A Department of Motor Vehicles intervention yields moderate increases in donor designation rates.

CONTEXT: Nearly all persons (37% of public) who have a joined an organ donor registry in the United States have done so through their Department of Motor Vehicles (DMV) office, which is an underused venue for organ donation campaigns. OBJECTIVE: To evaluate the effectiveness of a statewide DMV-based intervention to increase donor designation rates. DESIGN AND SETTING: Thirty DMV offices in Florida were randomly assigned to receive usual care (n = 15) or an organ donation intervention (n = 15). MEASUREMENT AND PRIMARY OUTCOME: Donor designation rates were assessed at baseline (before the intervention), during the intervention, and at follow-up. RESULTS: When baseline donor designation rate and region were controlled for, the intervention group showed a significantly higher aggregate monthly donor designation rate than the usual care group during the intervention phase of the study (P = .02). Donor designation rates did not differ significantly (P = .13) during the follow-up phase. Lower donor designations rates were significantly associated with DMV service regions with more minorities, less education, and lower income. CONCLUSION: We conclude that a comprehensive DMV-based intervention focused on staff education and direct interactions with the public could yield modest increases in donor designation rates.

Family initiated discussions about organ donation at the time of death.

Some family members initiate organ donation discussions before being approached by donor coordinators or healthcare providers. We examined differences between families that did vs. did not initiate organ donation discussions and factors predicting donation consent among those families that self-initiated the discussion. Next-of-kin of donor-eligible individuals (147 donors, 138 non-donors) from one organ procurement organization completed a telephone interview. Seventy-three families (25.6%) first mentioned organ donation, and 54 (74%) of them consented to donation. Several characteristics of the deceased and next-of-kin were associated with whether family members initiated the donation discussion with donation coordinators or healthcare providers. Moreover, family mention of donation was more likely to yield consent when the deceased was younger (OR=0.95, CI=0.92-0.99), next-of-kin was a registered donor (OR=3.86, CI=2.84-6.76), and when family was more satisfied with the healthcare team (OR=1.20, CI=1.04-1.39). Knowing the deceased's donation intentions and being exposed to positive organ donation messages are more likely to trigger families to raise donation with providers. Organ procurement organizations (OPOs) and healthcare providers should work collaboratively to develop strategies for how best to respond to families who initiate this conversation.

A novel tetraspanin fusion protein, peripherin-2, requires a region upstream of the fusion domain for activity.

Peripherin-2 (also known as peripherin/rds), a photoreceptor specific tetraspanin protein, is required to maintain normal cell structure through its role in renewal processes requiring membrane fusion. It is the first tetraspanin fusogen and has been shown to directly mediate fusion between disk membranes and opposing membranes to maintain the highly ordered structure of rod outer segments. Localized to the C terminus of human, bovine, and murine peripherin-2 is an amphiphilic fusion peptide domain (residues 312-326) and a highly conserved region upstream of this domain that we hypothesize is essential for fusogenic function. Our previous studies indicated that substitution of a threonine for a proline at position 296 within this highly conserved region enhanced fusion activity. In this study we wanted to determine whether this proline is essential with the introduction of three additional substitutions of proline with alanine, leucine, and glutamic acid. Wild type, P296T, P296A, P296L, and P296E mutants of peripherin-2 were expressed as His6-tagged full-length proteins in Madin-Darby canine kidney (MDCK) cells. All of the proteins were localized to intracellular membranes and detected as 42-kDa monomers by Western blot analysis. The wild type, P296A, and P296L assembled into core tetramers; in contrast the P296T and P296E formed higher order oligomers. Fusogenic activity of full-length protein expressed in MDCK membranes and purified protein reconstituted in model membrane liposomes was determined using fluorescence quenching techniques. Fusion activity was decreased in the P296L, P296A, and P296E mutants both in endogenous MDCK membranes and in model liposomes. Collectively, these results suggest that the proline at position 296 is necessary for optimal function.

Heterologous expression of WT and mutant photoreceptor peripherin/rds in Madin Darby canine kidney cells: an assessment of fusogenic function.

Peripherin/rds is proposed to function as a fusion protein within the rod outer segment and a fusion domain has been mapped to amino acids 311-325 within the C-terminus. To map regions within peripherin/rds required for membrane fusion a series of C-terminal mutants was analyzed. Madin Darby canine kidney cells were transiently transfected with an Xpress or FLAG epitope tagged peripherin/rds (wt) and three mutants of peripherin/rds. The mutants selected were a P296T mutant (replacement of the proline at position 296 with a threonine) and two C-terminal deletion mutants (one lacking the terminal 10 amino acids, Delta10 and one lacking the terminal 50 amino acids, Delta50). The wt protein, the P296T and Delta10 mutants were detected on SDS-PAGE as 84 kDa dimers, that resolved into 38-42 kDa monomers under reducing conditions. The Delta50 mutant showed a slightly increased mobility. The cellular localization of mutants differed from that of wt peripherin/rds. The wt Xpress-human and wt FLAG-bovine peripherin/rds were localized to both intracellular and plasma membranes. In contrast, the C-terminal deletion mutants were localized only to the intracellular membrane. The P296T mutant presented a still different pattern: initially the protein localized to intracellular membranes. Upon confluence, however, the localization appeared to become predominantly plasma membrane. To assess the fusion activity of the proteins, the cell membranes were fractionated using sucrose density gradient centrifugation and the various fractions identified based on immunoreactivity in Western blot analysis with Golgi (anti-rab 6) or plasma membrane (anti-ZO-3) specific marker proteins. All membrane fractions were assayed for fusion with ROS plasma membrane vesicles. The plasma membrane enriched fractions (isolated at densities of 1.08 and 1.125 g ml(-1)) containing tagged peripherin/rds and the Delta10 mutant promoted membrane fusion with ROS plasma membrane vesicles. In contrast, fusion was not detected with plasma membrane vesicles from mock-transfected cells or the Delta50 peripherin/rds deletion mutant. Fusion was enhanced in a less dense fraction enriched in the P296T mutant (isolated from the 1.04/1.02 interface) relative to wt. Fusion was dependent on the presence of peripherin/rds in the membranes and could be inhibited with trypsinolysis and competition studies with the bovine fusion peptide, PP-5. Peptide competition suggests that the fusion domain of human peripherin/rds is most likely identical to that characterized in bovine and corresponds to amino acid residues 312-326. The C-terminal deletion mutants have allowed us to predict the minimal region of the C-terminus necessary for fusion to include residues starting at number 335. In addition a second region important in the formation of a fusion competent peripherin/rds has been mapped to a region upstream of the fusion peptide domain.