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PubMed1997

Measuring the quality of health care: state of the art.

BACKGROUND: September 12-13, 1996, in Washington, DC, the Institute of Medicine, as part of its Special Initiative on Health Care Quality, held an invitational conference to illustrate exemplary quality measurement and to discuss the results. Patient reports, innovative uses of outcome measures for quality improvement, risk adjustment, assessment in integrated health plans and health care settings, clinical guidelines, and projects on disseminating information on quality measurement techniques and tools were among the topics represented. IMPROVING MEDICATION USE: Brent James described studies undertaken to reduce adverse drug events (ADEs). When implementing any measurement system where error is a possible factor, it is important to emphasize identifying problems for the purpose of learning, not judgment. Lucian Leape agreed that staff involved must feel that the purpose of the study is to examine system problems, not individuals' mistakes. David Classen described a nonproprietary computerized disease-management program designed to reduce ADEs in infectious diseases. "A QUALITY VISION": Robert Brook said that the relationship between cost or resources devoted to care and quality is not well understood and is certainly not simple. He also said that although investments in measurement strategies are needed to make them better, that doesn't mean we shouldn't attempt to use the measurements we have now. CLOSING ADDRESS: Mark Chassin said that the presentations at the conference provided evidence that should allow us to conclude beyond a reasonable doubt that quality can be measured-with a degree of scientific precision equal to that of most of the measures used to take care of patients every day.

Health Services Research↗

PubMed1999

Detecting adverse drug reactions to improve patient outcomes.

Adverse drug reactions and inappropriate administration of medications account for poor outcomes for patients. They place patients in life-threatening situations, lead to increased health care costs, extend length of stay in hospitals, as well as increasing litigation. This paper will highlight the incidence of adverse drug events (ADE) in health care and show the low rate of detection within conventional medical records. I will also show how electronic medical records (EMR) improve detection of ADE, enhance clinician compliance to their management, improve patient outcomes, and reduce health care costs.

Adverse Drug Reaction Reporting Systems↗

PubMed2006

How many hospital pharmacy medication dispensing errors go undetected?

BACKGROUND: Hospital pharmacies dispense large numbers of medication doses for hospitalized patients. A study was conducted at an academic tertiary care hospital to characterize the incidence and severity of medication dispensing errors in a hospital pharmacy. METHODS: Direct observation of dispensing processes was undertaken to determine presence of errors with review by a physician panel to determine severity. RESULTS: A total of 140,755 medication doses filled by pharmacy technicians were observed during a seven-month period, and 3.6% (5075) contalned errors. The hospital pharmacist detected only 79% of these errors during routine verification; thus, 0.75% of doses filled would have left the phannacy with undetected errors. Overall, 23.5% of undetected errors were potential adverse drug events (ADEs), of which 28% were serious and 0.8% were life threatening. The most common potential ADEs were incorrect medications (36%), incorrect strength (35%), and incorrect dosage form (21%). DISCUSSION: Given the volume of medications dispensed, even a low rate of drug distribution process translates into a large number of errors with potential to harm patients. Pharmacy distribution systems require further process redesign to achieve the highest possible level of safety and reliability.

Academic Medical Centers↗

PubMed2002

Regional and temporal changes in AIDS in Europe before HAART.

In a prospective observational study 4,485 patients from 46 clinical centres in 17 European countries were followed between April 1994 and November 1996. Information on AIDS-defining events (ADEs) were collected together with basic demographic data, treatment history and laboratory results. The centres were divided into four geographical regions (north, central, south-west and south-east) so that it was possible to identify any existing regional differences in ADEs. The regional differences that we observed included a higher risk of all forms of Mycobacterium tuberculosis infections (Tb) and wasting disease in the south-west and an increased risk of infections with the Mycobacterium avium complex (MAC) in the north. In Cox multivariable analyses, where north was used as the reference group, we observed hazard ratios of 6.87, 7.77, 2.29 and 0.16 (P < 0.05 in all cases) for pulmonary Tb, extrapulmonary Tb, wasting disease and MAC respectively in the south-west. Pneumocystis carinii pneumonia (PCP) was less commonly diagnosed in the central region (RH = 0.51, 95% CI 0 32-0.79, P = 0.003) and most common in the south-east (RH = 1.04, 95% CI 0.71-1.51, P = 0.85). Comparisons with a similar 'AIDS in Europe' study that concentrated on the early phase of the epidemic reveal that most of the regional differences that were observed in the 1980s still persist in the mid-1990s.

