TCERG1L allelic variation is associated with cisplatin-induced hearing loss in childhood cancer, a PanCareLIFE study.

In children with cancer, the heterogeneity in ototoxicity occurrence after similar treatment suggests a role for genetic susceptibility. Using a genome-wide association study (GWAS) approach, we identified a genetic variant in TCERG1L (rs893507) to be associated with hearing loss in 390 non-cranial irradiated, cisplatin-treated children with cancer. These results were replicated in two independent, similarly treated cohorts (n = 192 and 188, respectively) (combined cohort: P = 5.3 × 10-10, OR 3.11, 95% CI 2.2-4.5). Modulating TCERG1L expression in cultured human cells revealed significantly altered cellular responses to cisplatin-induced cytokine secretion and toxicity. These results contribute to insights into the genetic and pathophysiological basis of cisplatin-induced ototoxicity.

The global spectrum of protein-coding pharmacogenomic diversity.

Differences in response to medications have a strong genetic component. By leveraging publically available data, the spectrum of such genomic variation can be investigated extensively. Pharmacogenomic variation was extracted from the 1000 Genomes Project Phase 3 data (2504 individuals, 26 global populations). A total of 12 084 genetic variants were found in 120 pharmacogenes, with the majority (90.0%) classified as rare variants (global minor allele frequency <0.5%), with 52.9% being singletons. Common variation clustered individuals into continental super-populations and 23 pharmacogenes contained highly differentiated variants (FST>0.5) for one or more super-population comparison. A median of three clinical variants (PharmGKB level 1A/B) was found per individual, and 55.4% of individuals carried loss-of-function variants, varying by super-population (East Asian 60.9%>African 60.1%>South Asian 60.3%>European 49.3%>Admixed 39.2%). Genome sequencing can therefore identify clinical pharmacogenomic variation, and future studies need to consider rare variation to understand the spectrum of genetic diversity contributing to drug response.

Modified diadenosine tetraphosphates with dual specificity for P2Y1 and P2Y12 are potent antagonists of ADP-induced platelet activation.

BACKGROUND: Diadenosine 5',5'''-P(1),P(4)-tetraphosphate (Ap(4)A), a natural compound stored in platelet dense granules, inhibits ADP-induced platelet aggregation. Ap(4)A inhibits the platelet ADP receptors P2Y(1) and P2Y(12), is a partial agonist of P2Y(12), and is a full agonist of the platelet ATP-gated ion channel P2X1. Modification of the Ap(4)A tetraphosphate backbone enhances inhibition of ADP-induced platelet aggregation. However, the effects of these Ap(4)A analogs on human platelet P2Y(1), P2Y(12) and P2X1 are unclear. OBJECTIVE: To determine the agonist and antagonist activities of diadenosine tetraphosphate analogs towards P2Y(1), P2Y(12), and P2X1. METHODS: We synthesized the following Ap(4)A analogs: P(1),P(4)-dithiotetraphosphate; P(2),P(3)-chloromethylenetetraphosphate; P(1)-thio-P(2),P(3)-chloromethylenetetraphosphate; and P(1),P(4)-dithio-P(2),P(3)-chloromethylenetetraphosphate. We then measured the effects of these analogs on: (i) ADP-induced platelet aggregation; (ii) P2Y(1)-mediated changes in cytosolic Ca(2+); (iii) P2Y(12)-mediated changes in vasodilator-stimulated phosphoprotein phosphorylation; and (iv) P2X1-mediated entry of extracellular Ca(2+). RESULTS: Ap(4)A analogs with modifications in the phosphate backbone inhibited both P2Y(1) and P2Y(12), and showed no agonist activity towards these receptors. The dithio modification increased inhibition of P2Y(1), P2Y(12), and platelet aggregation, whereas the chloromethylene modification increased inhibition of P2Y(12) and platelet aggregation, but decreased P2Y(1) inhibition. Combining the dithio and chloromethylene modifications increased P2Y(1) and P2Y(12) inhibition. As compared with Ap(4)A, each modification decreased agonist activity towards P2X1, and the dual modification completely eliminated P2X1 agonist activity. CONCLUSIONS: As compared with Ap(4)A, tetraphosphate backbone analogs of Ap(4)A have diminished activity towards P2X1 but inhibit both P2Y(1) and P2Y(12) and, with greater potency, inhibit ADP-induced platelet aggregation. Thus, diadenosine tetraphosphate analogs with dual receptor selectivity may have potential as antiplatelet drugs.

