Health status in young adults two decades after pediatric liver transplantation.

We conducted a cross-sectional study of patients who underwent pediatric liver transplant (LT) between 1988 and 1992 to evaluate long-term health status. Survivors completed socio-demographic, medical and Health-Related Quality of Life (HRQOL) surveys by mail including the SF-36v2, PedsQL™4.0 Generic Core Scale, PedsQL™ Cognitive Functioning Scale and PedsQL™3.0 Transplant Module. SF-36 scores were converted to SF6D-based utilities and risk factors for lower outcomes were assessed. Eighty-five of 171 patients had survived. Fifty-six were contacted with a response rate of 66%. Median age at LT was 0.86 years (IQR 0.58-3.0) and 64.3% had biliary atresia. Mean age at survey was 23.0 ± 4.4 years: 62% attended college, 68% lived with parents and 80% of those over 23 were employed. Patient health utilities were lower than norms (0.75 ± 0.12 vs. 0.82 ± 0.18, p < 0.01) and correlated with unemployment (p < 0.042), hospitalizations (p < 0.005) and lower education level (p < 0.016). Lower PedsQL™3.0 Transplant Module and PedsQL™ 4.0 Generic Core Scale scores correlated with unemployment (p = 0.006, p = 0.009) and hospitalizations (p = 0.006, p = 0.02). Pediatric transplant recipients who survive to adulthood have lower physical HRQOL, measurable transplant-related disability and lower health utility. Transplantation is life saving; however, physical and psychological sequelae continue to affect health status up to two decades later.

A novel histone H4 mutant defective in nuclear division and mitotic chromosome transmission.

The histone proteins are essential for the assembly and function of th e eukaryotic chromosome. Here we report the first isolation of a temperature-sensitive lethal histone H4 mutant defective in mitotic chromosome transmission Saccharomyces cerevisiae. The mutant requires two amino acid substitutions in histone H4: a lethal Thr-to-Ile change at position 82, which lies within one of the DNA-binding surfaces of the protein, and a substitution of Ala to Val at position 89 that is an intragenic suppressor. Genetic and biochemical evidence shows that the mutant histone H4 is temperature sensitive for function but not for synthesis, deposition, or stability. The chromatin structure of 2 micrometer circle minichromosomes is temperature sensitive in vivo, consistent with a defect in H4-DNA interactions. The mutant also has defects in transcription, displaying weak Spt- phenotypes. At the restrictive temperature, mutant cells arrest in the cell cycle at nuclear division, with a large bud, a single nucleus with 2C DNA content, and a short bipolar spindle. At semipermissive temperatures, the frequency of chromosome loss is elevated 60-fold in the mutant while DNA recombination frequencies are unaffected. High-copy CSE4, encoding an H3 variant related to the mammalian CENP-A kinetochore antigen, was found to suppress the temperature sensitivity of the mutant without suppressing the Spt- transcription defect. These genetic, biochemical, and phenotypic results indicate that this novel histone H4 mutant defines one or more chromatin-dependent steps in chromosome segregation.

A novel CYP2D6 allele with an abolished splice recognition site associated with the poor metabolizer phenotype.

A novel loss-of function allele of the CYP2D6 gene was characterized in a PM individual using exon-by-exon PCR-SSCP analysis. This allele, we termed CYP2D6(F), harbours four mutations including a new mutation (D6-F) which abolishes the splice acceptor site of the 1st intron and results in a premature stop codon. DNA samples from a large population of healthy unrelated volunteers were tested for D6-F using a PCR-assay we developed for the specific identification of the mutation in genomic DNA. The prevalence of D6-F was very low. However, its identification combined with that of the previously reported gene inactivating mutations would further increase the phenotype prediction rate by genotyping.

An efficient strategy for detection of known and new mutations of the CYP2D6 gene using single strand conformation polymorphism analysis.

