Adenovirus mediated expression "in vivo" of the chemokine receptor CXCR1.

A major hurdle in the structural analysis of membrane proteins is the expression of a functional and homogeneous form of the protein. Except for rhodopsin, most G protein-coupled receptors (GPCRs) are endogenously expressed at very low levels. Heterologous expression of GPCRs in bacteria, yeast, insect cells or mammalian cell lines often yields proteins with large amounts of misfolded proteins and heterogeneous posttranslational modifications. Here, we report a novel mammalian "in vivo" system for the expression of the chemokine receptor CXCR1. This receptor was expressed in liver of mice infected with adenovirus encoding CXCR1. Liver plasma membranes from infected mice displayed high-levels of (125)I-labeled human interleukin-8 (IL-8) binding. The pharmacological profile of the recombinant CXCR1 expressed "in vivo" was similar to those expressed in neutrophils. We found that the incorporation of the detergent solubilized CXCR1 into phospholipid vesicles in the presence of Gi/Go proteins is required for the reconstitution of (125)I-IL-8 binding. On the basis of the presence of the several endogenous His residues and glycosylation moieties in CXCR1 we fractionated the detergent-solubilized plasma membranes by employing Ni- and Concanavalin A-based chromatography. Fractions enriched with CXCR1 were monitored by (125)I-IL-8-bound to the receptor and Western blots with anti-CXCR1 antibodies. This robust expression system could be readily applied for the expression of GPCRs and other eukaryotic membrane proteins.

Identification of Genes Responding to Low-Dose Arsenite Using HiCEP.

ABSTRACT Chronic ingestion of arsenic in polluted food and water can cause various human disorders including skin and lung cancers. Sensitive biomarkers from human tissue/cells could help to prevent chronic intoxication with low-dose arsenite. Using High-Coverage Expression Profiling (HiCEP), an Amplified Fragment Length Polymorphism (AFLP)-based gene expression profiling technique, we analyzed the expression of approximately 11,000 genes in human lung fibroblasts (HFLIII) and compared the profiles between cells, treated and untreated with 1 muM sodium arsenite (NaAsO(2)). Hundreds of genes appeared upregulated and downregulated more than two-fold, 2 h after the treatment. Marked induction was found (>4.4-fold) in a few genes including HMOX1, INHBA, and ANKRD11. Induction of the HMOX1 was detected with a dose of arsenite at as low as 0.3 muM (0.04 ppm) and reached its maximum at 4 h after the treatment. The arsenite-induced HMOX1 expression was attenuated by the promoted glutathione (GSH) synthesis by N-acetyl-L-cysteine (NAC). However, it was not affected by pretreating the cells with general radical scavengers, consistent with the fact that ionizing radiation at either high-or low-doses has never induced HMOX1 in the same assay system. Thus, induction of HMOX1 gene is highly sensitive and also selective against arsenite in the cells. The present process could provide a useful strategy for exploring biomarkers that might help in assessing the known and unknown risks of any natural and artificial toxic reagents.

Radiation-induced apoptosis in peritoneal resident macrophages of C3H mice: selective involvement of superoxide anion, but not other reactive oxygen species.

Remarkably, apoptosis was induced by gamma-ray-irradiation in peritoneal resident macrophages (PRM) of C3H mice, but not other strains of mice. The mechanism of this strain-specific apoptosis induction was studied. Apoptosis in PRM was detected microscopically. Various radical scavengers were examined to identify the critical radicals involved in apoptosis induction. Intracellular peroxide levels were measured with a redox-sensitive dye, 2',7'-dichlorofluorescin diacetate (DCFH). Superoxide dismutase or catalase was introduced into the cells using commercially available Hemagglutinating Virus of Japan (HVJ) envelope vector kit. The enzyme activity of superoxide dismutase was also measured. Radiation-induced apoptosis in C3H mouse PRM was significantly suppressed by treatment with a pharmacological scavenger of superoxide anion, Tiron, but not with other radical scavengers. Intracellular peroxide levels were not elevated by irradiation at doses high enough to induce apoptosis maximally. Radiation-induced apoptosis in C3H mouse PRM was markedly suppressed by superoxide dismutase introduced into the cells using the HVJ envelope vector, but not catalase. The enzyme activity of superoxide dismutase in C3H mouse PRM was comparable with that in B6 mouse PRM. It was concluded that superoxide played the major role in radiation-induced apoptosis in the C3H mouse PRM and that cellular responses downstream or unrelated to superoxide might be responsible for the strain difference in radiation-induced apoptosis of mouse PRM.

A deficiency in DNA repair and DNA-PKcs expression in the radiosensitive BALB/c mouse.

We have studied the efficiency of DNA double strand break (DSB) rejoining in primary cells from mouse strains that show large differences in in vivo radiosensitivity and tumor susceptibility. Cells from radiosensitive, cancer-prone BALB/c mice showed inefficient end joining of gamma ray-induced DSBs as compared with cells from all of the other commonly used strains and F1 hybrids of C57BL/6 and BALB/c mice. The BALB/c repair phenotype was accompanied by a significantly reduced expression level of DNA-PKcs protein as well as a lowered DNA-PK activity level as compared with the other strains. In conjunction with published reports, these data suggest that natural genetic variation in nonhomologous end joining processes may have a significant impact on the in vivo radiation response of mice.

