Immune responses induced by intranasal imiquimod and implications for therapeutics in rhinovirus infections.

Notwithstanding the progress recently made in immunology and virology, there is yet no effective, specific treatment for the common cold. Symptomatic treatment is minimally effective. An anecdotal report of rapid clearing of the common cold of recent onset after intranasal application of imiquimod in several subjects by one of the authors, made us test the hypothesis that this treatment works through the secretion of interferon by the nasal mucosa. We decided to do an animal study in primates (Indian Macaca Mulata): 5 treatment and 3 control animals were used. Imiquimod or placebo was massaged into the nares of the animals and periodic samples of post-nasal fluid were taken and measurements for Interferon alpha (IFNalpha) and Tumor Necrosis Factor alpha (TNFalpha) were made by ELISA methods, and kinetic studies. mRNA IFNalpha was also isolated and analyzed by quantitative competitive RT-PCR. The internal standard was constructed to be complementary to and compete with oligonucleotide primers and for amplification of target sequences. One intranasal application of imiquimod rapidly (1-4 Hours) induced high levels of mRNA for IFNalpha, and minimal levels in the control animals. Rapid induction of INFalpha, and proportional increase of TNFalpha sustained for 4 and 6 hours respectively were noted. No adverse reactions to treatment were found in macaques during this short period of intranasal imiquimod usage (except in one macaque with a short period of lacrimation). No animal had cytotoxic effects when examined at 6 hr, 12 hr, 24 hr or 48 hr, except one animal, which had an episode of lacrimation for 6 hr post treatment. Thus both safety and efficacy of short treatment with imiquimod is proven in this animal model. Proof of principle for intranasal treatment of the common cold with imiquimod is shown. We think that this work will encourage a number of double blind clinical trials to confirm the effectiveness of the intranasal treatment of the common cold with imiquimod.

HTLV-I and HTLV-II virus expression increase with HIV-1 coinfection.

Coinfections with HIV-1 and HTLV-I or HTLV-II have been associated with unique immunophenotypes and an increased risk for development of neurodegenerative conditions. These findings may result from an increased HTLV-I or II viral burden in dually infected individuals. To investigate this possibility, HTLV-I/II tax/rex messenger RNA and viral antigen expression in peripheral blood mononuclear cells (PBMCs) were measured in 37 HTLV-I- or HTLV-II-infected subjects with or without HIV-1 coinfection. Tax/rex messenger RNA was detected in 14 of 24 PBMC samples from dually infected subjects, compared with only 1 of 13 PBMC samples from singly infected subjects (58% versus 7%; p < .003). The reverse transcription-polymerase chain reaction (RT-PCR) assay correlated with HTLV-I/II viral antigen detection in PBMC cultures but not with HIV-1 viral load levels in plasma. These findings may provide clues regarding the pathophysiologic consequences of HIV/HTLV-I and HIV/HTLV-II coinfections.

The role of the membrane-spanning and extra-membranous regions of the iron-sulfur protein in its assembly into the cytochrome bc1 complex of yeast mitochondria.

The assembly of six deletion mutants of the Rieske iron-sulfur protein into the cytochrome bc1 complex was investigated by immunoprecipitation from detergent-solubilized mitochondria with specific antisera against either the iron-sulfur protein or the intact cytochrome bc1 complex. After import, the mutant proteins lacking residues 41-55 or 66-78, located at the membrane-spanning region of the protein, and residues 182-196 located at the C-terminus of the protein, were assembled in vitro into the bc1 complex approximately 50% as effectively as the wild type iron-sulfur protein suggesting that these regions of the iron-sulfur protein may not be critical for the assembly. By contrast, only trace amounts of the mutant proteins lacking residues 80-95, 122-135, 138-153 located in the extra-membranous region of the iron-sulfur protein were assembled into the bc1 complex. After import in vitro into mitochondria isolated from a cytochrome b-deficient yeast strain, the mutants lacking residues 41-55 and 182-196 were assembled as efficiently as the wild type; however, the mutants lacking residues 55-66 and 66-78 were assembled less efficiently in the absence of cytochrome b suggesting that the hydrophobic membrane-spanning region, residues 55-78, of the iron-sulfur protein, may interact with cytochrome b during the assembly of the bc1 complex.

Assembly of deletion mutants of the Rieske iron-sulfur protein into the cytochrome bc1 complex of yeast mitochondria.

