Foreigners traveling to the U.S. for transplantation may adversely affect organ donation: a national survey.

The aims of this study were (1) to determine attitudes among the American public regarding foreigners coming to the United States for the purposes of transplantation, and (2) to investigate the impact this practice might have on the public's willingness to donate organs. A probability-based national sample of adults age > or =18 was asked whether people should be allowed to travel to the United States to receive a transplant, and whether this practice would discourage the respondents from becoming an organ donor. Among 1049 participants, 30% (95% CI 25-34%) felt that people should not be allowed to travel to the United States to receive a deceased donor transplant, whereas 28% felt this would be acceptable in some cases. Thirty-eight percent (95% CI 33-42%) indicated that this practice might prevent them from becoming an organ donor. In conclusion, deceased-donor transplantation of foreigners is opposed by many Americans. Media coverage of this practice has the potential to adversely affect organ donation.

Attitudes of the American public toward organ donation after uncontrolled (sudden) cardiac death.

Concerns about public support for organ donation after cardiac death have hindered expansion of this practice, particularly rapid organ recovery in the context of uncontrolled (sudden) cardiac death (uDCD). A nationally representative Internet-based panel was provided scenarios describing donation in the context of brain death, controlled cardiac death and uncontrolled cardiac death. Participants were randomized to receive questions about trust in the medical system before or after the rapid organ recovery scenario. Among 1631 panelists, 1049 (64%) completed the survey. Participants expressed slightly more willingness to donate in the context of controlled and uncontrolled cardiac death than after brain death (70% and 69% vs. 66%, respectively, p < 0.01). Eighty percent of subjects (95% CI 77-84%) would support having a rapid organ recovery program in their community, though 83% would require family consent or a signed donor card prior to invasive procedures for organ preservation. The idea of uDCD slightly decreased trust in the medical system from 59% expressing trust to 51% (p = 0.02), but did not increase belief that a signed donor card would interfere with medical care (28% vs. 32%, p = 0.37). These findings provide support for the careful expansion of uDCD, albeit with formal consent prior to organ preservation.

Geographic variation in organ availability is responsible for disparities in liver transplantation between Hispanics and Caucasians.

The aims of this study were to determine whether disparities in waiting list outcomes exist for Hispanics and African Americans during the post-MELD era, and to investigate interactions between disparities and geography. Scientific Registry of Transplant Recipients data were used to compare Hispanics and African Americans to Caucasians listed between 2003 and 2008. Endpoints included (i) receipt of a liver transplant and (ii) death or removal from the waiting list for being too sick or medically unsuitable. Adjustment for possible confounders was performed using multivariate Cox regression, with adjustment for geographic variation using a fixed-effects multilevel model. In multivariate analysis, African Americans have similar hazard of transplantation and death/removal as Caucasians during the post-MELD era. However, Hispanics are less likely to receive a transplant than Caucasians despite adjustment for potential confounders (HR 0.80, 95% CI 0.77-0.83), while having a similar hazard of death/removal. This effect disappeared after adjusting for unequal regional distribution of Hispanics, who represent 8% of patients in donation service areas (DSAs) having median waiting times of < or = 155 days versus 19% in DSAs with median waiting times of >155 days. In conclusion, disparities in liver transplantation exist for Hispanics during the post-MELD era, caused by geographic variation in organ availability.

The sfr6 mutation in Arabidopsis suppresses low-temperature induction of genes dependent on the CRT/DRE sequence motif.

The sfr mutations, which result in sensitivity to freezing after cold acclimation, define genes that are required for freezing tolerance. We tested plants homozygous for mutations sfr2 to sfr7 for cold-induced gene expression and found that sfr 6 plants were deficient in cold-inducible expression of the genes KIN1, COR15a, and LTI78, which all contain the C repeat/dehydration-responsive element (CRT/DRE) motif in their promoters. Similarly, sfr 6 plants failed to induce KIN1 normally in response to either osmotic stress or the application of abscisic acid. In contrast, cold-inducible expression of genes CBF1, CBF2, CBF3, and ATP5CS1, which lack the CRT/DRE motif, was not affected. The freezing-sensitive phenotype that defines sfr 6 also was found to be tightly linked to the gene expression phenotype. To determine whether the failure of cold induction of CRT/DRE-containing genes in sfr 6 was due to altered low-temperature calcium signaling, cold-induced cytosolic-free calcium ([Ca2+]cyt) elevations were investigated in the sfr 6 mutant, but these were found to be indistinguishable from those of the wild type. We discuss the possibilities that CRT/DRE binding proteins (such as CBF1) require activation to play a role in transcription and that the SFR6 protein is a vital component of their activation.