To detect mutations in the cytochrome P450 CYP2D6 gene (CYP2D6), we developed a strategy based on single-strand conformation polymorphism (SSCP) analysis of the gene amplified by polymerase chain reaction (PCR). The efficiency of the method was evaluated by analysing DNA samples from extensive metabolizers (EM) and poor metabolizers (PM) of debrisoquine. Haplotypes, alleles and mutations of CYP2D6 had previously been characterized in each individual using PCR assays, Xba I restriction fragment length polymorphism (RFLP) and sequencing. PCR-SSCP results were in complete agreement with those obtained using established methods. All previously characterized mutations were associated with particular shifts in the electrophoretic mobility of DNA fragments allowing their identification. We further tested the efficiency of PCR-SSCP for detecting new CYP2D6 mutations. DNA from a PM subject presumed to carry an unknown non-functional mutant allele of CYP2D6 was amplified and bands with aberrant migration patterns were observed on SSCP gels. Sequence analysis of the corresponding DNA fragments revealed the causative mutations. In this way, a novel non-functional allele of the gene, carrying three previously reported mutations and a new mutation in the third exon which results in a premature termination codon, was characterized. Finally, CYP2D6 SSCP analysis was performed on DNA amplified with fluorescent primers and an automated DNA sequencer was used for SSCP analysis of products. We conclude that the PCR-SSCP approach is a powerful method of identifying simultaneously known and new mutations of the CYP2D6 gene.

Short report: severe malaria associated with blood group.

The ABO blood groups are not linked to the incidence of simple malaria infection but have been associated with rosette formation. In an effort to see if clinically severe malaria is associated with blood group, 489 patients were studied in Zimbabwe. Patients with malaria and group A blood had lower hemoglobin levels and more risk of coma than did infected patients with other blood groups. In this population, severe malaria is associated with blood group.

PIP: Clinical experience in Zimbabwe suggests that severe malaria is more frequent in individuals in the non-O blood groups. In a study conducted by the authors in 1995--a relatively light malaria season--the 27 patients with non-O blood had a mean hemoglobin level of 11.7 g/dl compared with 12.3 g/dl in the 26 patients with group O blood and were more likely to have jaundice or central nervous system symptoms. The present study evaluated 489 patients with positive malaria smears recruited from the Sanyati Baptist Hospital in Kadoma, Zimbabwe, during the 1996 rainy season. Overall, 266 had group O blood, 104 had group A, 103 had group B, and 16 had group AB. The mean hemoglobin levels were 11.8 +or- 2.8, 11.2 +or- 2.6, 11.4 +or- 2.4, and 12.4 +or- 2.7 g/dl, respectively. Coma was more frequent in patients with group A blood (9/104) than in those with non-A blood (11/385). Again, these findings suggest patients with group A blood are at greatest risk of clinically severe malaria. Cerebral malaria has been linked to the ability of Plasmodium falciparum to trigger formation of red blood cell rosettes, especially in those with group A blood. It is unclear, however, whether blood group, through its influence on rosette formation, is causally related to severe malaria or merely serves as a marker for other host-parasite interactions that provoke the severe manifestations of malaria.

Performance of 133 compounds in the lambda prophage induction endpoint of the Microscreen assay and a comparison with S. typhimurium mutagenicity and rodent carcinogenicity assays.

The Microscreen assay was developed as a means of testing very small samples, as in complex mixture fractionation. It is a multi-endpoint assay which utilizes E. coli WP2s(lambda). Exposure takes place to serial dilutions of the test compound in microtitre wells (250 microliters) followed by sampling from wells in which growth has occurred ('non-toxic wells'). Although a number of different endpoints can be measured, only the prophage induction endpoint (the first one developed) has been extensively tested. Results with 133 compounds are presented. These include 111 compounds which have been tested in the S. typhimurium assay and 66 compounds for which both rodent bioassay and S. typhimurium assay data exists. The concordance for the Microscreen assay and the S. typhimurium assay was 71%. For this group of compounds, the sensitivity of the Microscreen assay in detecting carcinogens was 76% compared with 58% for the S. typhimurium assay. However, the S. typhimurium assay was somewhat more specific (69%) compared with the Microscreen (56%). The overall association between carcinogenicity and Microscreen results was statistically significant (p = 0.029), whereas for the S. typhimurium assay the association with carcinogenicity was non-significant (p = 0.086). The Microscreen assay was able to detect halogenated compounds better than the S. typhimurium assay. The Microscreen assay should prove useful in complex mixture fractionation, or in other situations where sample size is limiting.