Psychiatric genetics in South Africa: cutting a rough diamond.

Psychiatric disorders place a considerable healthcare burden on South African society. Incorporating genetic technologies into future treatment plans offers a potential mechanism to reduce this burden. This review focuses on psychiatric genetic research that has been performed in South African populations with regards to obsessive-compulsive disorder, schizophrenia and bipolar disorder. Preliminary findings from these studies suggest that data obtained in developed countries cannot necessarily be extrapolated to South African population groups. Psychiatric genetic studies in South Africa seem to involve relatively low-cost methodologies and only a limited number of large national collaborative studies. Future research in South Africa should therefore aim to incorporate high-throughput technologies into large scale psychiatric studies through the development of collaborations. On a global level, the vast majority of psychiatric genetic studies have been performed in non-African populations. South Africa, as the leading contributor to scientific research in Africa, may provide a foundation for addressing this disparity and strengthening psychiatric genetic research on the continent. Although the elucidation of the genetic architecture of psychiatric disorders has proved challenging, examining the unique genetic profiles found in South African populations could provide valuable insight into the genetics of psychiatric disorders.

Thymidine kinase and uridine-cytidine kinase from Entamoeba histolytica: cloning, characterization, and search for specific inhibitors.

Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Although anti-amoebic drugs such as metronidazole, emetine, chloroquine and nitazoxanide are generally effective, there is always potential for development of drug resistance. In order to find novel targets to control E. histolytica proliferation we cloned, expressed and purified thymidine kinase (Eh-TK) and uridine-cytidine kinase (Eh-UCK) from E. histolytica. Eh-TK phosphorylates thymidine with a Km of 0.27 microm, whereas Eh-UCK phosphorylates uridine and cytidine with Km of 0.74 and 0.22 mM, respectively. For both enzymes, ATP acts as specific phosphate donor. In order to find alternative treatments of E. histolytica infection we tested numerous nucleoside analogues and related compounds as inhibitors and/or substrates of Eh-TK and Eh-UCK, and active compounds against E. histolytica in cell culture. Our results indicate that inhibitors or alternative substrates of the enzymes, although partially reducing protozoan proliferation, are reversible and not likely to become drugs against E. histolytica infections.

Improving the quality of outpatient care for older patients with diabetes: lessons from a comparison of rural and urban communities.

OBJECTIVE: Our goal was to compare the quality of diabetic care received by patients in rural and urban communities. STUDY DESIGN: We performed a retrospective analysis of claims data captured by the Medicare program. POPULATION: We included all fee-for-service Medicare patients 65 years and older living in the state of Washington who had 2 or more physician encounters for diabetes care during 1994. OUTCOME MEASURES: The outcomes were the extent to which patients received 3 specific recommended services: glycated hemoglobin determination, cholesterol measurement, and eye examination. RESULTS: A total of 30,589 Medicare patients (8.4%) were considered to have diabetes; 29.1% lived in rural communities. Generalists provided most diabetic care in all locations. Patients living in small rural towns received almost half their outpatient care in larger communities. Patients living in large rural towns remote from metropolitan areas were more likely to have received the recommended tests than patients in all other groups. Patients who saw an endocrinologist at least once during the year were more likely to have received the recommended tests. CONCLUSIONS: Large rural towns may provide the best conditions for high-quality care: They are vibrant, rapidly growing communities that serve as regional referral centers and have an adequate-but not excessive-supply of both generalist and specialist physicians. Generalists provide most diabetic care in all settings, and consultation with an endocrinologist may improve adherence to guidelines.

