A high-content subtractive screen for selecting small molecules affecting internalization of GPCRs.

G-protein-coupled receptors (GPCRs) are pivotal in cellular responses to the environment and are common drug targets. Identification of selective small molecules acting on single GPCRs is complicated by the shared machinery coupling signal transduction to physiology. Here, we demonstrate a high-content screen using a panel of GPCR assays to identify receptor selective molecules acting within the kinase/phosphatase inhibitor family.

Automated genome-wide visual profiling of cellular proteins involved in HIV infection.

Recent genome-wide RNAi screens have identified >842 human genes that affect the human immunodeficiency virus (HIV) cycle. The list of genes implicated in infection differs between screens, and there is minimal overlap. A reason for this variance is the interdependence of HIV infection and host cell function, producing a multitude of indirect or pleiotropic cellular effects affecting the viral infection during RNAi screening.

Visual genome-wide RNAi screening to identify human host factors required for Trypanosoma cruzi infection.

The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical infection that affects millions of people in the Americas. Current chemotherapy relies on only two drugs that have limited efficacy and considerable side effects. Therefore, the development of new and more effective drugs is of paramount importance.

High-content classification of nucleocytoplasmic import or export inhibitors.

Transcription factors of the nuclear factor kappa B family are the paradigm for signaling dependent nuclear translocation and are ideally suited to analysis through image-based chemical genetic screening. The authors describe combining high-content image analysis with a compound screen to identify compounds affecting either nuclear import or export. Validation in silico and in vitro determined an EC(50) for the nuclear export blocker leptomycin B of 2.

Four-dimensional imaging of transvacuolar strand dynamics in tobacco BY-2 cells.

The vacuole is a characteristic organelle of plant cells and fulfills several important functions related to metabolism and growth of the cell. To shed light on the details of vacuolar structural changes in plant cells, we explored the three-dimensional organization and dynamics of living Nicotiana tabacum L. cv.

Actin-based motility of endosomes is linked to the polar tip growth of root hairs.

Plant tip growth has been recognized as an actin-based cellular process requiring targeted exocytosis and compensatory endocytosis to occur at the growth cone. However, the identity of subcellular compartments involved in polarized membrane trafficking pathways remains enigmatic in plants. Here we characterize endosomal compartments in tip-growing root hair cells.

Expression pattern of fibroblast growth factors (FGFs), their receptors and antagonists in primary endothelial cells and vascular smooth muscle cells.

Fibroblast growth factors (FGFs) are important angiogenic growth factors. While basic FGF (FGF2) is well established as a potent inducer of angiogenesis much less is known about other FGFs possibly expressed by EC. We investigated the expression of all known FGFs, their main tyrosine kinase receptors and antagonists by RT-PCR analysis in human umbilical vascular endothelial cells (HUVECs) to obtain a complete expression profile of this important growth factor system in model endothelial cells (EC).

Auxin inhibits endocytosis and promotes its own efflux from cells.

One of the mechanisms by which signalling molecules regulate cellular behaviour is modulating subcellular protein translocation. This mode of regulation is often based on specialized vesicle trafficking, termed constitutive cycling, which consists of repeated internalization and recycling of proteins to and from the plasma membrane. No such mechanism of hormone action has been shown in plants although several proteins, including the PIN auxin efflux facilitators, exhibit constitutive cycling.

Endosome-to-cytosol transport of viral nucleocapsids.

During viral infection, fusion of the viral envelope with endosomal membranes and nucleocapsid release were thought to be concomitant events. We show here that for the vesicular stomatitis virus they occur sequentially, at two successive steps of the endocytic pathway. Fusion already occurs in transport intermediates between early and late endosomes, presumably releasing the nucleocapsid within the lumen of intra-endosomal vesicles, where it remains hidden.

Neointimal smooth muscle cells display a proinflammatory phenotype resulting in increased leukocyte recruitment mediated by P-selectin and chemokines.

Leukocyte recruitment is crucial for the response to vascular injury in spontaneous and accelerated atherosclerosis. Whereas the mechanisms of leukocyte adhesion to endothelium or matrix-bound platelets have been characterized, less is known about the proadhesive role of smooth muscle cells (SMCs) exposed after endothelial denudation. In laminar flow assays, neointimal rat SMCs (niSMCs) supported a 2.

Syncytial fusion of human trophoblast depends on caspase 8.

Differentiation of human placental villous trophoblast includes syncytial fusion of cytotrophoblast forming syncytiotrophoblast. Early stages of the apoptosis cascade were described to be involved in this differentiation process. We investigated the role of the initiator caspase 8 in syncytial fusion in vitro, cultivating placental villous explants with or without caspase 8 antisense oligonucleotides or peptide inhibitors for up to 120 h.

An unusual internal ribosomal entry site of inverted symmetry directs expression of a potato leafroll polerovirus replication-associated protein.

Potato leafroll polerovirus (PLRV) genomic RNA acts as a polycistronic mRNA for the production of proteins P0, P1, and P2 translated from the 5'-proximal half of the genome. Within the P1 coding region we identified a 5-kDa replication-associated protein 1 (Rap1) essential for viral multiplication. An internal ribosome entry site (IRES) with unusual structure and location was identified that regulates Rap1 translation.

Molecular farming of recombinant antibodies in plants.

