A broad absorption band with peak maximum at 272 nm was observed which was due to the charge transfer between Eu3+ ions and neighboring oxygen anions. A deep red emission at the peak wavelength of 612 nm was observed which could be attributed to the 5D0→7F2 transition in Eu3+ ions. The highest luminance for Y1-x-yGdyNbO4:Eux3+ under 254 nm excitation was achieved at Eu3+ concentration of 18 mol.% (x=0.18) and Gd3+ concentration of 8.2 mol.% (y=0.082). The luminance of Y0.738Gd0.082NbO4:Eu3+0.18 was higher than that of a typical commercial phosphor Y2O3:Eu3+ and the CIE chromaticity coordinate was (0.6490, 0.3506), which was deeper than that of Y2O3:Eu3+. The particle size of the synthesized phosphors was controlled by the NaCl flux and particle size as high as 8 μm with uniform size distribution of particles was obtained.     

Identification of determinants on a dualtropic human immunodeficiency virus type 1 envelope glycoprotein that confer usage of CXCR4

The chemokine receptors CCR5 and CXCR4, in combination with CD4, mediate cellular entry of macrophage-tropic (M-tropic) and T-cell-tropic strains of human immunodeficiency virus type 1 (HIV-1), respectively, while dualtropic viruses can use either receptor. We have constructed a panel of chimeric viruses and envelope glycoproteins in which various domains of the dualtropic HIV-1(DH12) gp160 were introduced into the genetic background of an M-tropic HIV-1 isolate, HIV-1(AD8). These constructs were employed in cell fusion and virus infectivity assays using peripheral blood mononuclear cells, MT4 T cells, primary monocyte-derived macrophages, or HOS-CD4 cell lines, expressing various chemokine receptors, to assess the contributions of different gp120 subdomains in coreceptor usage and cellular tropism. As expected, the dualtropic HIV-1(DH12) gp120 utilized either CCR3, CCR5, or CXCR4, whereas HIV-1(AD8) gp120 was able to use only CCR3 or CCR5. We found that either the V1/V2 or the V3 region of HIV-1(DH12) gp120 individually conferred on HIV-1(AD8) the ability to use CXCR4, while the combination of both the V1/V2 and V3 regions increased the efficiency of CXCR4 use. In addition, while the V4 or the V5 region of HIV-1(DH12) gp120 failed to confer the capacity to utilize CXCR4 on HIV-1(AD8), these regions were required in conjunction with regions V1 to V3 of HIV-1(DH12) gp120 for efficient utilization of CXCR4. Comparison of virus infectivity analyses with various cell types and cell fusion assays revealed assay-dependent discrepancies and indicated that events occurring at the cell surface during infection are complex and cannot always be predicted by any one assay.     

Utilization of chemokine receptors, orphan receptors, and herpesvirus-encoded receptors by diverse human and simian immunodeficiency viruses

Human immunodeficiency virus type 1 (HIV-1) requires both CD4 and a coreceptor to infect cells. Macrophage-tropic (M-tropic) HIV-1 strains utilize the chemokine receptor CCR5 in conjunction with CD4 to infect cells, while T-cell-tropic (T-tropic) strains generally utilize CXCR4 as a coreceptor. Some viruses can use both CCR5 and CXCR4 for virus entry (i.e., are dual-tropic), while other chemokine receptors can be used by a subset of virus strains. Due to the genetic diversity of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) and the potential for chemokine receptors other than CCR5 or CXCR4 to influence viral pathogenesis, we tested a panel of 28 HIV-1, HIV-2, and SIV envelope (Env) proteins for the ability to utilize chemokine receptors, orphan receptors, and herpesvirus-encoded chemokine receptor homologs by membrane fusion and virus infection assays. While all Env proteins used either CCR5 or CXCR4 or both, several also used CCR3. Use of CCR3 was strongly dependent on its surface expression levels, with a larger number of viral Env proteins being able to utilize this coreceptor at the higher levels of surface expression. ChemR1, an orphan receptor recently shown to bind the CC chemokine I309 (and therefore renamed CCR8), was expressed in monocyte and lymphocyte cell populations and functioned as a coreceptor for diverse HIV-1, HIV-2, and SIV Env proteins. Use of ChemR1/CCR8 by SIV strains was dependent in part on V3 loop sequences. The orphan receptor V28 supported Env-mediated cell-cell fusion by four T- or dual-tropic HIV-1 and HIV-2 strains. Three additional orphan receptors failed to function for any of the 28 Env proteins tested. Likewise, five of six seven-transmembrane-domain receptors encoded by herpesviruses did not support Env-mediated membrane fusion. However, the chemokine receptor US28, encoded by cytomegalovirus, did support inefficient infection by two HIV-1 strains. These findings indicate that additional chemokine receptors can function as HIV and SIV coreceptors and that surface expression levels can strongly influence coreceptor use.