Characterization of the cell membrane-associated products of the Neuregulin 4 gene.

The NRG4 gene is a member of a family of four genes that encode a class of epidermal growth factors. This gene has been reported to express a protein designated here as NRG4A1. We describe here a novel splice variant of the NRG4 gene, NRG4A2, which encodes a C-terminal region containing a predicted type I PDZ-binding peptide. Both NRG4A1 and NRG4A2 were shown to be expressed on the cell surface, as expected by the presence of a predicted transmembrane sequence, and were modified at a single N-linked glycosylation site in the extracellular domain. Significant stabilization of expression of both proteins was seen in the presence of the proteosome inhibitor MG-132 suggesting that they are normally degraded by this system. N-terminal cleavage was inhibited in both isotypes by the broad-spectrum matrix metalloproteinase inhibitor, galardin (GM 6001). A glycosylated, secreted form of NRG4A1 was detected in the cell medium which showed biological activity in two assays, phosphorylation of the HER4 receptor and stimulation of neurite formation in PC-12 cells stably expressing HER4. Transfection and expression of green fluorescent protein-tagged proteins and immunofluorescent staining with specific anti-peptide antibodies showed that NRG4A1 is localized to membrane ruffles, while NRG4A2 has a more punctate membrane distribution.

99mTc-SnF2 colloid "LLK": particle size, morphology and leucocyte labelling behaviour.

99mTc-SnF2 colloid (Radpharm LLK) leucocyte labelling agent is used in whole blood, exploiting phagocytosis. The objectives of this work were to optimize leucocyte labelling in leucocyte-enriched plasma, and to investigate: (i) the effect of temperature and other factors on labelling efficiency; (ii) the selectivity for different leucocyte types; (iii) the viability of the labelled cells and efflux of the radiolabel; and (iv) the physical characteristics of the colloid. Density gradient centrifugation was used to investigate the labelling efficiency, cell selectivity and efflux, Trypan blue to study the viability, and laser scattering, electron microscopy and membrane filtration to investigate particle size and morphology. Particles appeared as loose, coiled, chain-like aggregates of much smaller particles (<0.05 microm). The aggregate diameter ranged from <0.1 to >5 microm and increased with time. The distribution of radioactivity amongst the particle sizes varied widely. The labelling efficiency in leucocyte-rich plasma was enhanced at 37 degrees C compared to room temperature, and by centrifuging during labelling. The selectivity for different leucocyte types varied markedly between batches and blood samples, in some cases showing preference for mononuclear cells and in others for granulocytes. Viability was excellent and comparable with 99mTc-hexamethylpropyleneamine oxime (99mTc-HMPAO)-labelled cells. A significant fraction of radiolabel, comparable to that observed with 99mTc-HMPAO, was lost from leucocytes during incubation in vitro over 4 h. Thus, 99mTc-SnF2 is a convenient, efficient labelling agent for leucocytes, but shows variable cell selectivity which may be linked to particle size variability, and there is significant efflux of radioactivity from labelled cells.

Structural and biochemical characterisation of the paraflagellar rod of Crithidia fasciculata.

The trypanosomatid Crithidia fasciculata possesses an intraflagellar structure known as the paraflagellar or paraxial rod which runs from a point 1 to 2 micrometer distal to the basal body to the flagellar tip. In longitudinal section the paraflagellar rod was composed of three "sets" of parallel filaments arranged in a lattice. In cross section it consisted of two electron dense "plaques", one near the flagellar membrane, the other near the axoneme, separated by 6 to 7 fibrous elements. The position of the paraflagellar rod in relation to the axonemal central pair remained static along the length of the flagellum and was the same in all flagella examined. The paraflagellar rod was anchored to the axoneme by a regular array of 5 to 7 nm diameter links. These rod/axoneme links were sensitive to trypsin digestion enabling the rod to be separated from the axoneme. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the paraflagellar rod consisted mainly of two proteins, PFR1 (76 000 Daltons) and PFR2 (68 000 Daltons). The isoelectric points of these two proteins were remarkably similar. A PFR-enriched fraction was obtained by prolonged dialysis of demembranated flagella against a low concentration buffer. The paraflagellar rod and the central pair of singlet microtubules went into solution, leaving only the outer doublets intact. The relevance of these results to the study of the role of the paraflagellar rod in flagellar motility were discussed.