AIDS-Related Opportunistic Infections↗

PubMed2000

Mechanisms of inactivation of human S-adenosylhomocysteine hydrolase by 5',5',6',6'-tetradehydro-6'-deoxy-6'-halohomoadenosines.

In an effort to design more specific and potent inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase, we investigated the mechanisms by which 5',5',6', 6'-tetradehydro-6'-deoxy-6'-halohomoadenosines (X = Cl, Br, I) inactivated this enzyme. The 6'-chloro (a) and 6'-bromo (b) acetylenic nucleoside analogues produced partial ( approximately 50%) loss of enzyme activity with a concomitant ( approximately 50%) reduction of E-NAD(+) to E-NADH. In addition, Ade and halide ions were released from the inhibitors in amounts suggestive of a process involving enzyme catalysis. AdoHcy hydrolase, which was inactivated with compound a, was shown to contain 2 mol of the inhibitor covalently bound to Lys318 of two subunits of the homotetramer. These data suggest that the enzyme-mediated water addition at the 5' position of compound a or b produces an alpha-halomethyl ketone intermediate, which is then attacked by a proximal nucleophile (i.e., Lys318) to form the enzyme-inhibitor covalent adduct (lethal event); in a parallel pathway (nonlethal event), addition of water at the 6' position produces an acyl halide, which is released into solution and chemically degrades into Ade, halide ion, and sugar-derived products. In contrast, compound c completely inactivated AdoHcy hydrolase by converting 2 equiv of E-NAD(+) to E-NADH and causing the release of 2 equiv of E-NAD(+) into solution. Four moles of the inhibitor was shown to be tightly bound to the tetrameric enzyme. These data suggest that compound c inactivates AdoHcy hydrolase by a mechanism similar to the acetylenic analogue of Ado described previously by Parry et al. [(1991) Biochemistry 30, 9988-9997].

Adenine↗

PubMed1994

Purification of, generation of monoclonal antibodies to, and mapping of phosphoribosyl N-formylglycinamide amidotransferase.

5'-Phosphoribosyl N-formylglycinamide (FGAR) amidotransferase (EC 6.3.5.3) catalyzes the fourth reaction in the de novo synthesis of purines, that is, the conversion of FGAR to 5'-phosphoribosyl N-formylglycinamidine (FGAM). This is the only step of the pathway for which a vertebrate gene has not been cloned. FGAR amidotransferase has been highly purified from Chinese hamster ovary (CHO) cells, and this preparation has been used to generate monoclonal antibodies in mice. Two of these antibodies, designated BD4 and DD2, have been shown to recognize a 150-kDa protein in CHO-K1 cells that is of very low abundance in Ade-B cells, a CHO line in which FGAR amidotransferase activity is undetectable. Furthermore, the protein recognized by these antibodies is 5-10-fold more abundant in Azr cells. The CHO Azr cell line was made resistant to azaserine, a potent inhibitor of FGAR amidotransferase, and displays a 5-10-fold increase in FGAR amidotransferase activity over the parental K1 line. FGAR amidotransferase activity and the 150-kDa protein recognized by both monoclonal antibodies were found to immunoprecipitate concomitantly using antibody BD4. Monoclonal antibody DD2 cross-reacted with a human protein of identical molecular mass. A number of Ade-B/human hybrid cells were generated by somatic cell fusion and subsequent 5-bromo-2-deoxyuridine segregation. Analysis of these lines, together with two independently generated human/mouse hybrid cell lines, by both cytogenetics and immunoblotting with antibody DD2 revealed that the human FGAR amidotransferase gene is located on chromosome 17p.