Treatment of fracture-dislocation of the proximal interphalangeal joint using the Suzuki external fixator.

We describe a simple technique for fracture-dislocations of the proximal interphalangeal joint. Eight fingers with a fracture-dislocation were treated with a self-manufactured dynamic external fixator, allowing early mobilization. The fixator consists of pins and rubbers. The clinical and radiographic outcome was evaluated and recorded. A near-normal function was obtained in four patients. The average total active motion was 82 degrees. Radiographic reduction was maintained. This external fixator is an inexpensive and simple technique for these difficult fracture-dislocations. Early intervention (before two weeks post-trauma) is recommended.

The relationship between malaria and HIV.

OBJECTIVE: To determine if there is an association between HIV and malaria infection. DESIGN: A cross sectional survey. SETTING: Sanyati Rural District, a malarious endemic area of Zimbabwe. SUBJECTS: 338 volunteers aged 15 months to 76 years. MAIN OUTCOME MEASURES: Prevalence of Malaria and HIV. RESULTS: The prevalence of malaria and HIV was 26.6% and 26.3% respectively. There was no association between prevalence of HIV and malaria. CONCLUSION: There is no association between malaria and HIV.

Liver transplantation: living-related donations.

The LRLT procedure appears to be a promising alternative and adjunct to conventional liver transplantation. The advantages to this technique are a donor for each recipient, an improved quality of graft, and a reduction in pretransplant mortality in children. The procedure is not without risks, but the overall benefits seem to outweigh these risks, and only future experience will tell otherwise. Will there continue to be a need for such a procedure? We believe there will be, because the number of candidates needing transplantation will continue to grow and the demand for cadaveric organs will never be met. However, the procedure is extremely labor and resource intensive, and it is our belief that only a handful of large major medical centers will be able to adopt a live-donor program.

Theoretical study of the SNV reaction of trichloroethylene (TCE) and CH3S- as a model for glutathione conjugation of TCE.

Trichloroethylene (TCE), a major environmental pollutant, is activated to mutagenic and nephrotoxic intermediates through a glutathione (GSH) conjugation pathway. Three product isomers of GSH-TCE conjugation, having potentially different toxicities, are theoretically possible: cis- or trans-S-(1, 2-dichlorovinyl)glutathione (cis- or trans-1,2-DCVG, respectively) or 2,2-DCVG. This study involved application of ab initio molecular orbital theory to computing potential energy profiles (PEPs) and predicting product outcome of the reaction of CH3S- with TCE as a model for GSH-TCE conjugation in biological systems. A goal of this study was to determine the extent to which a body of chemical knowledge pertaining to nucleophilic vinylic substitution (SNV) reactions, of which the GSH-TCE conjugation is a representative example, is relevant to this biological conjugation problem. PEPs were computed for all studied species at the HF/6-31+G level of theory; electron correlation effects were estimated at the MP2/6-31+G and MP4/6-31+G levels, and the influence of solvation was estimated using the PS-GVB solvation model. Multiple proposed reaction pathways were considered, including conjugation at the C1 or C2 site on TCE, by in-plane (sigma) or out-of-plane (pi) approach of the nucleophile. Some aspects of the MP2 and HF PEPs were found to differ significantly. However, on the basis of comparison of activation barriers, calculations at all levels of theory predict preference for C2 conjugation over C1 conjugation and formation of the trans-1,2-DCVM product over the cis-1,2-DCVM product. These predictions are consistent with GSH-TCE conjugation results from in vivo experiments. In contrast, relative product energies appear to be a poor indicator of the product outcome for this system. Hence, theoretical consideration of the reaction chemistry in the vicinity of the site of nucleophilic addition appears to be necessary and sufficient to predict the outcome of the enzyme-mediated GSH-TCE conjugation.

