AuthorsHe M, Stoevesandt O, Palmer EA, Khan F, Ericsson O, Taussig MJ,
JournalNat Methods
PubMed ID18204456
'We describe a method, DNA array to protein array (DAPA), which allows the ''printing'' of replicate protein arrays directly from a DNA array template using cell-free protein synthesis. At least 20 copies of a protein array can be obtained from a single DNA array. DAPA eliminates the need for separate ... More
Comparison of hydroxylated print additives on antibody microarray performance.
AuthorsWu P, Grainger DW,
JournalJ Proteome Res
PubMed ID17081047
'Various hydroxylated additives were added to antibody print buffers at different concentrations to stabilize printed antibodies during normal array spot desiccation on commercial polymer-coated microarray slides. Polyvinyl alcohol addition to print buffers produced the most regular spot morphologies, homogeneous intra-spot antibody distribution, uniform fluorescence intensity, and improved analyte capture activity, ... More
The tetraspanin CD9 mediates lateral association of MHC class II molecules on the dendritic cell surface.
AuthorsUnternaehrer JJ, Chow A, Pypaert M, Inaba K, Mellman I
JournalProc Natl Acad Sci U S A
PubMed ID17190803
'We have found that MHC class II (MHC II) molecules exhibit a distinctive organization on the dendritic cell (DC) plasma membrane. Both in DC lysates and on the surface of living cells, I-A and I-E molecules engaged in lateral interactions not observed on other antigen-presenting cells such as B blasts. ... More
Development of homogeneous binding assays based on fluorescence resonance energy transfer between quantum dots and Alexa Fluor fluorophores.
AuthorsNikiforov TT, Beechem JM
JournalAnal Biochem
PubMed ID16860286
'We studied the fluorescence resonance energy transfer (FRET) between quantum dots emitting at 565, 605, and 655 nm as energy donors and Alexa Fluor fluorophores with absorbance maxima at 594, 633, 647, and 680 nm as energy acceptors. As a first step, we prepared covalent conjugates between all three types ... More
Restriction of receptor movement alters cellular response: physical force sensing by EphA2.
AuthorsSalaita K, Nair PM, Petit RS, Neve RM, Das D, Gray JW, Groves JT,
JournalScience
PubMed ID20223987
'Activation of the EphA2 receptor tyrosine kinase by ephrin-A1 ligands presented on apposed cell surfaces plays important roles in development and exhibits poorly understood functional alterations in cancer. We reconstituted this intermembrane signaling geometry between live EphA2-expressing human breast cancer cells and supported membranes displaying laterally mobile ephrin-A1. Receptor-ligand binding, ... More
Cell-specific subcellular localization of soluble epoxide hydrolase in human tissues.
AuthorsEnayetallah AE, French RA, Barber M, Grant DF
JournalJ Histochem Cytochem
PubMed ID16314446
'Soluble epoxide hydrolase (sEH) is a phase-I xenobiotic metabolizing enzyme having both an N-terminal phosphatase activity and a C-terminal epoxide hydrolase activity. Endogenous hydrolase substrates include arachidonic acid epoxides, which have been involved in regulating blood pressure and inflammation. The subcellular localization of sEH has been controversial. Earlier studies using ... More
Crossed and uncrossed retinal projections to the hamster circadian system.
AuthorsMuscat L, Huberman AD, Jordan CL, Morin LP
JournalJ Comp Neurol
PubMed ID14566946
'The hamster suprachiasmatic nucleus (SCN), site of the circadian clock, has been thought to be equally and completely innervated by each retina. This issue was studied in animals that had received an injection of the tracer cholera toxin subunit B (CTb) conjugated to Alexa 488 into the vitreous of one ... More
Counting low-copy number proteins in a single cell.
We have designed a microfluidic device in which we can manipulate, lyse, label, separate, and quantify the protein contents of a single cell using single-molecule fluorescence counting. Generic labeling of proteins is achieved through fluorescent-antibody binding. The use of cylindrical optics enables high-efficiency (approximately 60%) counting of molecules in micrometer-sized ... More
Asymmetric T lymphocyte division in the initiation of adaptive immune responses.
AuthorsChang JT, Palanivel VR, Kinjyo I, Schambach F, Intlekofer AM, Banerjee A, Longworth SA, Vinup KE, Mrass P, Oliaro J, Killeen N, Orange JS, Russell SM, Weninger W, Reiner SL
JournalScience
PubMed ID17332376
A hallmark of mammalian immunity is the heterogeneity of cell fate that exists among pathogen-experienced lymphocytes. We show that a dividing T lymphocyte initially responding to a microbe exhibits unequal partitioning of proteins that mediate signaling, cell fate specification, and asymmetric cell division. Asymmetric segregation of determinants appears to be ... More
A microengraving method for rapid selection of single cells producing antigen-specific antibodies.
AuthorsLove JC, Ronan JL, Grotenbreg GM, van der Veen AG, Ploegh HL
JournalNat Biotechnol
PubMed ID16699501
Monoclonal antibodies that recognize specific antigens of interest are used as therapeutic agents and as tools for biomedical research. Discovering a single monoclonal antibody requires retrieval of an individual hybridoma from polyclonal mixtures of cells producing antibodies with a variety of specificities. The time required to isolate hybridomas by a ... More
MicroRNA expression profiling using microarrays.
