In the first extracellular domain of E-cadherin, heterophilic interactions, but not the conserved His-Ala-Val motif, are required for adhesion.
AuthorsRenaud-Young M, Gallin WJ
JournalJ Biol Chem
PubMed ID12154084
The classical cadherins, definitive proteins of the cadherin superfamily, are characterized functionally by their ability to mediate calcium-dependent cell aggregation in vitro. To test hypothetical mechanisms of adhesion, we have constructed two mutants of the chicken E-cadherin protein, one with the highly conserved His-Ala-Val (HAV) sequence motif reversed to Val-Ala-His ... More
Apparent cytosolic calcium gradients in T-lymphocytes due to fura-2 accumulation in mitochondria.
AuthorsQuintana A, Hoth M
JournalCell Calcium
PubMed ID15193858
'Fura-2 is the most common dye to measure cytosolic Ca2+ concentrations ([Ca2+]i). To facilitate simultaneous imaging of many cells while preserving their cytosolic environment, fura-2 is often loaded into the cytosol in its membrane-permeant ester form. It has been reported that small amounts of fura-2 accumulate in intracellular compartments, an ... More
Balancing the activation state of the endothelium via two distinct TGF-beta type I receptors.
AuthorsGoumans MJ, Valdimarsdottir G, Itoh S, Rosendahl A, Sideras P, ten Dijke P
JournalEMBO J
PubMed ID11927558
'The generation of mice lacking specific components of the transforming growth factor-beta (TGF-beta) signal tranduction pathway shows that TGF-beta is a key player in the development and physiology of the cardiovascular system. Both pro- and anti-angiogenic properties have been ascribed to TGF-beta, for which the molecular mechanisms are unclear. Here ... More
Tracking individual kinesin motors in living cells using single quantum-dot imaging.
AuthorsCourty S, Luccardini C, Bellaiche Y, Cappello G, Dahan M
JournalNano Lett
PubMed ID16834436
'We report a simple method using semiconductor quantum dots (QDs) to track the motion of intracellular proteins with a high sensitivity. We characterized the in vivo motion of individual QD-tagged kinesin motors in living HeLa cells. Single-molecule measurements provided important parameters of the motor, such as its velocity and processivity, ... More
Adenovirus-facilitated nuclear translocation of adeno-associated virus type 2.
AuthorsXiao W, Warrington KH, Hearing P, Hughes J, Muzyczka N
JournalJ Virol
PubMed ID12388712
'We examined cytoplasmic trafficking and nuclear translocation of adeno-associated virus type 2 (AAV) by using Alexa Fluor 488-conjugated wild-type AAV, A20 monoclonal antibody immunocytochemistry, and subcellular fractionation techniques followed by DNA hybridization. Our results indicated that in the absence of adenovirus (Ad), AAV enters the cell rapidly and escapes from ... More
Incorporation of monoclonal antibodies into cells by osmotic permeabilization. Effect on cellular metabolism.
AuthorsChakrabarti R, Pfeiffer NE, Wylie DE, Schuster SM
JournalJ Biol Chem
PubMed ID2566602
'Incorporation of asparagine synthetase-specific monoclonal antibodies into L5178Y D10/R (L-asparaginase-resistant) murine lymphoma cells by osmotic lysis of pinocytic vesicles was used to evaluate the potential of the technique for macromolecular incorporation for metabolic studies. Nonspecific effects of the incorporation procedure included temporary inhibition of protein and DNA synthesis by 80-85% ... More
RNA interference by osmotic lysis of pinosomes: liposome-independent transfection of siRNAs into mammalian cells.
AuthorsGruber J, Boese G, Tuschl T, Osborn M, Weber K
JournalBiotechniques
PubMed ID15283206
'The osmotic lysis of pinosomes procedure has been adapted to deliver small interfering RNAs (siRNAs) into cells in culture. Under hypertonic conditions, siRNAs were internalized into pinosomes. A subsequent osmotic shock in hypotonic buffer disrupted the pinosomes and caused the release of siRNAs into the cell cytoplasm. Both steps could ... More
Continuous photobleaching in vesicles and living cells: a measure of diffusion and compartmentation.