Antibodies represent a large proportion of therapeutic drugs currently in development. In most cases, they are produced in mammalian cell lines or transgenic animals because these have been shown to fold and assemble the proteins correctly and generate authentic glycosylation patterns. However, such expression systems are expensive, difficult to scale up and there are safety concerns due to potential contamination with pathogenic organisms or oncogenic DNA sequences.

'Molecular farming' of antibodies in plants.

'Molecular farming' is the production of valuable recombinant proteins in transgenic organisms on an agricultural scale. While plants have long been used as a source of medicinal compounds, molecular farming represents a novel source of molecular medicines, such as plasma proteins, enzymes, growth factors, vaccines and recombinant antibodies, whose medical benefits are understood at a molecular level. Until recently, the broad use of molecular medicines was limited because of the difficulty in producing these proteins outside animals or animal cell culture.

Targeting tryptophan decarboxylase to selected subcellular compartments of tobacco plants affects enzyme stability and in vivo function and leads to a lesion-mimic phenotype.

Tryptophan decarboxylase (TDC) is a cytosolic enzyme that catalyzes an early step of the terpenoid indole alkaloid biosynthetic pathway by decarboxylation of L-tryptophan to produce the protoalkaloid tryptamine. In the present study, recombinant TDC was targeted to the chloroplast, cytosol, and endoplasmic reticulum (ER) of tobacco (Nicotiana tabacum) plants to evaluate the effects of subcellular compartmentation on the accumulation of functional enzyme and its corresponding enzymatic product. TDC accumulation and in vivo function was significantly affected by the subcellular localization.

Uptake of a fluorescent marker in plant cells is sensitive to brefeldin A and wortmannin.

We assessed FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl)pyridinium dibromide] as a fluorescent endocytosis marker in intact, walled plant cells. At 4 degrees C, FM1-43 stained the plasma membrane, and after 30 to 120 min of incubation at 26 degrees C, FM1-43 labeled cytoplasmic vesicles and then the vacuole. Fluorimetric quantitation demonstrated dye uptake temperature sensitivity (approximately 65% reduction at 16 degrees C, >90% at 4 degrees C).

Molecular farming of pharmaceutical proteins.

Molecular farming is the production of pharmaceutically important and commercially valuable proteins in plants. Its purpose is to provide a safe and inexpensive means for the mass production of recombinant pharmaceutical proteins. Complex mammalian proteins can be produced in transformed plants or transformed plant suspension cells.

Antibody production by molecular farming in plants.

"Molecular farming" is the production of pharmaceutical proteins in transgenic plants and has great potential for the production of therapeutic anti-cancer antibodies and recombinant therapeutic proteins. Plants make fully functional recombinant human or animal antibodies. Cultivating transgenic plants on an agricultural scale will produce almost unlimited supplies of recombinant proteins for uses in medicine.

Towards molecular farming in the future: pichia pastoris-based production of single-chain antibody fragments.

This review article focuses on the use of the methylotrophic yeast Pichia pastoris as a recombinant protein-expression system. P. pastoris is a useful system for the expression of milligram-to-gram quantities of a protein, which can be scaled up to fermentation to meet greater demands.

Towards molecular farming in the future: transient protein expression in plants.

Molecular farming in plants can be achieved by stable or transient expression of a recombinant protein. Transient expression of recombinant proteins in plants can rapidly provide large amounts of the proteins for detailed characterization. It is fast, flexible and can be carried out at field scale using viral vectors, but it lacks the increases in production volume that can be achieved easily with stable transgenic crops.

Towards molecular farming in the future: using plant-cell-suspension cultures as bioreactors.

Plant-suspension cells are an in vitro system that can be used for recombinant protein production under carefully controlled certified conditions. Plant-suspension cells can be grown in shake flasks or fermenters to produce secondary metabolites, like vincristine and vinblastine, and to produce recombinant proteins after transformation. This review article focuses on discussing the generation of transformed suspension-cell lines expressing recombinant proteins, like antibodies, and recombinant-protein downstream processing and purification.

Towards molecular farming in the future: moving from diagnostic protein and antibody production in microbes to plants.

Molecular farming of pharmaceuticals in plants has the potential to provide almost unlimited amounts of recombinant proteins for use in disease diagnosis and therapy. Transgenic plants are attracting interest as bioreactors for the inexpensive production of large amounts of safe, functional, recombinant macromolecules, such as blood substitutes, vaccines and antibodies. In some cases, the function of expressed recombinant proteins can be rapidly analysed by expression in microbes or by transient expression in intact or virally infected plants.

Molecular farming of recombinant antibodies in plants.

'Molecular farming' is the production of recombinant proteins in plants. It is intended to harness the power of agriculture to cultivate and harvest transgenic plants producing recombinant therapeutics. Molecular farming has the potential to provide virtually unlimited quantities of recombinant antibodies for use as diagnostic and therapeutic tools in both health care and the life sciences.

Protein phosphorylation during phagosome maturation.

Phagolysosome biogenesis is driven by a series of interactions between phagosomes and organelles of the biosynthetic and endocytic pathways. The presence of endocytic markers on phagosomes suggests that phagosomes and endosomes share common structural and functional characteristics. In that line of thought, protein phosphorylation has been shown to be involved in regulatory aspects of the fusion properties of endosomes and other vacuolar organelles.