Polytopic membrane protein folding and assembly in vitro and in vivo.

The insertion and folding of proteins in biological membranes during protein synthesis in vivo is fundamental to membrane biogenesis. At present, however, certain molecular aspects of this process can only be understood by complementary studies in vitro. We bring together in vitro and in vivo results, highlighting how the studies inform each other and increase our knowledge of the folding and assembly of polytopic membrane proteins.

Direct block of the cystic fibrosis transmembrane conductance regulator Cl(-) channel by niflumic acid.

Niflumic acid is widely used to inhibit Ca(2+) -activated Cl(-) channels. However, the chemical structure of niflumic acid resembles that of diphenylamine-2-carboxylate, a drug that inhibits the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. To investigate how niflumic acid inhibits CFTR Cl(-) channel, we studied recombinant wild-type human CFTR in excised inside-out membrane patches.

A kinetic model of intermediate formation during assembly of cholera toxin B-subunit pentamers.

Cholera toxin is the most important virulence factor produced by Vibrio cholerae. The pentameric B-subunit of the toxin can bind to GM1-ganglioside receptors, leading to toxin entry into mammalian cells. Here, the in vitro disassembly and reassembly of CtxB(5) (the B subunit pentamer of cholera toxin) is investigated.

Regulation of epidermal growth factor receptor traffic by the small GTPase rhoB.

Members of the Rho family of small GTPases control cell adhesion and motility through dynamic regulation of the actin cytoskeleton. Although twelve family members have been identified, only three of these - RhoA, Rac and Cdc42 - have been studied in detail. RhoA regulates the formation of focal adhesions and the bundling of actin filaments into stress fibres.

Imaging Ca2+ concentration changes at the secretory vesicle surface with a recombinant targeted cameleon.

Regulated exocytosis involves the Ca(2+)-triggered fusion of secretory vesicles with the plasma membrane, by activation of vesicle membrane Ca(2+)-binding proteins [1]. The Ca(2+)-binding sites of these proteins are likely to lie within 30 nm of the vesicle surface, a domain in which changes in Ca2+ concentration cannot be resolved by conventional fluorescence microscopy. A fluorescent indicator for Ca2+ called a yellow 'cameleon' (Ycam2) - comprising a fusion between a cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13 and an enhanced yellow-emitting GFP - which is targetable to specific intracellular locations, has been described [2].

Real-time imaging of gene expression in single living cells.

Recent advances in reporter gene technologies are now allowing us to image gene transcription at the single cell level, using either fluorescence or luminescence microscopy. Here, the basis of these techniques is outlined and their advantages and disadvantages in various biological systems are discussed.

TGF-beta expression in the human colon: differential immunostaining along crypt epithelium.

Samples of colorectal carcinoma, adenoma and normal colorectal mucosa were examined for the expression of TGF-beta by immunohistochemistry. Immunoreactivity for TGF-beta was present in 52 out of a total of 58 samples of normal mucosa examined. In adenomas and carcinomas TGF-beta expression was observed in eight out of ten and 46 out of 48 samples respectively and was largely restricted to epithelial cells.

Heterotypic and homotypic re-inoculation of mice already latently infected with herpes simplex virus type 1.

Mice were infected at 4 weeks of age with a type 1 strain of herpes simplex virus (HSV) and re-infected 4 weeks later with either a type 1 or a type 2 strain of HSV. The virus used for first infection could be distinguished from that used later since it was resistant to phosphonoformic acid and formed syncytial plaques. Sites used for the second inoculation were as follows: at the site of primary infection, at a different site within the same dermatome or in the equivalent dermatome on the opposite side (also called "remote" site).