Animals↗

PubMed1994

(E)-5',6'-didehydro-6'-deoxy-6'-fluorohomoadenosine: a substrate that measures the hydrolytic activity of S-adenosylhomocysteine hydrolase.

(E)-5',6'-Didehydro-6'-deoxy-6'-fluorohomoadenosine (EDDFHA), which is a poor substrate for the oxidative activity of S-adenosyl-L-homocysteine (AdoHcy) hydrolase and thus a poor mechanism-based inhibitor was shown to be a good substrate for the hydrolytic activity of this enzyme. Incubation of EDDFHA with AdoHcy hydrolase (NAD+ form) produces a large molar excess of hydrolytic products [e.g., fluoride ion, adenine (Ade) derived from chemical degradation of homoadenosine 6'-carboxaldehyde (HACA), and 6'-deoxy-6'-fluoro-5'-hydroxyhomoadenosine (DFHHA)] accompanied by a slow irreversible inactivation of the enzyme. The enzyme inactivation was shown to be time-dependent, biphasic, and concomitant with the reduction of the enzyme-bound NAD+ (E.NAD+) to E-NADH. The reaction of EDDFHA with AdoHcy hydrolase was shown to proceed by three pathways: pathway a, water attack at the 6'-position of EDDFHA and elimination of fluoride ion results in the formation of HACA, which degrades chemically to form Ade; pathway b, water attack at the 5'-position of EDDFHA results in the formation of DFHHA; and pathway c, oxidation of EDDFHA results in formation of the NADH form of the enzyme (inactive) and 3'keto-EDDFHA, which could react with water at either the C5' or C6' positions. The partition ratios among the three pathways were determined to be k3':k6':k5' = 1:29:79 with one lethal event (enzyme inactivation) occurring every 108 nonlethal turnovers.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗

PubMed1982

S-Adenosylhomocysteinase: mechanism of inactivation by 2'-deoxyadenosine and interaction with other nucleosides.

S-Adenosylhomocysteinase (SAHase), a tetrameric enzyme, is inactivated by 2'-deoxyadenosine (2'dAdo) in a time-dependent process [Hirshfield, M. S. (1979) J. Biol. Chem. 254, 22-25]. It has been proposed that inactivation involves oxidation of 2'dAdo at C-3' by enzyme-bound nicotinamide adenine dinucleotide (NAD), subsequent proton abstraction at C-2', and elimination of adenine. This results in irreversible formation of enzyme-bound NADH and of adenine (Ade) and inactivation [Abeles, R. H., TAshjian, A. H., Jr., & Fish, S (1980) Biochem. Biophys. Res. Commun. 95, 612-617]. It has now been established that upon inactivation of SAHase with deoxy[2'(R)-3H]adenosine, 3H2O is formed. This is consistent with the proposed mechanism and of 3H2O release shows that maximally two of the four subunits participate in the reaction that results in 3H2O release. Reaction of SAHase with 2'dAdo results in reduction of two of the enzyme-bound NAD molecules. However, all four NAD molecules can be reduced by NaBH4, but only two are reduced to C-4 NADH. When the enzyme is inactivated with adenine-labeled 2'dAdo, radioactivity corresponding to 0.5-1.0 mumol of 2'dAdo binds tightly per micromole of subunit. This radioactive material is not removed from the enzyme by extensive dialysis but can be displaced by unlabeled 2'dAdo or Ade. After denaturation of the complex, radioactive material is released. Of this material 80-90% is adenine and less than 1% 2'dAdo. 2'dAdo also binds tightly to the enzyme reduced with NaBH4. Upon denaturation mostly adenine (80-90%) is released. Reaction of [2'-3H]2'dAdo with enzyme reduced with NaBH4 does not result in 3H2O formation. We conclude that the enzyme catalyzes the release of adenine from 2'dAdo by two mechanisms: One involves formation of 3'keto-2'dAdo and subsequent elimination of adenine. The other does not involve oxidation of 2'dAdo and probably is a hydrolytic process. It is proposed that the ability of the enzyme to carry out the hydrolytic process is a direct consequence of the manner in which 2'dAdo as well as the normal substrate binds to the enzyme, i.e., hydrogen-bond interaction of the protein with the adenine moiety and distortion of the ribose ring. When adenine-labeled adenosine is added to the enzyme, radioactivity corresponding to 0.5 mumol/mumol of subunit is associated with the protein after gel filtration. Of the radioactive material bound to the protein, 20% is adenine, 15% is adenosine, and the remaining radioactivity is present in unidentified compounds. The adenine bound to the enzyme does not participate in the catalytic process, and we conclude that it is bound to two of the subunits that do not participate in catalysis. Possible, these two subunits have a regulatory function. SAHase probably consists of two nonequivalent pairs of subunits. Only one pair participates in catalysis, but all four subunits probably bind Ado and 2'dAdo. We have confirmed the fact that the carbocyclic analogue of adenosine inactivates SAHase [Guranowski, A., Montgomery, J. A., 110-115]...