Cold stress: manipulating freezing tolerance in plants.

A single transcription factor can trigger induction of the freezing-tolerant state in Arabidopsis. Is such a factor all that is lacking in non-hardy plants?

Increased DT-diaphorase activity in transformed and tumorigenic pancreatic acinar cells.

Pancreatic acinar cells from rats treated in vitro with 5-azacytidine and/or transfected with an activated c-H-ras demonstrated transformation and tumorigenic phenotypes. DT-diaphorase (NAD(P)H:quinone oxidoreductase) activity was determined in these non-tumorigenic (3AP) and tumorigenic cells (T3AP and T5AM). T5AM cells were those treated with 5-azacytidine and further treated with N'-methyl-N'-nitro-nitrosoguanidine. Higher levels of enzyme activity were found in transformed cells when compared to that in control cells (> 15-fold, 3AP cells; > 40-fold, T3AP cells; > 20-fold T5AM cells). In contrast, NADPH-cytochrome c reductase activity was decreased in transformed cells (> 10-fold, 3AP cells; > 20-fold, T3AP cells; > 10-fold, T5AM cells). These studies demonstrated that pancreatic acinar cells are capable of undergoing alterations in enzyme activity patterns when transformed and that DT-diaphorase may be a good marker for malignant transformation.

Properties of engineered antifreeze peptides.

Eight antifreeze-like peptides were produced by cleavage from engineered chimeric proteins. One was homologous to an antifreeze peptide of the winter flounder; the others differed in length and/or sequence. The homologous peptide and all those of equal or greater length were able to inhibit recrystallization. The longer peptides were so hydrophobic that their identification required modification of the usual protocols for high pressure liquid chromatography. Their elution positions were correlated to their hydrophobicities and their lengths. Additional naturally occurring antifreezes may be identifiable with this knowledge.

Enhanced survival of yeast expressing an antifreeze gene analogue after freezing.

Yeast, like most organisms, survives poorly under freezing conditions. It has been proposed that after rapid cooling yeast suffers a loss in viability from the recrystallization of intracellular ice. Antifreeze proteins found in the blood of certain polar fishes have been shown to be potent inhibitors of ice recrystallization at very low concentrations. We have examined the feasibility of protecting rapidly cooled yeast cells from freezing damage by inhibiting the recrystallization of intracellular ice through in vivo expression of an antifreeze analogue gene. A chemically synthesized gene encoding a protein similar to but differing from the antifreeze proteins of the fish Pseudopleuronectes americanus (winter flounder) was genetically fused to the 3' end of a truncated staphylococcal Protein A gene. When the fused gene was expressed in the budding yeast Saccharomyces cerevisiae, its cells were shown to produce a new chimeric protein that inhibited the recrystallization of ice in vitro. Yeast cells expressing the chimeric antifreeze protein showed a twofold increase in survival after rapid freezing (95 degrees C/min to -196 degrees C) and moderate rates of warming (26 to 64 degrees C/min) compared to cells lacking the chimeric protein.

Inhibition of recrystallization in ice by chimeric proteins containing antifreeze domains.

Using synthetic DNA, we assembled a gene encoding a protein identical in sequence to one of the antifreeze proteins produced by the fish Pseudopleuronectes americanus (winter flounder). To address the relationship between structure and function, we also assembled genes encoding proteins varying in sequence and length. The synthetic genes were cloned into a bacterial expression vector to generate translational fusions to the 3' end of a truncated staphylococcal protein A gene; the chimeric proteins encoded by these fusions, varying only in their antifreeze domains, were isolated from Escherichia coli. The antifreeze domains conferred the ability to inhibit ice recrystallization, which is characteristic of naturally occurring antifreeze proteins, on the chimeric proteins. The chimeric proteins varied in their effectiveness of inhibiting ice recrystallization according to the number of 11-amino acid repeats present in the antifreeze moiety. A protein with only two repeats lacked activity, while the inhibitory activity increased progressively for proteins containing three, four, and five repeats. Some activity was lost upon removal of either the salt bridge or the carboxyl-terminal arginine, but surprisingly, not when both features were absent together.

Rates of assembly and degradation of bacterial ice nuclei.