The assembly of two deletion mutants of the Rieske iron-sulfur protein into the cytochrome bc1 complex was investigated after import in vitro into mitochondria isolated from a strain of yeast, JPJ1, from which the iron-sulfur protein gene (RIP) had been deleted. The assembly process was investigated by immunoprecipitation of the labeled iron-sulfur protein or the two deletion mutants from detergent-solubilized mitochondria with specific antisera against either the iron-sulfur protein or the bc1 complex (complex III) [Fu and Beattie (1991). J. Biol. Chem. 266, 16212-16218]. The deletion mutants lacking amino acid residues 55-66 or residues 161-180 were imported into mitochondria in vitro and processed to the mature form via an intermediate form. After import in vitro, the protein lacking residues 161-180 was not assembled into the complex, suggesting that the region of the iron-sulfur protein containing these residues may be involved in the assembly of the protein into the bc1 complex; however, the protein lacking residues 55-66 was assembled in vitro into the bc1 complex as effectively as the wild type iron-sulfur protein. Moreover, this mutant protein was present in the mitochondrial membrane fraction obtained from JPJ1 cells transformed with a single-copy plasmid containing the gene for this protein lacking residues 55-66. This deletion mutant protein was also assembled into the bc1 complex in vivo, suggesting that the hydrophobic stretch of amino acids, residues 55-66, is not required for assembly of the iron-sulfur protein into the bc1 complex; however, this association did not lead to enzymatic activity of the bc1 complex, as the Rieske FeS cluster was not epr detectable in these mitochondria.

Import of subunit VII of the cytochrome bc1 complex into yeast mitochondrial.

The import of subunit VII, the so-called ubiquinol binding protein of the cytochrome bc1 complex of yeast mitochondria, has been investigated by subcloning the gene for subunit VII into the expression vector pSP64. The precursor to subunit VII synthesized in vitro by a transcription/translation system had the same molecular weight as the mature form of this protein present in mitochondria, confirming earlier reports that subunit VII does not contain a cleavable presequence. The subunit VII precursor was imported efficiently into a protease-resistant compartment of isolated yeast mitochondria in a reaction dependent upon a membrane potential and the presence of ATP both in the mitochondrial matrix and in the extramitochondrial space. After import, the radiolabeled subunit VII was bound to the mitochondrial inner membrane through protein-protein interactions and could not be detected in the matrix fraction or in the intermembrane space. Subunit VII was also imported into mitoplasts, where the protein was associated with the inner membrane facing the matrix side of the membrane. By contrast, after import into mitochondria obtained from yeast cells lacking cytochrome b, subunit VII was localized in the supernatant fraction, suggesting that the presence of cytochrome b may be required for the association of subunit VII with the inner mitochondrial membrane.

Sequences of the iron-sulfur protein precursor necessary for its import and two-step processing in yeast mitochondria.

Gene fusion techniques were used to examine whether the presequence of the iron-sulfur protein contains sufficient information for the import of attached mouse cytosolic enzyme dihydrofolate reductase into isolated yeast mitochondria and its subsequent two-step processing. Genes encoding amino-terminal segments of the iron-sulfur precursor protein including the proposed first presequence (residues 1-21), the complete presequence (residues 1-30), and various lengths of the precursor protein from 31 to 160 residues were fused, in frame, to dihydrofolate reductase. All of the fusion proteins, after synthesis in an in vitro transcription-translation system, were imported into a protease-resistant compartment of mitochondria where a single proteolytic cleavage was observed at the first processing site. The second cleavage, however, was not observed after import of any of the chimeric proteins, suggesting that the second cleavage may be strongly influenced by the presence of the passenger protein. A deletion mutant of the iron-sulfur protein precursor lacking residues 161-180 underwent two proteolytic cleavages similar to those observed for the wild-type iron-sulfur protein after import into mitochondria. These results suggest that the complete sequence of the mature form of the iron-sulfur protein including the amino acid residues involved in binding the iron sulfur clusters is not necessary for the second cleavage to occur. In addition, the hydrophobic sequence present in residues 55-66 of the precursor protein which has been suggested to anchor the iron-sulfur protein to the inner membrane, was not necessary for the import and two-step processing of the protein, since a deletion mutant lacking residues 55-66 was processed correctly.

Direct interaction between the internal NADH: ubiquinone oxidoreductase and ubiquinol:cytochrome c oxidoreductase in the reduction of exogenous quinones by yeast mitochondria.