Adenine↗

PubMed1987

Two-dimensional 1H NMR of gene 5 protein indicates that only two aromatic rings interact significantly with oligodeoxynucleotide bases.

The interaction of gene 5 protein (G5P) with oligodeoxynucleotides is investigated by 1H NMR methods, principally two-dimensional nuclear Overhauser effect spectroscopy (NOESY). Aromatic resonances of G5P are specifically assigned from crystallographic data, while the low-field resonances of nucleotides are assigned with sequential or other procedures. Chemical shift changes that accompany binding of d(pA)4, d(A)4, d(pT)4, and d(pA)8, combined with specific protein-nucleotide nuclear Overhauser effects (NOEs) obtained from NOESY spectra, suggest that Phe-73 and Tyr-26 are the only aromatic residues that stack significantly with nucleotide bases. Chemical shift data also imply a role for Leu-28, though this has not been confirmed with intermolecular NOEs. Binding of all four oligonucleotides causes marked upfield movements (0.1-0.6 ppm) of G5P NOESY cross peaks belonging to Tyr-26, Leu-28, and Phe-73. Most other G5P spin systems, notably those of Tyr-34 and Tyr-41, do not appear to be significantly affected. In the d(pA)4-G5P complex an intermolecular NOE is observed between Tyr-26 and H1' of Ade-1, while Phe-73 has NOEs with the H2, H8, and H1' protons of Ade-2 and -3. Intramolecular NOEs seem to follow a similar pattern in the partially cooperative d(pA)8-G5P complex, though specific nucleotide resonance assignments are not possible in this case. Binding causes relatively small chemical shift changes for the base resonances in adenylyl nucleotides, suggesting that there is some, but not complete, unstacking of the bases.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMedDate not supplied

Development of auxotrophic agrobacterium tumefaciens for gene transfer in plant tissue culture.

Auxotrophic strains of Agrobacterium tumefaciens were generated for use in liquid co-culture with plant tissue for transient gene expression. Twenty-one auxotrophs were recovered from 1,900 tetracycline-resistant insertional mutants generated with a suicide vector transposon mutagenesis system. Twelve of these auxotrophs were characterized on a nutrient matrix. Isolates were screened for growth in plant cell and root culture, and three auxotrophs were identified that had limited growth: adenine (ade-24), leucine (leu-27), and cysteine (cys-32). Ade-24 displayed poor T-DNA delivery in a transient expression test delivering GUS from a binary vector, while cys-32 displayed the best ability to deliver DNA of these three auxotrophs. The growth yield of cys-32 on cysteine was assessed to provide a quantitative basis for co-culture nutrient supplementation. The utility of cys-32 for delivering T-DNA to plant tissues is demonstrated, where an 85-fold enhancement in GUS expression over wild-type A. tumefaciens was achieved.