The kinetics of ice-nucleus assembly from newly synthesized nucleation protein were observed following induction of nucleation gene expression in the heterologous host Escherichia coli. Assembly was significantly slower for the small proportion of ice nuclei active above -4.4 degrees C; this was consistent with the belief that these nuclei comprise the largest aggregates of nucleation protein. The kinetics of nucleus degradation were followed after inhibiting protein synthesis. Nucleation activity and protein showed a concerted decay, indicating that most of the functional ice nuclei are in equilibrium with a single cellular pool of nucleation protein. A minority of the ice nuclei decayed much more slowly than the majority; presumably their nucleation protein was distinct either by virtue of different structure or different subcellular compartmentalization, or because of its presence in a metabolically distinct subpopulation of cells.

Clustering of ice nucleation protein correlates with ice nucleation activity.

Antibodies raised against a synthetic peptide specifically detect ice nucleation proteins from Pseudomonas species in Western blots. In immunofluorescent staining of whole bacteria, the antibodies reveal the protein in clusters, as indicated by patches of intense fluorescence in Escherichia coli cells heterologously expressing Pseudomonas ice nucleation genes. The abundance, size, and brightness of the clusters vary considerably from cell to cell. Their varying sizes may explain the variability in activity of bacterial ice nuclei. Growth at lower temperatures produces more ice nuclei, and gives brighter and more frequent patches, than growth at 37 degrees C. The observed clustering may thus reflect formation of functional ice nucleation sites in vivo. The presence of ice nucleation protein in clusters is also correlated with alterations in cell morphology.

Deletion mutagenesis of the ice nucleation gene from Pseudomonas syringae S203.

The ice nucleation gene inaZ, from Pseudomonas syringae S203, was manipulated to produce a series of defined rearrangements in its coding sequence without changing the reading frame. The effects of these mutations on the ice nucleation phenotype were determined in a heterologous host, Escherichia coli K12. Deletions which disrupted the periodicity of 16 codons, in a repetitive region of inaZ, caused the frequencies of ice nuclei in the bacterial population to be significantly depressed; the nuclei with thresholds at warmer temperatures were most affected. In contrast, when the periodicity was left intact, deletions and duplications in the same region had only slight effects on nucleation activity. Deletions removing part or all of one of the nonrepetitive regions (that encoding the amino-terminal domain of the InaZ protein) did not abolish nucleation activity, but caused it to be limited to cooler threshold temperatures. In contrast, the non-repetitive carboxy-terminal domain of the InaZ protein was shown to be essential for ice nucleation at all temperatures. The differential requirements (for periodicity, and for the amino-terminus) in forming nuclei with different thresholds may be significant for understanding what determines the threshold temperature of an ice nucleus.

Nonlinear relationship between concentration and activity of a bacterial ice nucleation protein.

The expression level of an ice nucleation gene (inaZ) was varied in Escherichia coli to observe the relationship between activity and gene product. The ice nucleation activity increased as the 2nd to 3rd power of the membrane concentration of the inaZ gene product, implying that molecules of InaZ protein interact cooperatively in groups of two to three at the rate-limiting step of ice nucleus assembly. The 2nd to 3rd power relationship was independent of the threshold temperature at which ice nucleation was measured and was consistent over a 500-fold range of protein concentration. Such a relationship indicates that the same rate-limiting step must be common to the formation of ice nuclei displaying all the various threshold temperatures within a bacterial population. Observations of Pseudomonas syringae, expressing the inaZ gene at various levels, were consistent with a similar relationship and hence a similar mechanism of ice nucleus assembly in Pseudomonas.

Genetic determinants for catabolite induction of antibiotic biosynthesis in Pseudomonas fluorescens HV37a.

Antibiotic biosynthesis is regulated by glucose in Pseudomonas fluorescens HV37a. Fusions between antibiotic biosynthetic operons (afu operons) and the Escherichia coli lac operon were isolated to evaluate the genetic determinants for the regulation of antibiotic biosynthesis. Four afu transcriptional units were defined, afuE, afuR, afuAB, and afuP. The afuE and afuR transcripts were promoted divergently at one locus and were catabolite induced, by 250-fold and 5-fold, respectively; the afuAB and afuP transcriptional units were not linked to the others and were not catabolite induced. Thus, regulation of afuE and afuR operon transcription is apparently the mechanism whereby glucose regulates antibiotic biosynthesis. Catabolite induction of the afuE and afuR transcriptional unit was dependent on the products of the afuA, afuB, and afuP genes. Expression of the afuE transcriptional unit was altered quantitatively in afuE mutants. Apparently the afuE transcriptional unit is regulated, at least in part, by its own gene products. Under inducing conditions, expression of the afuE, afuR, and afuP transcriptional units increased rapidly during a 6-h period.

An iron-antagonized fungistatic agent that is not required for iron assimilation from a fluorescent rhizosphere pseudomonad.

