Parents in transition: Experiences of parents of young people with a liver transplant transferring to adult services.

Predictors of successful transition from pediatric to adult services include ability to self-manage and engage with healthcare services. Parents have a key role in healthcare management throughout childhood and adolescence including encouraging development of self-management skills in their children. Transition to adult services can be challenging for parents and young people, yet parents' views regarding transition remain largely unexplored. Nine parents of pediatric liver transplant recipients (15.2-25.1 yr) participated in semistructured interviews. Interviews were analyzed using IPA. Analysis revealed three key themes: "emotional impact of transplantation," "protection vs. independence," and "ending relationships and changing roles." Parents expressed the dichotomous nature of the desire to promote independence in their child while still maintaining control and protection, and discussed how changing roles and relationships were difficult to navigate. Parents are important facilitators of young people's development of self-management skills for successful transfer to adult services. Parents should be supported to move from a "managerial" to a "supervisory" role during transition to help young people engage independently with the healthcare team. Findings support the development of interventions for parents to emphasize their role in transition and guide the transfer of self-management skills from parent to young person.

"Are these adult doctors gonna know me?" Experiences of transition for young people with a liver transplant.

Excellent survival rates in paediatric LTx have resulted in increasing numbers of young people transferring from paediatric to adult care. Understanding the mechanisms of successful transition is imperative for ensuring good long-term outcomes and developing services for young people. Semi-structured interviews were conducted with 17 young people (10 females; age range: 15.2-25.1 years). Eight were within 1 year of transferring to adult services; nine had transferred. Interviews were analysed using IPA. Analysis revealed two major themes in both pre- and post-transfer groups: "relationships with healthcare professionals" and "continuity of care." Young people experienced difficulty ending relationships with paediatric clinicians and forming new relationships with adult clinicians. They expressed frustrations over a perceived lack of continuity of care after transfer and a fear of the unknown nature of adult services. The importance of a holistic approach to care was emphasized. Interventions are needed to support young people in transition, particularly in ending relationships in paediatric care and forming new relationships in adult care. Young people need help to develop strategies to cope with the different approaches in adult services. Interventions to provide clinicians with skills to communicate and engage with young people are imperative.

Expression and characterization of human pancreatic preprocarboxypeptidase A1 and preprocarboxypeptidase A2.

We are investigating the potential utility of human carboxypeptidases A in antibody-directed enzyme prodrug therapy (ADEPT). Hybridization screening of a human pancreatic cDNA library with cDNA probes that encoded either rat carboxypeptidase A1 (rCPA1) or carboxypeptidase A2 (rCPA2) was used to clone the human prepro-CPA homologs. After expression of the respective pro-hCPA cDNA in Saccharomyces cerevisiae, the enzymes were purified to homogeneity by a combination of hydrophobic and ion-exchange chromatography. Purified hCPA1 and hCPA2 migrate as a single protein band with M(r) 34,000 when subjected to gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions. Kinetic studies of the purified enzymes with hippuryl-L-phenylalanine resulted in kcat/Km values of 57,000 and 19,000 M-1 s-1 for hCPA1 and hCPA2, respectively. Using the ester substrate, hippuryl-D, L-phenyllactate, we found unique esterase/ peptidase specific activity ratios among hCPA1, hCPA2, rCPA1, and bovine CPA (bCPA) ranging from 13 to 325. Two potential ADEPT substrates, methotrexate-alpha-phenylalanine (MTX-Phe) and methotrexate-alpha-(1-naphthyl)alanine (MTX-naphthylAla) were also analyzed. The kcat/Km values for MTX-Phe were 440,000 and 90,000 M-1 s-1 for hCPA1 and hCPA2, respectively, and for MTX-naphthylAla these values were 1400 and 1,400,000 M-1 s-1 for hCPA1 and hCPA2, respectively. The kinetic data show that hCPA2 has a larger substrate binding site than the hCPA1 enzyme. Differences between hCPA1 and hCPA2 were also observed in thermal stability experiments at 60 degrees C where the half-life for thermal denaturation of hCPA2 is eightfold longer than that for hCPA1. These experiments indicate that hCPA1 and hCPA2 are potential candidates for use in a human-based ADEPT approach.

Selective inhibition of topoisomerases from Pneumocystis carinii compared with that of topoisomerases from mammalian cells.

