Separation of the arterial wall from blood contact using hydrogel barriers reduces intimal thickening after balloon injury in the rat: The roles of medial and luminal factors in arterial healing

The objective of this study was to clarify the relative roles of medial versus luminal factors in the induction of thickening of the arterial intima after balloon angioplasty injury. Platelet-derived growth factor (PDGF) and thrombin, both associated with thrombosis, and basic fibroblast growth factor (bFGF), stored in the arterial wall, have been implicated in this process. To unequivocally isolate the media from luminally derived factors, we used a 20-μm thick hydrogel barrier that adhered firmly to the arterial wall to block thrombus deposition after balloon-induced injury of the carotid artery of the rat. Thrombosis, bFGF mobilization, medial repopulation, and intimal thickening were measured. Blockade of postinjury arterial contact with blood prevented thrombosis and dramatically inhibited both intimal thickening and endogenous bFGF mobilization. By blocking blood contact on the two time scales of thrombosis and of intimal thickening, and by using local protein release to probe, by reconstitution, the individual roles of PDGF-BB and thrombin, we were able to conclude that a luminally derived factor other than PDGF or thrombin is required for the initiation of cellular events leading to intimal thickening after balloon injury in the rat. We further conclude that a luminally derived factor is required for mobilization of medial bFGF.

Replication-dependent Histone Gene Expression Is Related to Cajal Body (CB) Association but Does Not Require Sustained CB Contact

Interactions between Cajal bodies (CBs) and replication-dependent histone loci occur more frequently than for other mRNA-encoding genes, but such interactions are not seen with all alleles at a given time. Because CBs contain factors required for transcriptional regulation and 3′ end processing of nonpolyadenylated replication-dependent histone transcripts, we investigated whether interaction with CBs is related to metabolism of these transcripts, known to vary during the cell cycle. Our experiments revealed that a locus containing a cell cycle-independent, replacement histone gene that produces polyadenylated transcripts does not preferentially associate with CBs. Furthermore, modest but significant changes in association levels of CBs with replication-dependent histone loci mimic their cell cycle modulations in transcription and 3′ end processing rates. By simultaneously visualizing replication-dependent histone genes and their nuclear transcripts for the first time, we surprisingly find that the vast majority of loci producing detectable RNA foci do not contact CBs. These studies suggest some link between CB association and unusual features of replication-dependent histone gene expression. However, sustained CB contact is not a requirement for their expression, consistent with our observations of U7 snRNP distributions. The modest correlation to gene expression instead may reflect transient gene signaling or the nucleation of small CBs at gene loci.

Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: Use of a photoactivatable reagent

Interaction of light-activated rhodopsin with transducin (T) is the first event in visual signal transduction. We use covalent crosslinking approaches to map the contact sites in interaction between the two proteins. Here we use a photoactivatable reagent, N-[(2-pyridyldithio)-ethyl], 4-azido salicylamide. The reagent is attached to the SH group of cytoplasmic monocysteine rhodopsin mutants by a disulfide-exchange reaction with the pyridylthio group, and the derivatized rhodopsin then is complexed with T by illumination at λ >495 nm. Subsequent irradiation of the complex at λ310 nm generates covalent crosslinks between the two proteins. Crosslinking was demonstrated between T and a number of single cysteine rhodopsin mutants. However, sites of crosslinks were investigated in detail only between T and the rhodopsin mutant S240C (cytoplasmic loop V-VI). Crosslinking occurred predominantly with Tα. For identification of the sites of crosslinks in Tα, the strategy used involved: (i) derivatization of all of the free cysteines in the crosslinked proteins with N-ethylmaleimide; (ii) reduction of the disulfide bond linking the two proteins and isolation of all of the Tα species carrying the crosslinked moiety with a free SH group; (iii) adduct formation of the latter with the N-maleimide moiety of the reagent, maleimido-butyryl-biocytin, containing a biotinyl group; (iv) trypsin degradation of the resulting Tα derivatives and isolation of Tα peptides carrying maleimido-butyryl-biocytin by avidin-agarose chromatography; and (v) identification of the isolated peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We found that crosslinking occurred mainly to two C-terminal peptides in Tα containing the amino acid sequences 310–313 and 342–345.