The reduction of duroquinone (DQ) and 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB) by NADH and ethanol was investigated in intact yeast mitochondria with good respiratory control ratios. In these mitochondria, exogenous NADH is oxidized by the NADH dehydrogenase localized on the outer surface of the inner membrane, whereas the NADH produced by ethanol oxidation in the mitochondrial matrix is oxidized by the NADH dehydrogenase localized on the inner surface of the inner membrane. The reduction of DQ by ethanol was inhibited 86% by myxothiazol; however, the reduction of DQ by NADH was inhibited 18% by myxothiazol, suggesting that protein-protein interactions between the internal (but not the external) NADH: ubiquinone oxidoreductase and ubiquinol:cytochrome c oxidoreductase (the cytochrome bc1 complex) are involved in the reduction of DQ by NADH. The reduction of DQ and DB by NADH and ethanol was also investigated in mutants of yeast lacking cytochrome b, the iron-sulfur protein, and ubiquinone. The reduction of both quinone analogues by exogenous NADH was reduced to levels that were 10 to 20% of those observed in wild-type mitochondria; however, the rate of their reduction by ethanol in the mutants was equal to or greater than that observed in the wild-type mitochondria. Furthermore, the reduction of DQ in the cytochrome b and iron-sulfur protein lacking mitochondria was myxothiazol sensitive, suggesting that neither of these proteins is an essential binding site for myxothiazol. The mitochondria from the three mutants also contained significant amounts of antimycin- and myxothiazol-insensitive NADH:cytochrome c reductase activity, but had no detectable succinate:cytochrome c reductase activity. These results suggest that the mutants lacking a functional cytochrome bc1 complex have adapted to oxidize NADH.

Import of the iron-sulfur protein of the cytochrome b.c1 complex into yeast mitochondria.

The yeast gene for the Rieske iron-sulfur protein of the cytochrome b.c1 complex was subcloned into the expression vector, pSP64, then transcribed and translated in vitro in a reticulocyte lysate in the presence of [35S]methionine. Import studies in vitro of the newly synthesized precursor form of the iron-sulfur protein into isolated yeast mitochondria revealed that the precursor form of the iron-sulfur protein is processed into the mature form via an intermediate form. After the import reaction at 18 or 27 degrees C, treatment of mitochondria with exogenous protease indicated that both intermediate and mature forms had been internalized into mitochondria where they were resistant to digestion by external protease. Import and processing of the iron-sulfur protein into mitochondria also occurred at temperatures ranging from 2 to 27 degrees C in a temperature-dependent manner. Processing of the precursor form to the intermediate form appeared to be less sensitive to temperature than the processing of the intermediate form to the mature form. Moreover, at temperatures of 12 degrees C or lower, the mature form produced was completely digested by exogenous protease suggesting that it was assembled incorrectly in the membrane and not assembled into the b.c1 complex. The successive disappearance of first the mature form and then the intermediate form of the iron-sulfur protein by increasing concentrations of the metal chelators, EDTA and o-phenanthroline, suggested that two different proteases requiring divalent metal ions are involved in the two-step processing of the presequence of the iron-sulfur protein. Furthermore, mitoplasts containing only the matrix/inner membrane fraction were able to import and process the precursor form of the iron-sulfur protein indicating that both proteolytic processing events occur in the matrix/inner membrane fraction.

Electron transfer through center o of the cytochrome b-c1 complex of yeast mitochondria involves subunit VII, the ubiquinone-binding protein.

The role of subunit VII, the ubiquinone-binding protein of the cytochrome b-c1 complex, in electron transfer reactions was investigated in yeast mitochondria. Preincubation of submitochondrial particles with specific antibody against subunit VII prior to addition of either succinate, NADH, or the reduced form of the decyl analogue of ubiquinol resulted in an approximately 40% increase in the extent of cytochrome c1 reduction compared with controls containing preimmune serum. Addition of antimycin, an inhibitor of center i, to submitochondrial particles resulted in a 21% decrease in the rate and a 36% decrease in the extent of cytochrome c1 reduction by succinate. Preincubation of submitochondrial particles with the antibody against subunit VII prior to addition of antimycin resulted in an increase in both the rate and extent of cytochrome c1 reduction to the levels observed in the control without inhibitor. The addition of myxothiazol (an inhibitor of center o), myxothiazol plus antimycin, or alkyl hydroxynaphthoquinone (an inhibitor analogue of ubiquinone) resulted in an almost complete inhibition in both the rate and extent of cytochrome c1 reduction; however, preincubation with the antibody against subunit VII prior to addition of these inhibitors resulted in a significant increase in cytochrome c1 reduction. These results confirm our previous report (Japa, S., Zhu, Q. S., and Beattie, D. S. (1987) J. Biol. Chem. 262, 5441-5444) that subunit VII is involved in electron transfer reactions at center o of the b-c1 complex. We suggest that the binding of antibody to subunit VII inhibits the transfer of electrons to cytochrome b-566. Consequently, two electrons are transferred to the iron-sulfur protein and cytochrome c1 through an antimycin-insensitive pathway. Moreover, the antibody may change the conformation of subunit VII, such that the myxothiazol and hydroxynaphthoquinone binding sites are partially blocked thus permitting electron flow to cytochrome c1.