Type I and II topoisomerase activities were partially purified from Pneumocystis carinii. The catalytic (strand-passing) activities of both enzymes were selectively inhibited by members of a series of dicationic-substituted bis-benzimidazoles compared with those of topoisomerases of mammalian (calf thymus) origin. The most active inhibitors of the parasite enzymes were also highly effective in an in vivo animal model of P. carinii pneumonia. Selected dicationic-substituted bis-benzimidazoles also strongly inhibited the induction of the topoisomerase I- and II-mediated cleavable complex, suggesting that the biologically active DNA minor groove-binding molecules inhibit the enzyme-DNA binding step of the topoisomerase reaction sequence. The apparent selectivities for the parasite enzymes and the low levels of toxicity to mammalian cells for the biologically active bis-benzimidazoles suggest that these compounds hold promise as effective therapeutic agents in the treatment of a life-threatening AIDS-related disease, P. carinii pneumonia.

2,4-Diamino-5-benzylpyrimidines as antibacterial agents. 13. Some alkenyl derivatives with high in vitro activity against anaerobic organisms.

A series of 2,4-diamino-5-(3,5-dialkenyl-4-methoxy- or -4-hydroxybenzyl)pyrimidines was prepared from [(allyloxy)benzyl]pyrimidines by Claisen rearrangements, and the resulting allyl phenols were further modified by methylation and rearrangement to 1-propenyl analogues. Analogous 3,4-dimethoxy-5-alkenyl derivatives were prepared by similar techniques. High in vitro antibacterial activity was obtained against certain anaerobic organisms, such as Bacteroides species and Fusobacterium, which was equal to or better than the control, metronidazole, in several cases. The profile was similar against Neisseria gonorrhoeae and Staphylococcus aureus. The 3,5-bis(1-propenyl)-4-methoxy derivative 8 was 1 order of magnitude more active against Escherichia coli dihydrofolate reductase than its saturated counterpart, and it was also more active than trimethoprim, 1. However, it was considerably less active in vitro against the Gram-negative organisms. The 3,4-dimethoxy-5-alkenyl, -5-alkyl, and -5-alkoxy analogues had very high broad-spectrum antibacterial activity. However, pharmacokinetic studies of four of the compounds in dogs and rats and in vivo studies with an abdominal sepsis model in rats showed no advantages over trimethoprim.

DNA intercalation and inhibition of topoisomerase II. Structure-activity relationships for a series of amiloride analogs.

Among its many properties, amiloride is a DNA intercalator and topoisomerase II inhibitor. Previous work has indicated that the most stable conformation for amiloride is a planar, hydrogen-bonded, tricyclic structure. To determine whether the ability of amiloride to intercalate into DNA and to inhibit DNA topoisomerase II was dependent on the ability to assume a cyclized conformation, we studied the structure-activity relationship for 12 amiloride analogs. These analogs contained structural modifications which could be expected to allow or impede formation of a cyclized conformation. Empirical assays consisting of biophysical, biochemical, and cell biological approaches, as well as computational molecular modeling approaches, were used to determine conformational properties for these molecules, and to determine whether they intercalated into DNA and inhibited topoisomerase II. Specifically, we measured the ability of these compounds to 1) alter the thermal denaturation profile of DNA, 2) modify the hydrodynamic behavior of DNA, 3) inhibit the catalytic activity of purified DNA topoisomerase II in vitro, 4) promote the topoisomerase II-dependent cleavage of DNA, and 5) inhibit functions associated with DNA topoisomerase II in intact cells. Results indicated that only those analogs capable of cyclization could intercalate into DNA and inhibit topoisomerase II. Thus, the ability of amiloride and the 12 analogs studied to intercalate into DNA and to inhibit topoisomerase II appears dependent on the ability to exist in a planar, hydrogen-bonded, tricyclic conformation.

In vitro cleavable-complex assay to monitor antimicrobial potency of quinolones.

Seven quinolones were evaluated to determine whether their ability to generate the DNA gyrase-mediated cleavable complex correlated with their ability to inhibit the catalytic activity of purified DNA gyrase and inhibit the growth of Escherichia coli. The rank order of potency of these drugs in the cleavable-complex assay was essentially the same as in the DNA supercoiling-inhibition assay. It required 2- to 10-fold-lower drug concentrations to generate the cleavable complex than to inhibit E. coli DNA gyrase. With the newer fluoroquinolones, a 25- to 100-fold-greater concentration was required for DNA gyrase inhibition than for cell growth inhibition, suggesting a more subtle interaction between these inhibitors and DNA gyrase than mere enzyme inhibition.

Amiloride intercalates into DNA and inhibits DNA topoisomerase II.

