DNA augments antigenicity of mycobacterial DNA-binding protein 1 and confers protection against Mycobacterium tuberculosis infection in mice

Mycobacterium consists up to 7% of mycobacterial DNA-binding protein 1 (MDP1) in total cellular proteins. Host immune responses to MDP1 were studied in mice to explore the antigenic properties of this protein. Anti-MDP1 IgG was produced after infection with either bacillus Calmette-Guérin or Mycobacterium tuberculosis in C3H/HeJ mice. However, the level of Ab was remarkably low when purified MDP1 was injected. MDP1 is considered to be associated with DNA in nucleoid, which contains immunostimulatory CpG motif. Therefore, we examined coadministration of MDP1 and DNA derived from M. tuberculosis. Consequently, this procedure significantly enhanced the production of MDP1-specific IgG. Five nanograms of DNA was enough to enhance MDP1-specific IgG production in the administration of 5 microg of MDP1 into mice. Strong immune stimulation by such a small amount of DNA is noteworthy, because >1,000- to 100,000-fold doses of CpG DNAs are used for immune activation. A synthetic peptide-based study showed that B cell epitopes were different between mice administered MDP1 alone and those given a mixture of MDP1 and DNA, suggesting that DNA alters the three-dimensional structure of MDP1. Coadministration of DNA also enhanced MDP1-specific IFN-gamma production and reduced the bacterial burden of a following challenge of M. tuberculosis, showing that MDP1 is a novel vaccine target. Finally, we found that MDP1 remarkably enhanced TLR9-dependent immune stimulation by unmethylated CpG oligo DNA in vitro. To our knowledge, MDP1 is the first protein discovered that remarkably augments the CpG-mediated immune response and is a potential adjuvant for CpG DNA-based immune therapies.     

Sequential immune escape and shifting of T cell responses in a long-term survivor of melanoma

Immune-mediated control of tumors may occur, in part, through lysis of malignant cells by CD8(+) T cells that recognize specific Ag-HLA class I complexes. However, tumor cell populations may escape T cell responses by immune editing, by preventing formation of those Ag-HLA complexes. It remains unclear whether the human immune system can respond to immune editing and recognize newly arising escape variants. We report an example of shifting immune responses to escape variants in a patient with sequential metastases of melanoma and long-term survival after surgery alone. Tumor cells in the first metastasis escaped immune recognition via selective loss of an HLA haplotype (HLA-A11, -B44, and -Cw17), but maintained expression of HLA-A2. In the second metastasis, immune escape from an immunodominant MART-1-specific T cell response was mediated by HLA class I down-regulation, resulting in a failure to present this epitope, but persistent presentation of a tyrosinase-derived epitope. Consequent to this modification in tumor Ag presentation, the dominant CTL response shifted principally toward a tyrosinase-targeted response, even though tyrosinase-specific CTL had been undetectable during the initial metastatic event. Thus, in response to immune editing of tumor cells, a patient's spontaneous T cell response adapted, gaining the ability to recognize and to lyse "edited" tumor targets. The observation of both immune editing and immune adaptation in a patient with long-term survival after surgery alone demonstrates an example of immune system reactivity to counteract the escape mechanism(s) developed by tumor cells, which may contribute to the clinical outcome of malignant disease.     

Separation of polyprenol and dolichol by monolithic silica capillary column chromatography

We attempted an analysis of naturally occurring polyprenol and dolichol using a monolithic silica capillary column in HPLC. First, the separation of the polyprenol mixture alone was performed using a 250 x 0.2 mm inner diameter (ID) octadecylsilyl (ODS)-monolithic silica capillary column. The resolution of the separation between octadecaprenol (prenol 18) and nonadecaprenol (prenol 19) exceeded by >or=2-fold the level recorded when using a conventional ODS-silica particle-packed column (250 x 4.6 mm ID) under the same elution conditions. Next, the mixture of the prenol type (polyprenol) and dolichol type (dihydropolyprenol) was subjected to this capillary HPLC system, and the separation of each homolog was successfully achieved. During the analysis of polyprenol fraction derived from Eucommia ulmoides leaves, dolichols were found as a single peak, including all-trans-polyprenol and cis-polyprenol previously identified. This sensitive high-resolution system is very useful for the analysis of compounds that are structurally close to polyprenols and dolichols and that have a low content.     

Association of active gamma-secretase complex with lipid rafts

Cholesterol has been implicated in the pathogenesis of Alzheimer's disease (AD). Although the underlying mechanisms are not yet clear, several studies have provided evidence for the involvement of cholesterol-rich lipid rafts in the production of amyloid beta peptide (Abeta), the major component of amyloid deposits in AD. In this regard, the gamma-secretase complex is responsible for the final cleavage event in the processing of beta-amyloid precursor protein (betaAPP), resulting in Abeta generation. The gamma-secretase complex is a multiprotein complex composed of presenilin, nicastrin (NCT), APH-1, and PEN-2. Recent reports have suggested that gamma-secretase activity is predominantly localized in lipid rafts, and presenilin and NCT have been reported to be localized in lipid rafts. In this study, various biochemical methods, including coimmunoprecipitation, in vitro gamma-secretase assay, and methyl-beta-cyclodextrin (MbetaCD) treatment, are employed to demonstrate that all four components of the active endogenous gamma-secretase complex, including APH-1 and PEN-2, are associated with lipid rafts in human neuroblastoma cells (SH-SY5Y). Treatment with statins, 3-hydroxy-3-methylglutaryl-CoA-reductase inhibitors, significantly decreased the association of the gamma-secretase complex with lipid rafts without affecting the distribution of flotillin-1. This effect was partially abrogated by the addition of geranylgeraniol. These results suggest that both cholesterol and protein isoprenylation influence the active gamma-secretase complex association with lipid rafts.     