AuthorsLiu CG, Calin GA, Volinia S, Croce CM,
JournalNat Protoc
PubMed ID18388938
Microarray technology is a powerful high-throughput tool capable of monitoring the expression of thousands of small noncoding RNAs at once within tens of samples processed in parallel in a single experiment. To conduct a genome-wide analysis of miRNA expression of normal and disease samples, such as cancer, and to distinguish ... More
Avoiding nonspecific interactions in studies of the plasma proteome: practical solutions to prevention of nonspecific interactions for label-free detection of low-abundance plasma proteins.
The molecular constitution of blood can be highly representative of the physiological state of an individual and offers an ideal target for studies of biomarkers. High-abundance plasma proteins, particularly albumin, dominate the plasma proteome, but it is the low-abundance proteins (such as cytokines) that are commonly associated with many pathophysiological ... More
Neuronal classification and marker gene identification via single-cell expression profiling of brainstem vestibular neurons subserving cerebellar learning.
AuthorsKodama T, Guerrero S, Shin M, Moghadam S, Faulstich M, du Lac S,
JournalJ Neurosci
PubMed ID22674258
Identification of marker genes expressed in specific cell types is essential for the genetic dissection of neural circuits. Here we report a new strategy for classifying heterogeneous populations of neurons into functionally distinct types and for identifying associated marker genes. Quantitative single-cell expression profiling of genes related to neurotransmitters and ... More
Identification of internalizing human single-chain antibodies targeting brain tumor sphere cells.
AuthorsZhu X, Bidlingmaier S, Hashizume R, James CD, Berger MS, Liu B,
JournalMol Cancer Ther
PubMed ID20587664
Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain tumor for which there is no curative treatment to date. Resistance to conventional therapies and tumor recurrence pose major challenges to treatment and management of this disease, and therefore new therapeutic strategies need to be developed. Previous ... More
DNA-gold nanorod conjugates for remote control of localized gene expression by near infrared irradiation.
AuthorsChen CC, Lin YP, Wang CW, Tzeng HC, Wu CH, Chen YC, Chen CP, Chen LC, Wu YC
JournalJ Am Chem Soc
PubMed ID16536544
Gold nanorods were attached to the gene of enhanced green fluorescence protein (EGFP) for the remote control of gene expression in living cells. The UV-vis spectroscopy, electrophoresis, and transmission electron microscopy (TEM) were used to study the optical and structural properties of the EGFP DNA and gold nanorod (EGFP-GNR) conjugates ... More
PNA-DNA hybridization study using labeled streptavidin by voltammetry and surface plasmon fluorescence spectroscopy.
AuthorsLiu J, Tiefenauer L, Tian S, Nielsen PE, Knoll W
JournalAnal Chem
PubMed ID16408929
Using ferrocene-streptavidin conjugates as amplifiers, we recently have demonstrated the simultaneous detection of DNA hybridization to peptide nucleic acid (PNA)-modified gold surfaces at the femtomole level by electrochemical and surface plasmon resonance techniques (Liu, J.; Tian, S.; Tiefenauer, L.; Nielsen, P. E.; Knoll, W. Anal. Chem. 2005, 77, 2756-2761). In ... More
Focal adhesion kinase plays a pivotal role in herpes simplex virus entry.
AuthorsCheshenko N, Liu W, Satlin LM, Herold BC
JournalJ Biol Chem
PubMed ID15994312
Development of strategies to prevent herpes simplex virus (HSV) infection requires knowledge of cellular pathways harnessed by the virus for invasion. This study demonstrates that HSV induces rapid phosphorylation of focal adhesion kinase (FAK) in several human target cells and that phosphorylation is important for entry post-binding. Nuclear transport of ... More
Orally tolerized T cells can form conjugates with APCs but are defective in immunological synapse formation.
AuthorsIse W, Nakamura K, Shimizu N, Goto H, Fujimoto K, Kaminogawa S, Hachimura S
JournalJ Immunol
PubMed ID16002680
Oral tolerance is systemic immune hyporesponsiveness induced by the oral administration of soluble Ags. Hyporesponsiveness of Ag-specific CD4 T cells is responsible for this phenomenon. However, the molecular mechanisms underlying the hyporesponsive state of these T cells are not fully understood. In the present study, we investigated the ability of ... More
Multiple residues in the transmembrane helix and connecting peptide of mouse tapasin stabilize the transporter associated with the antigen-processing TAP2 subunit.
AuthorsPapadopoulos M, Momburg F
JournalJ Biol Chem
PubMed ID17244610
The type I endoplasmic reticulum (ER) glycoprotein tapasin (Tpn) is essential for loading of major histocompatibility complex class I (MHC-I) molecules with an optimal spectrum of antigenic peptides and for stable expression of the heterodimeric, polytopic TAP peptide transporter. In a detailed mutational analysis, the transmembrane domain (TMD) and ER-luminal ... More