Rapid plasma membrane anchoring of newly synthesized p59fyn: selective requirement for NH2-terminal myristoylation and palmitoylation at cysteine-3.

The trafficking of Src family proteins after biosynthesis is poorly defined. Here we studied the role of dual fatty acylation with myristate and palmitate in biosynthetic transport of p59fyn. Metabolic labeling of transfected COS or NIH 3T3 cells with [35S]methionine followed by analysis of cytosolic and total membrane fractions showed that Fyn became membrane bound within 5 min after biosynthesis. Newly synthesized Src, however, accumulated in the membranes between 20-60 min. Northern blotting detected Fyn mRNA specifically in soluble polyribosomes and soluble Fyn protein was only detected shortly (1-2 min) after radiolabeling. Use of chimeric Fyn and Src constructs showed that rapid membrane targeting was mediated by the myristoylated NH2-terminal sequence of Fyn and that a cysteine at position 3, but not 6, was essential. Examination of G alpha(o)-, G alpha(s)-, or GAP43-Fyn fusion constructs indicated that rapid membrane anchoring is exclusively conferred by the combination of N-myristoylation plus palmitoylation of cysteine-3. Density gradient analysis colocalized newly synthesized Fyn with plasma membranes. Interestingly, a 10-20-min lag phase was observed between plasma membrane binding and the acquisition of non-ionic detergent insolubility. We propose a model in which synthesis and myristoylation of Fyn occurs on soluble ribosomes, followed by rapid palmitoylation and plasma membrane anchoring, and a slower partitioning into detergent-insoluble membrane subdomains. These results serve to define a novel trafficking pathway for Src family proteins that are regulated by dual fatty acylation.

Key parameters affecting the initial leaky effect of hemoglobin-loaded nanoparticles as blood substitutes.

In order to realize long-term carrying/delivering oxygen and minimize the adverse effects of free hemoglobin (Hb) in vivo, Hb is desired to be confined in Hb-loaded nanoparticles (HbP), a novel blood substitute with potential clinical applications, and thus functions as the native red blood cells (RBCs). However, the initial burst release of Hb ("leaky effect") greatly underscores the significance of this work. The study described here wants to disclose the key preparative parameters, including polymer, excipients in the inner aqueous phase and solvent profile, affecting the Hb release behavior (the initial 24 h) from HbP fabricated by commonly used solvent diffusion/evaporation double emulsion technique. The results demonstrate that PEGlytated polymers, regardless of two- or tri-block copolymers show slower release compared with the corresponding non-PEGlytated ones. The higher polymer concentration yields lower initial release. PEG200, added as excipient facilitates Hb burst effect to about 38.4%, almost 17% increase compared to the control ( approximately 21%), whereas, PVA and Poloxamer188, due to amphiphilic nature, can effectively attenuate this leakage to about 13.0 and 5.1%, respectively. The diffusion/extraction rate from oil phase and the subsequent evaporation rate from the aqueous continuous phase of solvents impose different influences on Hb release. To reduce the burst effect, the initial diffusion/extraction rate should be slow, whereas, the concomitant evaporation rate should be as fast as possible. The results obtained here will be guidance's for the future tailored design of more desirable polymersome nanoparticle blood substitutes.

Characterization, chromosomal mapping, and expression of different polyubiquitin genes in tissues from control and heat-shocked maize seedlings.

Polyubiquitin transcripts accumulate in plant and animal cells following a heat shock. Most species have a few to several polyubiquitin genes; within a species, the genes may differ in nucleotide (nt) sequence and (or) the number of 228-nt repeats encoding the ubiquitin monomer. This study examines three maize (inbred Oh43) polyubiquitin genes. Two of the genes, MubG9 and MubG5, possess five repeats; the third, MubG1 possesses seven repeats. Sequence analyses of the genomic clones, MubG9 and MubG1 and a cDNA clone, MubG5, reveal that they differ primarily from each other in their nt sequences 5' and 3' to their open reading frames. MubG1 contains a 1004-base pair (bp) intron in its 5' untranslated region. Using gene-specific probes, we show that the amount of polyribosome-associated mRNA transcripts from MubG9 isolated from 2- and 5-day old plumules and radicles is unchanged by heat shock. While the amount of transcript from MubG1 and MubG5 on the polyribosomes in plumules and radicles of 2-day-old seedlings is also unchanged by heat shock, the levels of these transcripts are elevated considerably in similar tissues from heat-shocked 5-day-old seedlings. Similar or identical gene-specific probes were employed to map the genes using the recombinant inbred method. MubG9 maps to chromosome 4L position 186; MubG1 maps to 5L104 and MubG5 to 4L188. The opportunity to use gene-specific probes extends the evidence for distinct modulation (time and tissue) of polyubiquitin gene expression in maize and provides the basis for locus assignment within the genome.