PDK1 is required for the hormonal signaling pathway leading to meiotic resumption in starfish oocytes

Meiotic resumption is generally under the control of an extracellular maturation-inducing hormone. It is equivalent to the G2-M phase transition in somatic cell mitosis and is regulated by cyclin B-Cdc2 kinase. However, the complete signaling pathway from the hormone to cyclin B-Cdc2 is yet unclear in any organism. A model system to analyze meiotic resumption is the starfish oocyte, in which Akt/protein kinase B (PKB) plays a key mediator in hormonal signaling that leads to cyclin B-Cdc2 activation. Here we show in starfish oocytes that when PDK1 activity is inhibited by a neutralizing antibody, maturation-inducing hormone fails to induce cyclin B-Cdc2 activation at the meiotic G2-M phase transition, even though PDK2 activity becomes detectable. These observations assign a novel role to PDK1 for a hormonal signaling intermediate toward meiotic resumption. They further support that PDK2 is a molecule distinct from PDK1 and Akt, and that PDK2 activity is not sufficient for the full activation of Akt in the absence of PDK1 activity.     

Paramagnetic NMR study of Cu(2+)-IDA complex localization on a protein surface and its application to elucidate long distance information

The paramagnetic metal chelate complex Cu(2+)-iminodiacetic acid (Cu(2+)-IDA) was mixed with ubiquitin, a small globular protein. Quantitative analyses of (1)H and (15)N chemical shift changes and line broadenings induced by the paramagnetic effects indicated that Cu(2+)-IDA was localized to a histidine residue (His68) on the ubiquitin surface. The distances between the backbone amide proton and the Cu(2+) relaxation center were evaluated from the proton transverse relaxation rates enhanced by the paramagnetic effect. These correlated well with the distances calculated from the crystal structure up to 20 A. Here, we show that a Cu(2+)-IDA is the first paramagnetic reagent that specifically localizes to a histidine residue on the protein surface and gives the long-range distance information.     

Novel mechanisms of pH sensitivity in tuna hemoglobin: a structural explanation of the root effect

The crystal structure of hemoglobin has been known for several decades, yet various features of the molecule remain unexplained or controversial. Several animal hemoglobins have properties that cannot be readily explained in terms of their amino acid sequence and known atomic models of hemoglobin. Among these, fish hemoglobins are well known for their widely varying interactions with heterotropic effector molecules and pH sensitivity. Some fish hemoglobins are almost completely insensitive to pH (within physiological limits), whereas others show extremely low oxygen affinity under acid conditions, a phenomenon called the Root effect. X-ray crystal structures of Root effect hemoglobins have not, to date, provided convincing explanations of this effect. Sequence alignments have signally failed to pinpoint the residues involved, and site-directed mutagenesis has not yielded a human hemoglobin variant with this property. We have solved the crystal structure of tuna hemoglobin in the deoxy form at low and moderate pH and in the presence of carbon monoxide at high pH. A comparison of these models shows clear evidence for novel mechanisms of pH-dependent control of ligand affinity.     

A lipid peroxidation-derived inflammatory mediator: identification of 4-hydroxy-2-nonenal as a potential inducer of cyclooxygenase-2 in macrophages

Cyclooxygenases (COXs) catalyze the conversion of arachidonic acid to eicosanoids, which mediate a variety of biological actions involved in vascular pathophysiology. In the present study, we investigated the role of lipid peroxidation products in the up-regulation of COX-2, an inducible isoform responsible for high levels of prostaglandin production during inflammation and immune responses. COX-2 was found to colocalize with 4-hydroxy-2-nonenal (HNE), a major lipid peroxidation-derived aldehyde, in foamy macrophages within human atheromatous lesions, suggesting that COX-2 expression may be associated with the accumulation of lipid peroxidation products within macrophages. To test the hypothesis that lipid peroxidation products might be involved in the regulation of prostanoid biosynthesis, we conducted a screen of oxidized fatty acid metabolites and found that, among the compounds tested, only HNE showed inducibility of the COX-2 protein in RAW264.7 macrophages. In addition, intraperitoneal administration of HNE resulted in an increase in cell numbers in the peritoneal cavity that was associated with significant increases in the peritoneal and tissue levels of COX-2 in mice. To understand the possible signaling mechanism underlying the inducing effect of HNE on COX-2 up-regulation, we examined the phosphorylation events that may lead to COX-2 induction and found that HNE did not stimulate the induction of nitric oxide synthase and activation of NF-kappaB but significantly activated p38 mitogen-activated protein kinase and its upstream kinase in RAW264.7 macrophages. Tyrosine kinases, such as the epidermal growth factor-like and Src family tyrosine kinases, appeared to mediate the stabilization of COX-2 mRNA via the p38 mitogen-activated protein kinase pathway. These findings suggest that HNE accumulated in macrophages/foam cells may represent an inflammatory mediator that plays a role in stimulation of the inflammatory response and contributes to the progression of atherogenesis.