Effects of stress management on pain behavior in rheumatoid arthritis.

OBJECTIVE: To examine the effects of stress management training on pain behavior exhibited by persons with rheumatoid arthritis (RA) and the relationship of change in pain behavior with certain patient characteristics as well as change in self-reported levels of pain. METHODS: Patients with RA (n = 131) were randomly assigned to 1 of 3 groups: a stress management group, an attention control group, or a standard care control group. The stress management and attention control groups received a 10-week intervention followed by a 15-month maintenance phase. RESULTS: The 3 groups did not differ significantly in the change in pain behavior at any of the assessment periods. However, persons with RA who had less disease activity tended to exhibit positive changes in pain behavior over time. Changes in self-reported pain were not significantly related to changes in pain behavior. CONCLUSION: The results indicate that stress management interventions do not reduce total pain behaviors exhibited by persons with RA. Changes in pain behaviors appear to be related to disease activity, age, and disease duration, but not to changes in self-reported measures of pain.

Novel nonsubstrate inhibitors of human thymidine phosphorylase, a potential target for tumor-dependent angiogenesis.

Thymidine phosphorylase/platelet-derived endothelial cell growth factor (TP/PD-ECGF) is an enzyme involved in thymidine metabolism and homeostasis, and its catalytic activity appears to play an important role in angiogenesis. Here we describe the cloning and expression of a His-tagged human TP/PD-ECGF and its assay with uracil and thymine analogues. We present the design, synthesis, and biological evaluation of novel 6-(phenylalkylamino)uracil derivatives which, at micromolar concentrations, inhibit both catabolic and anabolic reactions of human TP in vitro. These base analogues are not converted by the enzyme into the nucleoside form, thus representing pure nonsubstrate inhibitors of the enzyme.

In vitro antimicrobial activities of novel anilinouracils which selectively inhibit DNA polymerase III of gram-positive bacteria.

The 6-anilinouracils are novel dGTP analogs that selectively inhibit the replication-specific DNA polymerase III of gram-positive eubacteria. Two specific derivatives, IMAU (6-[3'-iodo-4'-methylanilino]uracil) and EMAU (6-[3'-ethyl-4'-methylanilino]uracil), were substituted with either a hydroxybutyl (HB) or a methoxybutyl (MB) group at their N3 positions to produce four agents: HB-EMAU, MB-EMAU, HB-IMAU, and MB-IMAU. These four new agents inhibited Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus faecalis, and Enterococcus faecium. Time-kill assays and broth dilution testing confirmed bactericidal activity. These anilinouracil derivatives represent a novel class of antimicrobials with promising activities against gram-positive bacteria that are resistant to currently available agents, validating replication-specific DNA polymerase III as a new target for antimicrobial development.

The effect of the doctor-patient relationship on emergency department use among the elderly.

OBJECTIVES: This study sought to determine the rate of emergency department use among the elderly and examined whether that use is reduced if the patient has a principal-care physician. METHODS: The Health Care Financing Administration's National Claims History File was used to study emergency department use by Medicare patients older than 65 years in Washington State during 1994. RESULTS: A total of 18.1% of patients had 1 or more emergency department visits during the study year; the rate increased with age and illness severity. Patients with principal-care physicians were much less likely to use the emergency department for every category of disease severity. After case mix, Medicaid eligibility, and rural/urban residence were controlled for, the odds ratio for having any emergency department visit was 0.47 for patients with a generalist principal-care physician and 0.58 for patients with a specialist principal-care physician. CONCLUSIONS: The rate of emergency department use among the elderly is substantial, and most visits are for serious medical problems. The presence of a continuous relationship with a physician--regardless of specialty--may reduce emergency department use.