Differential mechanisms of recognition and activation of interleukin-8 receptor subtypes.

We have probed an epitope sequence (His18-Pro19-Lys20-Phe21) in interleukin-8 (IL-8) by site-directed mutagenesis. This work shows that single and double Ala substitutions of His18 and Phe21 in IL-8 reduced up to 77-fold the binding affinity to IL-8 receptor subtypes A (CXCR1) and B (CXCR2) and to the Duffy antigen. These Ala mutants triggered neutrophil degranulation and induced calcium responses mediated by CXCR1 and CXCR2. Single Asp or Ser substitutions, H18D, F21D, F21S, and double substitutions, H18A/F21D, H18A/F21S, and H18D/F21D, reduced up to 431-fold the binding affinity to CXCR1, CXCR2, and the Duffy antigen. Interestingly, double mutants with charged residue substitutions failed to trigger degranulation or to induce wild-type calcium responses mediated by CXCR1. Except for the H18A and F21A mutants, all other IL-8 mutants failed to induce superoxide production in neutrophils. This study demonstrates that IL-8 recognizes and activates CXCR1, CXCR2, and the Duffy antigen by distinct mechanisms.

Structural biology of chemokine receptors.

Chemokine receptors are G protein-coupled receptors that mediate migration and activation of leukocytes as an important part of a protective immune response to injury and infection. In addition, chemokine receptors are used by HIV-1 to infect CD4 positive cells. The structural bases of chemokine receptor recognition and signal transduction are currently being investigated. High-resolution X-ray diffraction and NMR spectroscopy of chemokines indicate that all these peptides exhibit a common folding pattern, in spite of its low degree of primary-sequence homology. Chemokines' functional motifs have been identified by mutagenesis studies, and a possible mechanism for receptor recognition and activation is proposed, but high-resolution structure data of chemokine receptors is not yet available. Studies with receptor chimeras have identified the putative extracellular domains as the major selectivity determinants. Single-amino acid substitutions in the extracellular domains produce profound changes in receptor specificity, suggesting that motifs in these domains operate as a restrictive barrier to a common activation motif. Similarly HIV-1 usage of chemokine receptors involve interaction of one or more extracellular domains of the receptor with conserved and variable domains on the viral envelope protein gp 120, indicating a highly complex interaction. Elucidating the structural requirements for receptor interaction with chemokines and with HIV-1 will provide important insights into understanding the mechanisms of chemokine recognition and receptor activation. In addition, this information can greatly facilitate the design of effective immunomodulatory and anti-HIV-1 therapeutic agents.

Wortmannin inhibits repair of DNA double-strand breaks in irradiated normal human cells.

Wortmannin, a specific inhibitor of PI-3 kinase, was recently found to be an effective radiosensitizer in cells of various human and murine cell lines. Another study indicated that wortmannin inhibited repair of DNA double-strand breaks (DSBs) in irradiated Chinese hamster ovary cells using the neutral elution assay. To further clarify the mechanism behind radiosensitization by wortmannin, we have studied DSB repair in gamma-irradiated normal human fibroblasts using pulsed-field gel electrophoresis. The rejoining of DSBs in irradiated cells was significantly inhibited when 20 microM or more of wortmannin was added to the cells. The colony formation assay in cultures treated with wortmannin showed that the radiosensitization occurred in a manner that was dependent on the drug concentration. However, significant sensitization was observed only with a concentration of wortmannin of 20 microM or higher, reflecting the results of DSB rejoining studies. No marked reduction in plating efficiencies was observed for cells treated with wortmannin alone. The studies of the levels of expression of DNA-dependent protein kinase (DNA-PK) indicated that, while there were no significant changes in expression of Ku protein, the expression of the DNA-PK catalytic subunit (DNA-PKcs) was reduced markedly in cultures treated with wortmannin using an antibody against the C-terminus region of DNA-PKcs. In addition, no reduction in the levels of expression of DNA-PKcs was observed in cells treated with wortmannin using an antibody which recognizes a mid-region of this large protein. These results together with those of related studies suggest that wortmannin radiosensitizes normal human cells by inhibiting DSB repair and that this inhibition is a consequence of an inactivation of kinase activity and/or a structural change caused by binding of wortmannin to the C-terminus region of DNA-PKcs.

CXC chemokines suppress proliferation of myeloid progenitor cells by activation of the CXC chemokine receptor 2.