Agrobacterium tumefaciens↗

PubMed1996

Model of the Second Most Abundant Cisplatin-DNA Cross-Link: X-ray Crystal Structure and Conformational Analysis of cis-[(NH(3))(2)Pt(9-MeA-N7)(9-EtGH-N7)](NO(3)).2H(2)O (9-MeA = 9-Methyladenine; 9-EtGH = 9-Ethylguanine).

A model compound of the second most abundant DNA adduct of the antitumor agent cisplatin has been synthesized and structurally and spectroscopically characterized and its conformational behavior examined: cis-[(NH(3))(2)Pt(9-MeA-N7)(9-EtGH-N7)](NO(3))(2).2H(2)O (9-MeA = 9-methyladenine; 9-EtGH = 9-ethylguanine) crystallizes in the monoclinic system, space group P2(1)/n (No. 14) with a = 7.931(2), b = 11.035(3), c = 26.757(6) Å, beta = 94.94(2) degrees, and Z = 4. The two purine bases adopt a head-to-head orientation, with NH(2) of 9-MeA and CO of 9-EtGH being at the same side of the Pt coordination plane. A theoretical conformational analysis of the complex cis-[(NH(3))(2)Pt(Ade)(Gua)](2+) (Ade = adenine; Gua = guanine) based on molecular mechanics calculations of the nonbonded energy has revealed four minimum-energy zones similar to those derived previously for cis-[(NH(3))(2)Pt(Gua)(2)](2+) (Kozelka; et al. Eur. J. Biochem. 1992, 205, 895). This conformational analysis has allowed, together with the calculation of chemical shifts due to ring effects, the attribution of the two conformers observed for cis-[(NH(3))(2)Pt{d(ApG)}](+) by Dijt et al. (Eur. J. Biochem. 1989, 179, 344) to the two head-to-head conformational zones. The orientation of the two nucleobases in the crystal structure of cis-[(NH(3))(2)Pt(9-MeA)(9-EtGH)](2+) corresponds, according to our analysis, roughly to that preferentially assumed by the minor rotamer of cis-[(NH(3))(2)Pt{d(ApG)}](+).

Journal Article↗

PubMedDate not supplied

Identification and quantitation of benzo[a]pyrene-DNA adducts formed by rat liver microsomes in vitro.

The two DNA adducts of benzo[a]pyrene (BP) previously identified in vitro and in vivo are the stable adduct formed by reaction of the bay-region diol epoxide of BP (BPDE) at C-10 with the 2-amino group of dG (BPDE-10-N2dG) and the adduct formed by reaction of BP radical cation at C-6 with the N-7 of Gua (BP-6-N7Gua), which is lost from DNA by depurination. In this paper we report identification of several new BP-DNA adducts formed by one-electron oxidation and the diol epoxide pathway, namely, BP bound at C-6 to the C-8 of Gua (BP-6-C8Gua) and the N-7 of Ade (BP-6-N7Ade) and BPDE bound at C-10 to the N-7 of Ade (BPDE-10-N7Ade). The in vitro systems used to study DNA adduct formation were BP activated by horseradish peroxidase or 3-methylcholanthrene-induced rat liver microsomes, BP 7,8-dihydrodiol activated by microsomes, and BPDE reacted with DNA. Identification of the biologically-formed depurination adducts was achieved by comparison of their retention times on high-pressure liquid chromatography in two different solvent systems and by comparison of their fluorescence line narrowing spectra with those of authentic adducts. The quantitation of BP-DNA adducts formed by rat liver microsomes showed 81% as depurination adducts: BP-6-N7Ade (58%), BP-6-N7Gua (10%), BP-6-C8Gua (12%), and BPDE-10-N7Ade (0.5%). Stable adducts (19% of total) included BPDE-10-N2dG (15%) and unidentified adducts (4%). Microsomal activation of BP 7,8-dihydrodiol yielded 80% stable adducts, with 77% as BPDE-10-N2dG and 20% of the depurination adduct BPDE-10-N7Ade. The percentage of BPDE-10-N2dG (94%) was higher when BPDE was reacted with DNA, and only 1.8% of BPDE-10-N7Ade was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMedDate not supplied

Synthesis and structure determination of the adducts formed by electrochemical oxidation of the potent carcinogen dibenzo[a,I]pyrene in the presence of nucleosides.