Stress management in rheumatoid arthritis: what is the underlying mechanism?

OBJECTIVE: To test whether change in cognitive-behavioral variables (such as self-efficacy, coping strategies, and helplessness) is a mediator in the relation between cognitive behavior therapy and reduced pain and depression in persons with rheumatoid arthritis (RA). METHODS: A sample of patients with RA who completed a stress management training program (n = 47) was compared to a standard care control group (n = 45). A path analysis testing a model including direct effects of comprehensive stress management training on pain and depression and indirect effects via change in cognitive-behavioral variables was conducted. RESULTS: The path coefficients for the indirect effects of stress management training on pain and depression via change in cognitive-behavioral variables were statistically significant, whereas the path coefficients for the direct effects were found not to be statistically significant. CONCLUSION: Decreases in pain and depression following stress management training are due to beneficial changes in the arenas of self-efficacy (the belief that one can perform a specific behavior or task in the future), coping strategies (an individual's confidence in his or her ability to manage pain), and helplessness (perceptions of control regarding arthritis). There is little evidence of additional direct effects of stress management training on pain and depression.

Synthesis and molecular modeling of novel HSV1 uracil-DNA glycosylase inhibitors.

In a recent paper the first selective inhibitors of HSV1 uracil-DNA glycosylase (UDG) acting in the micromolar range have been reported. A 28.5 kDa catalytic fragment of HSV1 UDG has been crystallized in the presence of uracil, and the structure was recently solved. Starting with the optimized model of binding between 6-(4'-n-octylanilino)uracil (octAU) and UDG some new derivatives have been predicted to be active. In vitro studies with the novel synthetized compounds confirm the plausibility of the model and define the structure features for UDG inhibitors.

Inhibitors of DNA polymerase III as novel antimicrobial agents against gram-positive eubacteria.

6-Anilinouracils are selective inhibitors of DNA polymerase III, the enzyme required for the replication of chromosomal DNA in gram-positive bacteria (N. C. Brown, L. W. Dudycz, and G. E. Wright, Drugs Exp. Clin. Res. 12:555-564, 1986). A new class of 6-anilinouracils based on N-3 alkyl substitution of the uracil ring was synthesized and analyzed for activity as inhibitors of the gram-positive bacterial DNA polymerase III and the growth of gram-positive bacterial pathogens. Favorable in vitro properties of N-3-alkyl derivatives prompted the synthesis of derivatives in which the R group at N-3 was replaced with more-hydrophilic methoxyalkyl and hydroxyalkyl groups. These hydroxyalkyl and methoxyalkyl derivatives displayed K(i) values in the range from 0.4 to 2.8 microM against relevant gram-positive bacterial DNA polymerase IIIs and antimicrobial activity with MICs in the range from 0.5 to 15 microg/ml against a broad spectrum of gram-positive bacteria, including methicillin-resistant staphylococci and vancomycin-resistant enterococci. Two of these hydrophilic derivatives displayed protective activity in a simple mouse model of lethal staphylococcal infection.

Molecular modeling and synthesis of inhibitors of herpes simplex virus type 1 uracil-DNA glycosylase.

We recently reported the properties of the first selective inhibitors of herpes simplex virus type 1 (HSV1) uracil-DNA glycosylase (UDG), an enzyme of DNA repair that has been proposed to be required for reactivation of the virus from latency. 6-(4-Octylanilino)uracil (octAU) was the most potent inhibitor among a series of 6-(4-alkylanilino)uracils, acting in the micromolar range and without effect against human UDG. A 28.5-kDa catalytic fragment of HSV1 UDG has been crystallized in the presence of uracil, and the structure was recently solved. We have used the coordinates of this structure in order to study interaction of our inhibitors with the enzyme, and a model of binding between octAU and UDG has been derived. Starting with the optimized model, the activity of several octAU analogues was predicted, and the values compared favorably with experimental results found for the synthetic compounds. Several hydrophilic derivatives were predicted and found to be active as UDG inhibitors. These compounds will be useful to determine if UDG, like the viral thymidine kinase, is required for reactivation of HSV1 from latency in nerve cells.