Amiloride is capable of inhibiting DNA synthesis in mammalian cells in culture. Recent evidence indicates that the enzyme, DNA topoisomerase II, is probably required for DNA synthesis to occur in situ. In experiments to determine the mechanism of inhibition of DNA synthesis by amiloride, we observed that amiloride inhibited both the catalytic activity of purified DNA topoisomerase II in vitro and DNA topoisomerase II-dependent cell functions in vivo. Many compounds capable of inhibiting DNA topoisomerase II are DNA intercalators. Thus, we performed studies to determine if and how amiloride bound to DNA. Results indicated that amiloride 1) shifted the thermal denaturation profile of DNA, 2) increased the viscosity of linear DNA, and 3) unwound circular DNA, all behavior consistent with a DNA intercalation mechanism. Furthermore, quantitative and qualitative measurements of amiloride fluorescence indicated that amiloride (a) bound reversibly to purified DNA under conditions of physiologic ionic strength, and (b) bound to purified nuclei in a highly cooperative manner. Lastly, amiloride did not promote the cleavage of DNA in the presence of DNA topoisomerase II, indicating that the mechanism by which amiloride inhibited DNA topoisomerase II was not through the stabilization of a "cleavable complex" formed between DNA topoisomerase II, DNA, and amiloride. The ability of amiloride to intercalate with DNA and inhibit topoisomerase II is consistent with the proposed planar, hydrogen-bonded, tricyclic nature of amiloride's most stable conformation. Thus, DNA and DNA topoisomerase II must be considered as new cellular targets of amiloride action.

Antibacterial activity and mechanism of action of 3'-azido-3'-deoxythymidine (BW A509U).

The thymidine analog 3'-azido-3'-deoxythymidine (BW A509U; azidothymidine [AZT]) had potent bactericidal activity against many members of the family Enterobacteriaceae, including strains of Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Shigella flexneri, and Enterobacter aerogenes. AZT also had bactericidal activity against Vibrio cholerae and the fish pathogen Vibrio anguillarum. AZT had no activity against Pseudomonas aeruginosa, gram-positive bacteria, anaerobic bacteria, Mycobacterium tuberculosis, nontuberculosis mycobacteria, or most fungal pathogens. Several lines of evidence indicated that AZT must be activated to the nucleotide level to inhibit cellular metabolism: AZT was a substrate for E. coli thymidine kinase; spontaneously arising AZT-resistant mutants of E. coli ML-30 and S. typhimurium were deficient in thymidine kinase; and intact E. coli ML-30 cells converted [3H]AZT to its mono-, di-, and triphosphate metabolites. Of the phosphorylated metabolites, AZT-5'-triphosphate was the most potent inhibitor of replicative DNA synthesis in toluene-permeabilized E. coli pol A mutant cells. AZT-treated E. coli cultures grown in minimal medium contained highly elongated cells consistent with the inhibition of DNA synthesis. AZT-triphosphate was a specific DNA chain terminator in the in vitro DNA polymerization reaction catalyzed by the Klenow fragment of E. coli DNA polymerase I. Thus, DNA chain termination may explain the lethal properties of this compound against susceptible microorganisms.

In vitro and in vivo efficacy of the combination trimethoprim-sulfamethoxazole against clinical isolates of methicillin-resistant Staphylococcus aureus.

The in vitro susceptibilities of 16 independent, geographically distinct clinical isolates of methicillin-resistant Staphylococcus aureus to trimethoprim (TMP) in combination with sulfamethoxazole (SMX) were evaluated. Although methicillin-resistant S. aureus strains appear to be universally resistant to SMX, the combination TMP-SMX was found to be synergistic in vitro (in combination, the MICs of both drugs decreased 6- to 25-fold) as well as in vivo (5- to 6-fold reduction in TMP at 50% effective doses).

Increasing resistance to trimethoprim-sulfamethoxazole among isolates of Escherichia coli in developing countries.

Resistance of Escherichia coli to trimethoprim (TMP)-sulfamethoxazole remains at 3%-8% at many medical centers within the United States. In this study a 44% resistance rate was observed among E. coli isolated at a pediatric hospital in Santiago, Chile, and a 40% resistance rate at a general teaching hospital in Bangkok, Thailand. Most isolates were from urinary tract infections and showed high-level resistance (minimal inhibitory concentration of TMP greater than 1,000 micrograms/ml). Nineteen of 35 isolates tested transferred resistance to TMP; most cotransferred resistance to streptomycin and sulfonamides. Dihydrofolate reductase type I was detected by gene probing in 14 of 35 strains. Subsequent investigations in Brazil, Honduras, and Costa Rica revealed that this high rate of resistance was not an isolated phenomenon.

Isolation of a dihydrofolate reductase-deficient mutant of Escherichia coli.

A strain of Escherichia coli was isolated in which dihydrofolate reductase was not detected by an enzyme assay or by competition for antibody. This strain requires methionine, glycine, a purine, and thymidine for growth in addition to the auxotrophic requirements of the parent strain. It was found to be useful as a recipient of plasmids harboring dihydrofolate reductase genes.

