Molecular tracking devices quantify antigen distribution and archiving in the murine lymph node.

The detection of foreign antigens in vivo has relied on fluorescent conjugation or indirect read-outs such as antigen presentation. In our studies, we found that these widely used techniques had several technical limitations that have precluded a complete picture of antigen trafficking or retention across lymph node cell types. To address these limitations, we developed a 'molecular tracking device' to follow the distribution, acquisition, and retention of antigen in the lymph node.

Correction: DNA-catalyzed glycosylation using aryl glycoside donors.

Correction for 'DNA-catalyzed glycosylation using aryl glycoside donors' by Anthony R. Hesser et al., Chem.

DNA-catalyzed glycosylation using aryl glycoside donors.

We report the identification by in vitro selection of Zn(2+)/Mn(2+)-dependent deoxyribozymes that glycosylate the 3'-OH of a DNA oligonucleotide. Both β and α anomers of aryl glycosides can be used as the glycosyl donors. Individual deoxyribozymes are each specific for a particular donor anomer.

DNA Oligonucleotide 3'-Phosphorylation by a DNA Enzyme.

T4 polynucleotide kinase is widely used for 5'-phosphorylation of DNA and RNA oligonucleotide termini, but no natural protein enzyme is capable of 3'-phosphorylation. Here, we report the in vitro selection of deoxyribozymes (DNA enzymes) capable of DNA oligonucleotide 3'-phosphorylation, using a 5'-triphosphorylated RNA transcript (pppRNA) as the phosphoryl donor. The basis of selection was the capture, during each selection round, of the 3'-phosphorylated DNA substrate terminus by 2-methylimidazole activation of the 3'-phosphate (forming 3'-MeImp) and subsequent splint ligation with a 5'-amino DNA oligonucleotide.

Identification of Sequence-Selective Tyrosine Kinase Deoxyribozymes.

Deoxyribozymes (DNA enzymes) have been developed for a growing variety of chemical reactions, including with peptide substrates. We recently described the first tyrosine kinase deoxyribozymes, which lacked the ability to discriminate among peptide substrates on the basis of the amino acids surrounding the tyrosine residue. Those deoxyribozymes were identified by in vitro selection using a DNA-anchored peptide substrate in which the residues neighboring tyrosine were all alanine.

DNA catalysts with tyrosine kinase activity.

We show that DNA catalysts (deoxyribozymes, DNA enzymes) can phosphorylate tyrosine residues of peptides. Using in vitro selection, we identified deoxyribozymes that transfer the γ-phosphoryl group from a 5'-triphosphorylated donor (a pppRNA oligonucleotide or GTP) to the tyrosine hydroxyl acceptor of a tethered hexapeptide. Tyrosine kinase deoxyribozymes that use pppRNA were identified from each of N30, N40, and N50 random-sequence pools.

Spatial learning impairment, enhanced CDK5/p35 activity, and downregulation of NMDA receptor expression in transgenic mice expressing tau-tubulin kinase 1.

Tau-tubulin kinase-1 (TTBK1) is involved in phosphorylation of tau protein at specific Serine/Threonine residues found in paired helical filaments, suggesting its role in tauopathy pathogenesis. We found that TTBK1 levels were upregulated in brains of human Alzheimer' disease (AD) patients compared with age-matched non-AD controls. To understand the effects of TTBK1 activation in vivo, we developed transgenic mice harboring human full-length TTBK1 genomic DNA (TTBK1-Tg).

Calpain and proteasomal regulation of antiretroviral zinc finger protein OTK18 in human macrophages: visualization in live cells by intramolecular FRET.

As part of the innate immune defense against HIV infection, OTK18, a zinc finger protein, is upregulated in human macrophages and reduces viral replication through suppression of viral long-terminal repeat promoter activity. Although we know that the processing products of OTK18 accumulate in the cytoplasm of brain perivascular macrophages in advanced HIV encephalitis cases, the molecular mechanisms behind its post-translational processing are still poorly understood. To characterize OTK18 processing, we assessed a panel of protease inhibitors to identify the candidates involved in the OTK18 processing using human monocyte-derived macrophages (MDM) overexpressing OTK18 by recombinant adenoviral gene transfer.

Cytokine-mediated inhibition of fibrillar amyloid-beta peptide degradation by human mononuclear phagocytes.

Vaccination therapy of AD animal models and patients strongly suggests an active role of brain mononuclear phagocytes in immune-mediated clearance of amyloid-beta peptides (Abeta) in brain. Although Abeta uptake by macrophages can be regulated by pro- and anti-inflammatory cytokines, their effects on macrophage-mediated Abeta degradation are poorly understood. To better understand this mechanism of degradation, we examined whether pro- and anti-inflammatory cytokines affect the degradation of Abeta using primary cultured human monocyte-derived macrophages (MDM) and microglia using pulse-chase analysis of fibrillar and oligomer (125)I-Abeta40 and Abeta42.

Kinetic analysis of aggregated amyloid-beta peptide clearance in adult bone-marrow-derived macrophages from APP and CCL2 transgenic mice.

Accumulating evidence suggests that bone-marrow (BM)-derived mononuclear phagocytes have an important role in the clearance of soluble and aggregated amyloid-beta peptides (Abeta) in Alzheimer's disease (AD) brains. However, the exact kinetics of Abeta clearance in mononuclear phagocytes derived from transgenic animal models of AD expressing beta-amyloid precursor protein (APP) mutants have been poorly characterized. We have examined whether CCL2 and APP expression affects the clearance of Abeta in conjunction with our control, acetylated low-density lipoprotein (AcLDL), using primary cultured BM-derived macrophages derived from adult APP, CCL2, APP/CCL2, and control littermates.

Polyfluorinated bis-styrylbenzene beta-amyloid plaque binding ligands.

beta-Amyloid (Abeta) binding affinities and specificities for six bis-styrylbenzenes with multiple magnetically equivalent fluorine atoms in the form of a tetrafluorophenyl core or symmetrical trifluoromethyl and trifluoromethoxy groups were determined by means of fluorescence titrations with amyloid peptide Abeta1-40 and a novel in vitro fluorescence-based assay using APP/PS1 transgenic mouse brain sections. Bis-styrylbenzenes with a tetrafluorophenyl core had increased Abeta binding affinities compared to their monofluorophenyl or phenyl counterparts. Bis-styrylbenzenes with carboxylic acid functional groups had lower Abeta binding affinities than their neutral counterparts.

Interferon-gamma and tumor necrosis factor-alpha regulate amyloid-beta plaque deposition and beta-secretase expression in Swedish mutant APP transgenic mice.

Reactive astrocytes and microglia in Alzheimer's disease surround amyloid plaques and secrete proinflammatory cytokines that affect neuronal function. Relationship between cytokine signaling and amyloid-beta peptide (Abeta) accumulation is poorly understood. Thus, we generated a novel Swedish beta-amyloid precursor protein mutant (APP) transgenic mouse in which the interferon (IFN)-gamma receptor type I was knocked out (APP/GRKO).