6-Anilinouracil-based inhibitors of Bacillus subtilis DNA polymerase III: antipolymerase and antimicrobial structure-activity relationships based on substitution at uracil N3.

6-Anilinouracils (6-AUs) are dGTP analogues which selectively inhibit the DNA polymerase III of Bacillus subtilis and other Gram-positive bacteria. To enhance the potential of the 6-AUs as antimicrobial agents, a structure-activity relationship was developed involving substitutions of the uracil N3 position in two 6-AU platforms: 6-(3,4-trimethyleneanilino)uracil (TMAU) and 6-(3-ethyl-4-methylanilino)uracil (EMAU). Series of N3-alkyl derivatives of both 6-AUs were synthesized and tested for their ability to inhibit purified B. subtilis DNA polymerase III and the growth of B. subtilis in culture. Alkyl groups ranging in size from ethyl to hexyl enhanced the capacity of both platforms to bind to the polymerase, and with the exception of hexyl, they also significantly enhanced their antimicrobial potency. N3 substitution of the EMAU platform with more hydrophilic hydroxyalkyl and methoxyalkyl groups marginally enhanced anti-polymerase III activity but enhanced antibacterial potency severalfold. In sum, the results of these studies indicate that the ring N3 of 6-anilinouracils can tolerate substituents of considerable size and structural variety and, thus, can be manipulated to significantly enhance the antibacterial potency of this novel class of polymerase III-specific inhibitors.

A confirmatory factor analysis of the Center for Epidemiologic Studies Depression Scale in rheumatoid arthritis patients: additional evidence for a four-factor model.

OBJECTIVE: To examine the factor structure of the Center for Epidemiologic Studies Depression Scale (CES-D) in a sample of patients with rheumatoid arthritis (RA), testing all of the alternative models suggested by the previous evidence. METHODS: The CES-D was administered to a group of RA patients (n = 685) during a structured telephone interview. The telephone interview was repeated 6 months later (n = 537) and 12 months later (n = 453). Confirmatory factor analyses were conducted to test alternative models. RESULTS: The correlated 4-factor model and the second-order 4-factor model were the best fitting models. CONCLUSION: The factor structure of the CES-D previously found in the general population was replicated in an RA sample. The results are consistent with previous evidence of criterion contamination in the CES-D when used in an RA sample and provide support for the view that a single summary score may not be the most informative index of the CES-D.

Sensitivity of bacteriophage RB69 DNA polymerase to N2-(p-n-butylphenyl)-2'-deoxyguanosine nucleotides.

The response of bacteriophage RB69 DNA polymerase to N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP), related nucleotides and non-nucleoside inhibitors was measured and compared to values obtained for the closely related DNA polymerase from bacteriophage T4. Both enzymes showed similar responses to inhibitors in terms of Ki values and the ability to utilize BuPdGTP as a substrate. These results provide the relevance of using the recent crystal structure of RB69 DNA polymerase for analysis of BuPdGTP/B family DNA polymerase interactions.

Synthesis and biochemical study of N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-(alpha,beta-imido)triphosphate (BuPdGMPNHPP): a non-substrate inhibitor of B family DNA polymerases.

BuPdGMPNHPP was synthesized and assayed as a non-incorporable inhibitor of B family DNA polymerases. The derivative was synthesized by preparation of the imidophosphorane of BuPdG followed by reaction with orthophosphate using the imidazolide method. BuPdGMPNHPP inhibited human DNA polymerase alpha and T4 DNA polymerase 10 and 3.5-times more potently than BuPdGTP, respectively, and was not a substrate for either enzyme. BuPdGMPNHPP acts as an active site affinity probe that could find use in co-crystallization trials of B family DNA polymerases.