An additional allelic variant of the CYP2D6 gene causing impaired metabolism of sparteine.

The identification of a novel CYP2D6 allele from a healthy Caucasian poor metabolizer was achieved by using a previously described polymerase chain reaction/single-strand conformation polymorphism strategy. Among the four point mutations that this allele carries, a missense mutation in exon 1 (212 G-->A or D6-H) seems to be responsible for the loss of CYP2D6 function. Although the mutation D6-H has a low prevalence in a randomly selected population of healthy Caucasians, its identification should further increase the phenotype prediction rate by genotyping.

Structure-activity study of paracetamol analogues: inhibition of replicative DNA synthesis in V79 Chinese hamster cells.

Experimental and theoretical evidence pertaining to cytotoxic and genotoxic activity of paracetamol in biological systems was used to formulate a simple mechanistic hypothesis to explain the relative inhibition of replicative DNA synthesis by a series of 19 structurally similar paracetamol analogues, 5 of which were specifically analyzed for the current study. It was hypothesized that the observed activity variation of the paracetamol analogues was based on the relative abilities of these compounds to undergo H atom loss at the phenolic oxygen, and on the relative stabilities of the resulting free-radical species. Three calculated parameters were found to be relevant--the partial atomic charge on the ring carbon attached to the phenolic oxygen, the partial charge on the phenoxy radical oxygen, and the energy difference between the parent phenolic paracetamol analogue and the corresponding radical dissociation products. The variation in parameter values was significantly correlated with the relative inhibition of DNA synthesis and was easily rationalized in terms of the mechanistic hypothesis proposed. More specifically, competitive reaction with a tyrosyl radical species involving the transfer of a hydrogen atom at the active site of ribonucleotide reductase was suggested as the underlying mechanistic basis for the observed activity variation of the paracetamol analogues. Comparison of calculated parameters for a model tyrosyl species and the paracetamol analogues was entirely consistent with this view.

N-acetyltransferase 2 acetylation polymorphism: prevalence of slow acetylators does not differ between atopic dermatitis patients and healthy subjects.

The genetic polymorphism of human N-acetyltransferase 2 (NAT2) divides the human population into groups with rapid, intermediate and slow acetylator status. Slow acetylator status has been considered a predisposing factor for allergic diseases, lupus erythematosus, toxic epidermal necrolysis or Stevens-Johnson syndrome. The aim of this study was to investigate whether Caucasian patients suffering from atopic dermatitis differed from healthy individuals with regard to the genotype and phenotype of NAT2. Twenty unrelated healthy Caucasian volunteers (9 females and 11 males, aged from 22 to 59 years) and twenty unrelated Caucasian patients suffering from atopic dermatitis (9 females and 11 males, aged between 20 and 54 years) participated in this study. For each one, the NAT2 genotype was determined by polymerase chain reaction with DNA extracted from peripheral blood, using specific primers for the wild-type allele (wt) and the 3 most frequent mutated alleles of NAT2 (C481-->T, G590-->A and G857-->A). The NAT2 phenotype was evaluated with dapsone as a test substrate using high-pressure liquid chromatography. Statistical analysis was performed using the chi(2) test. Phenotype and genotype were distributed as follows: (1) of the healthy subjects, 60% were rapid acetylators (RA) and 40% were slow acetylators (SA); 10% of the RA and 15% of the SA were homozygous, 50% of the RA and 25% of the SA were heterozygous; (2) of the patients, 55% were RA, 40% were SA and 5% were intermediate acetylators (IA); 10% of the RA and 10% of the SA were homozygous, 45% of the RA and 35% of the SA were heterozygous. No significant statistical difference was found between the two groups for genotypes (p = 0.75) or phenotypes (p = 0.60). The phenotyping and genotyping results of healthy subjects were comparable to those found in previous studies. The absence of a significant statistical difference between healthy subjects and atopic dermatitis patients is in contrast to the results of previous studies. Some authors considered that allergic patients are mostly SAs. This could be explained by the fact that we only considered patients suffering from atopic dermatitis whereas, in other studies, patients suffered from different (one or several associated) allergic diseases. NAT2 polymorphism does not differ between patients suffering from atopic dermatitis and healthy subjects. The importance attributed to the SA status, which was previously considered a predisposing factor for allergic diseases such as atopic dermatitis, should be reviewed.