3'-Amino-2',3'-dideoxyribonucleosides of some pyrimidines: synthesis and biological activities.

3'-Amino-2',3'-dideoxyribonucleosides of thymine, uracil, and 5-iodouracil (1-3) were synthesized from the corresponding 2'-deoxyribonucleosides via the threo-3'-chloro and the erythro-3'-azido derivatives. Corresponding aminonucleosides of 5-bromouracil, 5-chlorouracil, and 5-fluorouracil (4-6) were synthesized enzymatically with 3'-amino-2',3'-dideoxythymidine as the aminopentosyl donor and thymidine phosphorylase (EC 2.4.2.4) as the catalyst. 3'-Amino-2',3'-dideoxycytidine (7) was synthesized by amination of the 3'-azido precursor of 3'-amino-2',3'-dideoxyuridine. The biological activity of 3'-amino-2',3'-dideoxy-5-fluorouridine (6) was notable among this group of aminonucleosides. It had an ED50 of 10 microM against adenovirus and was not appreciably cytotoxic to mammalian cells in culture. It also had activity against some Gram-positive bacteria but not against a variety of Gram-negative bacteria. The other aminonucleosides (1-5 and 7) lacked or exhibited weak antiviral and antibacterial activities. The only compounds in this group that were appreciably toxic to mammalian cells in culture were the thymidine and deoxycytidine analogues (1 and 7).

Monitoring of plasmid-encoded, trimethoprim-resistant dihydrofolate reductase genes: detection of a new resistant enzyme.

Using-gene-specific radiolabeled probe DNAs, we analyzed 42 clinical bacterial isolates with high-level trimethoprim (Tp) resistance for the presence of a type I or a type II plasmid-specified dihydrofolate reductase (DHFR) gene. Plasmid DNA from 17 strains harbored a type I DHFR, whereas 11 isolates contained plasmids that harbored a type II DHFR structural gene. The plasmid DNAs from five strains appeared to hybridize with both type I and type II DHFR probe DNAs. In addition, eight isolates had type I resistance determinants integrated into the chromosomes, presumably on transposon 7 (Tn7). Among the strains analyzed in this survey, none of the chromosomally located, Tp-insensitive reductases were of the type II class. Both the plasmid and chromosomal DNAs of one isolate showed no homology with either the type I or type II DHFR probe DNA. The plasmid harbored by this strain encoded a "new" Tp-resistant enzyme that differed significantly, both in molecular weight and with respect to trimethoprim and methotrexate inhibition kinetics, from the previously characterized plasmid-associated dihydrofolate reductases.

Molecular mechanisms of resistance to trimethoprim.

Resistance to inhibitors of dihydrofolate reductase arises from a variety of mechanisms involving enzyme alteration, cellular impermeability, enzyme overproduction, inhibitor modification, and loss of binding capacity. The mechanism of greatest clinical importance is the production of plasmid-encoded, trimethoprim-resistant forms of dihydrofolate reductase. At least two different types of these enzymes have been documented. The trimethoprim-resistant reductases differ from all other dihydrofolate reductases in molecular weight, subunit structure, kinetic properties, and binding of inhibitors. Colony hybridization techniques, developed for the detection of plasmid DNA coding for trimethoprim-resistant reductases, enable researchers to evaluate the prevalence and distribution of plasmid-borne resistance. Preliminary results obtained with a series of enzymatically characterized clinical isolates suggest that the colony hybridization technique may provide a convenient epidemiological tool for monitoring the dissemination of plasmid-borne resistance to trimethoprim.

Protein expression in Escherichia coli minicells containing recombinant plasmids specifying trimethoprim-resistant dihydrofolate reductases.

Deoxyribonucleic acid fragments containing the structural genes for several trimethoprim-resistant dihydrofolate reductases from naturally occurring plasmids were inserted into small cloning vehicles. The genetic expression of these hybrid plasmids was studied in purified Escherichia coli minicells. The type I dihydrofolate reductase, encoded by plasmid R483 and residing within transposon 7 (Tn7), had a subunit molecular weight of 18,000. The type II dihydrofolate reductase, specified by plasmid R67, had a subunit molecular weight of 9,000. These two enzymes were antigenically distinct in that anti-type II dihydrofolate reductase (R67) antibody did not cross-react with the type I (R483) protein. The trimethoprim-resistant reductase specified by plasmid R388 had a subunit molecular weight of about 10,500 and was immunologically related to the type II (R67) enzyme. A 9,000 subunit of the dihydrofolate encoded by the transposition element Tn402 was also antigenically related to the R67 reductase.