AuthorsDelon A, Usson Y, Derouard J, Biben T, Souchier C
JournalBiophys J
PubMed ID16428281
'We present a comprehensive and analytical treatment of continuous photobleaching in a compartment, under single photon excitation. In the very short time regime (t<0.1 ms), the diffusion does not play any role. After a transition (or short time regime), one enters in the long time regime (t>0.1-5 s), for which ... More
Tracking individual proteins in living cells using single quantum dot imaging.
'Single quantum dot imaging is a powerful approach to probe the complex dynamics of individual biomolecules in living systems. Due to their remarkable photophysical properties and relatively small size, quantum dots can be used as ultrasensitive detection probes. They make possible the study of biological processes, both in the membrane ... More
Production of beta-defensin-2 by human colonic epithelial cells induced by Salmonella enteritidis flagella filament structural protein.
AuthorsTakahashi A, Wada A, Ogushi K, Maeda K, Kawahara T, Mawatari K, Kurazono H, Moss J, Hirayama T, Nakaya Y
JournalFEBS Lett
PubMed ID11728477
'We recently showed that FliC of Salmonella enteritidis increased human beta-defensin-2 (hBD-2) expression, and now describe the signaling responsible pathway. FliC increased the intracellular Ca(2+) concentration ([Ca(2+)](in)) in Caco-2 cells. The [Ca(2+)](in) increase induced by FliC was prevented by U73122 and heparin, but not by chelating extracellular Ca(2+) or pertussis ... More
Localization of the C3-Like ADP-ribosyltransferase from Staphylococcus aureus during bacterial invasion of mammalian cells.
AuthorsMolinari G, Rohde M, Wilde C, Just I, Aktories K, Chhatwal GS
JournalInfect Immun
PubMed ID16714601
The C3stau2 exoenzyme from Staphylococcus aureus is a C3-like ADP-ribosyltransferase which possesses no specific receptor-binding domain or translocation unit required for entry in target cells where its substrate is located. Here we show that C3stau2 can reach its target after invasion of staphylococci in eukaryotic cells without needing translocation. ... More
Multianalyte single-cell analysis with multiple cell lines using a fiber-optic array.
AuthorsWhitaker RD, Walt DR,
JournalAnal Chem
PubMed ID17973503
A single-cell drug screening method is described that produces rich single-cell data and discriminates between single-cell responses from clonal populations stimulated with different agonists. Ligand-induced receptor activation is commonly detected by observing intracellular Ca2+ oscillations using high-throughput screening (HTS) methods. In most cases, HTS results in an average signal from ... More
Heterocellular contact at the myoendothelial junction influences gap junction organization.
AuthorsIsakson BE, Duling BR
JournalCirc Res
PubMed ID15961721
Heterocellular communication between vascular smooth muscle cells (VSMC) and endothelial cells (EC) at the myoendothelial junction (MEJ) is a critical part of control of the arteriolar wall. We have developed an in vitro model of the MEJ composed of primary cultures of murine EC and VSMC. Immunoctytochemistry and immunoblots demonstrated ... More
Coelenterate photoproteins as indicators of cytoplasmic free Ca2+ in small cells.
AuthorsCampbell AK, Dormer RL, Hallett MB
JournalCell Calcium
PubMed ID2861904
The Ca2+-activated photoproteins aequorin and obelin are capable of detecting rapid changes in free Ca2+ over the range 10nM-100uM. Whilst they have been used to quantify free Ca transients in giant cells for some time, their use in small mammalian cells has been restricted because of the difficulty of incorporating ... More
Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles.
AuthorsOkada CY, Rechsteiner M
JournalCell
PubMed ID6179631
We have developed a new procedure for introducing macromolecules into cultured mammalian cells based on osmotic lysis of pinocytic vesicles. Cells are first incubated in culture medium containing 0.5 M sucrose, 10% polyethylene glycol 1000 and the macromolecule to be transferred. Cells are then placed in medium diluted with 0.66 ... More
A quantitative single-cell assay for protein kinase B reveals important insights into the biochemical behavior of an intracellular substrate peptide.