IL-8 is one of the major mediators of the transendothelial migration of neutrophils from the circulation to the site of injury and infection. In this work we demonstrate that the CXC or alpha-chemokines, IL-8 and melanoma growth stimulatory activity (MGSA) induce myeloid suppression via direct action on progenitor cells, mediated by activation of the murine homologue of the CXC chemokine receptor-2 (CXCR2) or IL-8R B. We first show that proliferation of the IL-3-dependent murine myeloid progenitor cell line 32D is suppressed by human IL-8 and the functionally and structurally related peptide, MGSA. Second, we show for the first time the high endogenous expression of the murine CXCR2 in 32D cells, as demonstrated by Northern blot analysis, binding to [125I]macrophage inflammatory protein-2, and macrophage inflammatory protein-2-induced calcium responses in 32D cells. Third, we demonstrate that IL-8 and MGSA induce a rise in intracellular calcium in 32D cells. The IL-8-induced Ca2+ response is desensitizing, since a second dose of IL-8 did not trigger a second calcium response. Other chemokines, including neutrophil-activating protein-2, platelet factor-4, RANTES, and macrophage chemotactic protein-1, neither suppressed the proliferation of 32D cells nor induced a rise in intracellular calcium. Finally, the IC50 of IL-8- and MGSA-dependent suppression of proliferation of 32D cells is in good agreement with the EC50 of IL-8- and MGSA-dependent activation of neutrophil Mac-1 up-regulation and chemotaxis. Our studies are consistent with the idea that IL-8 and MGSA suppress the proliferation of 32D cells by activation of murine CXCR2.

A sandwich enzyme immunoassay for cardiac troponin I.

A sandwich enzyme immunoassay for cardiac troponin I (caTnI) is a description which can identify injuries to the heart. Bovine cTnI was purified from bovine cardiac muscle, then an antibody against cTnI was prepared. We confirmed by immunoblotting that the anti-cTnI antibody reacted only to proteins extracted from the heart and in addition, only to cTnI among them. A mouse monoclonal anti-cTnI IgG-coated polystyrene ball was incubated with cTnI, and subsequently with affinity-purified rabbit anti-cTnI Fab-peroxidase conjugate. Specifically bound peroxidase activity was assayed by fluorometry. The detection limit was 3 fmol (84 ng) per assay. The cross-reaction of the sandwich enzyme immunoassay in regard to proteins from other organs was investigated. A little cross-reaction was recognizable only when proteins from skeletal muscle were in high concentration, but we supposed that this assay could be applied to identify injuries to the heart. We developed a sandwich enzyme immunoassay for bovine cTnI. However, further studies will be aimed at developing a sandwich enzyme immunoassay for human cTnI.

Peroxidase labeling of IgMs fragment of ABO blood group specific mouse monoclonal IgM.

A method for peroxidase labeling of the monomeric subunit (IgMs) of ABO blood group specific mouse monoclonal IgM is described. IgM was purified from a commercial monoclonal anti-B blood grouping reagent by a combination of salt precipitation, euglobulin precipitation, and gel filtration. IgM was mildly reduced with L-cysteine to yield SH-bearing IgMs. Finally, IgMs was conjugated to horseradish peroxidase, into which SH-reacting maleimide groups had been introduced using N-succinimidyl 6-maleimidohexanoate, through the selective reaction between SH of IgMs and maleimide groups of peroxidase.

Different histochemical findings in the brain produced by mercuric chloride and methyl mercury chloride in rats.

The chemical form of mercury reactive by the histochemical technique was studied by using rats and mice treated with mercuric chloride (HgCl2) and methyl mercury chloride (MeHgCl). Mercury granules were demonstrated histochemically in the brain of HgCl2-treated rats with higher levels of inorganic mercury. However, mercury granules were not demonstrated in the brain of MeHgCl-treated rats in spite of a considerably high organic mercury level. In rats and mice with significant biotransformation of injected MeHg to inorganic mercury, the appearance of the peak inorganic mercury level and mercury granules in the brain seemed to occur on the same day. These results suggested that mercury granules in the brain represent inorganic mercury, but not organic mercury. In the brains showing mercury granules, the lowest level of inorganic mercury was 0.12 micrograms/g in HgCl2-treated rats, 0.14 micrograms/g in MeHgCl-treated rats, and 0.12 micrograms/g in MeHgCl-treated mice. These values were similar to the level of inorganic mercury in the brain from human autopsy cases. Mercury granules were demonstrated histochemically in nerve cells, choroid plexus and phagocytes in the brain of both HgCl2-and MeHgCl-treated rats, and in ependyma of MeHgCl-treated rats. The level of inorganic mercury of the brains showing mercury granules in phagocytes was lower in MeHgCl-treated rats than in HgCl2-treated rats. The numbers of mercury granules in nerve cells increased with the rising level of inorganic mercury in the brain of MeHgCl-treated rats.

Taxonomic study and fermentation of producing organism and antimicrobial activity of mildiomycin.

A taxonomic study of strain B-98891, which produced an antibiotic effective against powdery mildew of barley, identified it as Streptoverticillium rimofaciens. On agar media the antibiotic, which was named mildiomycin, was only weakly active against most fungi and bacteria tested. However, it inhibited some Mycobacterium and Rhodotorula, and it showed excellent control of powdery mildew of barley plants in greenhouse tests at concentrations between 31.2 and 62.5 ppm. Rhodotorula rubra IFO 0907 was selected as the test organism for in vitro assay of mildiomycin.