A nonsense mutation in the cytochrome P450 CYP2D6 gene identified in a Caucasian with an enzyme deficiency.

A novel mutation that generates a stop codon in the third exon of the gene encoding the cytochrome P-450 CYP2D6 was identified in a Caucasian having a deficiency of the isozyme, by means of single strand conformation polymorphism analysis of DNA fragments amplified by the polymerase chain reaction, followed by selective sequencing.

Synthesis and characterization of adducts of alachlor and 2-chloro-N-(2,6-diethylphenyl)acetamide with 2'-deoxyguanosine, thymidine, and their 3'-monophosphates.

Adducts of the preemergence herbicide 2-chloro-N-(methoxymethyl)-N-(2,6-diethylphenyl)-acetamide (alachlor) and 2-chloro-N-(2,6-diethylphenyl)acetamide (CDEPA) with 2'-deoxyguanosine, thymidine, 2'-deoxyguanosine 3'-monophosphate, and thymidine 3'-monophosphate have been synthesized and characterized. Under mildly basic conditions alachlor and CDEPA form N-1 adducts with 2'-deoxyguanosine and N-3 adducts with thymidine as a result of chlorine displacement. In addition, alachlor formed an N-7 adduct with 2'-deoxyguanosine, 7-[[(N-(methoxymethyl)-N-(2,6-diethylphenyl)carbamoyl]methyl]guani ne. N-1 adducts of alachlor and CDEPA with 2'-deoxyguanosine 3'-monophosphate and N-3 adducts with thymidine 3'-monophosphate are also described. In addition to spectroscopic data, structural proof included the dephosphorylation of each nucleotide adduct to its corresponding nucleoside adduct by nuclease P1. Alachlor and alachlor adducts but not CDEPA and CDEPA adducts exhibited rotational isomerism as evidenced by proton and 13C NMR studies. These rotamers were attributed to hindered rotation about the shortened N-carbonyl bond. Computational methods employing molecular mechanics and quantum mechanics were used to characterize the structures and energies of these rotamers to account for the patterns of duplicate NMR resonances observed.

Bone attachment to hydroxyapatite coated polymers.

Bone attachment to two classes of hydroxyapatite (HA) coated polymers was evaluated mechanically and histologically. Particulate HA was molded into the surface of thermoplastic implants and cast into the surface of thermoset implants. Coated and uncoated implants of both types were implanted in the distal femurs of rabbits for four and twelve weeks. After sacrifice, the bone/implant interface was evaluated mechanically via a push-out test with a servohydraulic test system. Paired statistical analysis revealed significantly greater shear strengths for coated vs. uncoated implants for both polymer systems at both time periods. Mixed mode of failure occurred with particle removal from the polymer surface and from the bone. One animal at each time period was saved for histology. Histology at both time periods indicated direct bone apposition to the HA coating as compared to a fibrous encapsulation about the uncoated implants. This was confirmed with backscatter scanning electron microscopy and scanning acoustic microscopy. Hydroxyapatite coatings significantly improve the bond strength between polymers and bone by allowing direct bone apposition and some mechanical interlocking with the bone.

Indications for liver transplantation in pediatric patients.

Orthotopic liver transplantation has become a widely accepted therapy for children with end-stage liver disease. Several factors need to be kept in mind when advocating this procedure in pediatric patients. These include the following: (1) Liver transplantation is a high-risk procedure with a 10-15% mortality in the best of circumstances. (2) Recipients require long-term drug administration and have a potential for chronic disability. (3) The procedure is extremely expensive. (4) The long-term survival remains unknown. (5) Newer techniques and procedures need to be carefully evaluated. These factors, and the indications and contraindications for this procedure are discussed in this overview.