AuthorsLi H, Sims CE, Kaluzova M, Stanbridge EJ, Allbritton NL
JournalBiochemistry
PubMed ID14769036
The introduction of peptides into living cells for the purpose of manipulating cellular biochemistry has become widespread throughout biology. However, little is known about the behavior of these short sequences of amino acids within cells, particularly those used as substrates or inhibitors for kinases and other enzymes. We utilized a ... More
Determining protease activity in vivo by fluorescence cross-correlation analysis.
AuthorsKohl T, Haustein E, Schwille P
JournalBiophys J
PubMed ID16055538
To date, most biochemical approaches to unravel protein function have focused on purified proteins in vitro. Whereas they analyze enzyme performance under assay conditions, they do not necessarily tell us what is relevant within a living cell. Ideally, cellular functions should be examined in situ. In particular, association/dissociation reactions are ... More
Relationship between vaccinia virus intracellular cores, early mRNAs, and DNA replication sites.
AuthorsMallardo M, Leithe E, Schleich S, Roos N, Doglio L, Krijnse Locker J
JournalJ Virol
PubMed ID11967332
Virus assembly, a late event in the life cycle of vaccinia virus (VV), is preceded by a number of steps that all occur in the cytoplasm of the infected host cell: virion entry, delivery of the viral core into the cytoplasm, and transcription from these cores of early mRNAs, followed ... More
Calcium transients induce spatially coordinated increases in traction force during the movement of fish keratocytes.
AuthorsDoyle A, Marganski W, Lee J
JournalJ Cell Sci
PubMed ID15126622
The coordination of protrusion with retraction is essential for continuous cell movement. In fish keratocytes the activation of stretch-activated calcium channels, and the resulting increase in intracellular calcium, trigger release of the rear cell margin when forward movement is impeded. Although it is likely that retraction involves a calcium-dependent increase ... More
Adult motor neuron apoptosis is mediated by nitric oxide and Fas death receptor linked by DNA damage and p53 activation.
AuthorsMartin LJ, Chen K, Liu Z
JournalJ Neurosci
PubMed ID16000635
The mechanisms of injury- and disease-related degeneration of motor neurons (MNs) need clarification. Unilateral avulsion of the sciatic nerve in the mouse induces apoptosis of spinal MNs that is p53 and Bax dependent. We tested the hypothesis that MN apoptosis is Fas death receptor dependent and triggered by nitric oxide ... More
Direct measurement of intracellular free Ca2+ in rat peritoneal macrophages: correlation with oxygen-radical production.
AuthorsHallett MB, Campbell AK
JournalImmunology
PubMed ID6414943
A novel method has been developed, based on osmotic lysis of intracellular pinocytotic vesicles, to introduce the Ca2+-activated photoprotein obelin into the cytoplasm of rat peritoneal macrophages. The change in osmolarity of the incubating medium necessary to induce lysis of the pinocytotic vesicles did not significantly affect the viability or ... More
Intracellular delivery strategies for antisense phosphorodiamidate morpholino oligomers.
AuthorsGhosh C, Iversen PL
JournalAntisense Nucleic Acid Drug Dev
PubMed ID10984120
Antisense oligonucleotides inhibit gene expression by interfering with transcription, translation, or splicing. They show great potential as gene-specific, nontoxic therapy for a wide variety of diseases. They are also powerful tools to study gene function as well as for validation of therapeutic targets. Even with compelling evidence of activity in ... More
Hypertonic sucrose inhibition of endocytic transport suggests multiple early endocytic compartments.
AuthorsPark RD, Sullivan PC, Storrie B
JournalJ Cell Physiol
PubMed ID3397386
Incubation of animal cells with hypertonic sucrose and polyethylene glycol (PEG) 1,000 renders endosomes sensitive in situ to hypotonic shock (Okada and Rechsteiner, 1982). We found that: 1) in vitro endosomes were osmotically insensitive; and 2) hypertonic sucrose inhibited transport from very early endosomes to lysosomes. Endocytic vesicles were labeled ... More