Fluorescent rhizosphere Pseudomonas sp. strain NZ130 promotes plant growth, and may do so in part because of its production of a growth inhibitory factor that is active against phytopathogenic fungi. Analysis of the inhibitory factor that is active against the phytopathogen Pythium ultimum showed that its activity is antagonized at iron concentrations above 10 microM. The iron-antagonized inhibitor was separated from the fluorescent siderophore of this pseudomonad by gel filtration. Mutants that lacked either the iron-antagonized inhibitor or the fluorescent siderophore were isolated. Results of complementation analysis of these mutants by use of a cosmid library indicated that distinct DNA sequences are required for the production of each factor. Analysis of isogenic mutant strains showed that the genetic requirements for the production of the iron-antagonized inhibitor and the fluorescent siderophore are different, and that only the fluorescent siderophore is required for iron assimilation. Fusions of these same sequences to a beta-galactosidase gene were used to show that the regions required for the production of both the fluorescent siderophore and the iron-antagonized inhibitor were iron-regulated.

Identification and purification of a bacterial ice-nucleation protein.

The protein product of a gene (inaZ) responsible for ice nucleation by Pseudomonas syringae S203 has been identified and purified after overexpression in Escherichia coli. The amino acid composition and the N-terminal sequence of the purified, denatured protein corresponded well with that predicted from the sequence of the inaZ gene. The product of inaZ was also found to be the major component in preparations of ice-nucleating, proteinaceous particles, obtained after extraction with and gel filtration in a mixture of urea and the nondenaturing detergent octyl beta-D-thioglucopyranoside. The activity of these preparations in the absence of added lipid implies that the protein participates directly in the nucleation process.

Molecular cloning of genetic determinants for inhibition of fungal growth by a fluorescent pseudomonad.

Pseudomonas fluorescens HV37a inhibits growth of the fungus Pythium ultimum in vitro. Optimal inhibition is observed on potato dextrose agar, a rich medium. Mutations eliminating fungal inhibition were obtained after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Mutants were classified by cosynthesis and three groups were distinguished, indicating that a minimum of three genes are required for fungal inhibition. Cosmids that contain wild-type alleles of the genes were identified in an HV37a genomic library by complementation of the respective mutants. This analysis indicated that three distinct genomic regions were required for fungal inhibition. The cosmids containing these loci were mapped by transposon insertion mutagenesis. Two of the cosmids were found to contain at least two genes each. Therefore, at least five genes in HV37a function as determinants of fungal inhibition.

Ice nucleation activity of Pseudomonas fluorescens: mutagenesis, complementation analysis and identification of a gene product.

A DNA fragment of 7.5 kb from Pseudomonas fluorescens MS1650 confers an ice nucleation phenotype when cloned in Escherichia coli. This DNA encodes a protein with an apparent mol. wt of 180 kd, which is found in both inner and outer membrane fractions of transformed E. coli cells. Insertion mutations throughout a 3.9-kb region cause deficiency in ice nucleation, and eliminate the 180-kd protein. Complementation is not observed between any pair of mutations, suggesting that the nucleating phenotype is encoded by a single transcriptional unit. Mutations in most parts of the 3.9-kb region are not completely deficient in phenotype: they still generate ice nuclei at low frequency. One insertion mutation was found to generate pseudowild revertants, which had undergone deletions of the entire insertion and some of the adjacent sequence; these could account for the incomplete deficiency. These deletions displayed depressed nucleation temperatures, but their nucleation frequencies were close to that of the wild-type gene.

Comparison of physical and genetic properties of palindromic DNA sequences.

Some viable palindromic DNA sequences were found to cause an increase in the recovery of genetic recombinants. Although these palindromes contained no Chi sites, their presence in cis caused apparent recA+-dependent recombination to increase severalfold. This biological property did not correlate with the physical properties of the palindromes' extrusion of cruciform structures in vitro. Thus, two unrelated palindromes with similar effects on recombination in both Escherichia coli and Pseudomonas syringae displayed quite different kinetics of cruciform formation. In plasmids of native superhelical density, one palindrome underwent rapid cruciform formation at 55 degrees C, whereas the other did not form detectable cruciforms at any temperature. A shorter palindrome with similarly rapid kinetics of cruciform formation did not affect recombination detectably. The lack of a clear relationship between physical and genetic properties was also demonstrated in the case of longer, inviable palindromes. Here we found that the degree of asymmetry required in vivo to rescue a long palindrome from inviability far exceeded that required to kinetically prohibit cruciform extrusion in vitro.