The iron-sulfur protein is necessary for the complete assembly of the low-molecular-weight subunits into the cytochrome b-c1 complex of yeast mitochondria.

A strain of yeast lacking the gene for the Rieske iron-sulfur protein (RIP) of the cytochrome b-c1 complex was used to study the assembly of this complex in the mitochondrial membrane. This strain lacks the mRNA for the iron-sulfur protein as evidenced by both Northern hybridization using a probe containing the coding region of the gene plus in vitro translation of total RNA followed by immunoprecipitation with a specific antibody against the iron-sulfur protein. In addition, isolated mitochondria from this strain lacked cytochrome c reductase activity with either succinate or the decyl analog of ubiquinol as substrate. Immunoblotting studies with antiserum against the cytochrome b-c1 complex revealed that mitochondria from the iron-sulfur protein-deficient strain have levels of core protein I, core protein II, and cytochrome c1 equal to those of wild-type mitochondria; however, a decrease in cytochrome b was evident from both immunoblotting and spectral analysis. Moreover, it is evident from the immunoprecipitates of radiolabeled mitochondria that the amounts of the low-molecular-weight subunits (17, 14, and 11 kDa) are decreased 53, 65, and 50%, respectively, in mitochondria lacking the iron-sulfur protein. These results suggest that the iron-sulfur protein is required for the complete assembly of the low-molecular-weight subunits into the cytochrome b-c1 complex.

Cytochrome b is necessary for the assembly of subunit VII in the cytochrome b-c1 complex of yeast mitochondria.

The synthesis and assembly of subunit VII, the Q-binding protein of the cytochrome b-c1 complex, into the inner mitochondrial membrane has been compared in wild-type yeast cells and in a mutant cell line lacking cytochrome b. Both immunoblotting and immunoprecipitation analysis with specific antiserum against subunit VII indicated that this subunit is not detectable in the mutant as compared to the wild-type mitochondria. However, labeling in vivo of the cytochrome b deficient yeast cells in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone clearly demonstrated that subunit VII was synthesized in the mutant cells to the same extent as in the wild-type cells. Incubation of subunit VII, synthesized in vitro in a reticulocyte lysate programmed with yeast RNA, with mitochondria isolated from both wild-type and cytochrome b deficient yeast cells revealed that the subunit VII was transported into the wild-type mitochondria into a compartment where it was resistant to digestion by exogenous proteinase K. By contrast, subunit VII was bound in lowered amounts to the cytochrome b deficient mitochondria where it remained sensitive to digestion by exogenous proteinase K, suggesting that the import of subunit VII may be impaired due to the lack of cytochrome b. Furthermore, subunit VII was synthesized both in vivo and in vitro with the same molecular mass as the mature form of this protein.

Subunit VII, the ubiquinone-binding protein, of the cytochrome b-c1 complex of yeast mitochondria is involved in electron transport at center o and faces the matrix side of the membrane.

The functional role and topographical orientation in the inner membrane of subunit VII, the ubiquinone-binding protein, of the cytochrome b-c1 complex of yeast mitochondria has been investigated. The apparent molecular weight of this subunit on sodium dodecyl sulfate-urea gels was calculated to be 15,500, while its amino acid composition was similar to that of the Q-binding proteins present in the cytochrome b-c1 complexes isolated from both beef heart and yeast mitochondria. The specific antibody obtained against subunit VII inhibited 30-47% of the ubiquinol-cytochrome c reductase activity in the isolated cytochrome b-c1 complex and in submitochondrial particles but had no effect on cytochrome c reductase activity in mitoplasts, mitochondria from which the outer membrane has been removed. Furthermore, the antibody against subunit VII strongly inhibited (74%) the reduction of cytochrome b by succinate in the presence of antimycin, an inhibitor of center i, but had no effect on cytochrome b reduction in the presence of myxothiazol, an inhibitor of center o. These results suggest that subunit VII, the Q-binding protein, is involved in electron transport at center o of the cytochrome b-c1 complex of the respiratory chain and that subunit VII is localized facing the matrix side of the inner mitochondrial membrane.