Because dibenzo[a,l]pyrene (DBP) is the most potent known carcinogenic aromatic hydrocarbon, reference adducts formed by reaction of deoxyribonucleosides with electrophilic intermediates of DBP are essential for identifying the structures of adducts formed in biological systems. Electrochemical oxidation of DBP in the presence of nucleosides leads to adducts from DBP.+. When 6.8 equiv of charge are consumed, three adducts are formed with dG: 7-(DBP-10-yl)Gua (89%), 8-(DBP-10-yl)dG (2%), and 8-(DBP-10-yl)Gua (2%). With 10 equiv of charge, however, only two adducts are formed: 7-(DBP-10-yl)Gua (89%) and 8-(DBP-10-yl)Gua (4%). Anodic oxidation of 8-(DBP-10-yl)dG yields 8-(DBP-10-yl)Gua. Anodic oxidation of DBP in the presence of G produces 7-(DBP-10-yl)Gua (27%) and 8-(DBP-10-yl)G (9%). Anodic oxidation of DBP in the presence of dA affords two adducts, N6-(DBP-10-yl)dA (28%) and 7-(DBP-10-yl)Ade (12%), whereas anodic oxidation in the presence of A produces only N6-(DBP-10-yl)A (24%). The structures of the adducts were elucidated by using UV, NMR, and MS. Formation of these adducts demonstrates that DBP.+ reacts at C-10 with nucleophiles. The most reactive nucleophilic groups for the Gua moiety are the N-7 and C-8, whereas for the Ade moiety they are N-7 and the 6-amino group.

Benzopyrenes↗

PubMed2002

A metabolic activation mechanism of 7H-dibenzo[c,g]carbazole via o-quinone. Part 2: covalent adducts of 7H-dibenzo[c,g]carbazole-3,4-dione with nucleic acid bases and nucleosides.

7H-dibenzo[c,g]carbazole (DBC) is a potent multispecies, multisite carcinogen present in the environment. The metabolic activation pathways of DBC are not completely known. It is hypothesized that DBC may be metabolically activated by oxidation to the reactive Michael acceptor o-quinones, which can form stable and depurinating DNA adducts. The synthesis of DBC-3,4-dione has been previously reported by this research group. In the present article, we describe the synthesis and chemical structural elucidation of nine DBC-nucleic acid adducts produced from reactions of DBC-3,4-dione with Ade, Cyt, 2'-deoxyguanosine (dGuo), 2'-deoxycytidine (dCyd), and Guo. Adducts were isolated from reaction mixtures by HPLC and analyzed using MS including elemental compositions and collision-activated dissociation (CAD), (1)H NMR, and two-dimensional chemical shift correlation spectroscopy (COSY) NMR. The adducts, 7-[3,4-dione-DBC-1-yl]-Ade, N4-[3,4-dione-DBC-1-yl]-Cyt, 5-[3,4-dione-DBC-1-yl]-Cyt, two conformational isomers of N2-[3,4-dihydroxy-DBC-1-yl]-dGuo, and two conformational isomers of N2-[3,4-dihydroxy-DBC-1-yl]-Guo, were characterized. Two adducts from reactions of DBC-3,4-dione with dCyd were identified by MS but not fully characterized by NMR due to instability of the adducts. Under similar conditions, the reactions of DBC-3,4-dione with Gua and 2'-deoxyadenosine (dAdo) did not result in an identifiable adduct. Liver DNA adducts from mice treated topically with DBC-3,4-dione (100 microg) in dimethyl sulfoxide/acetone (15/85, 100 microL) were identified with 32P-postlabeling. The major adduct chromatographically matched one of the adducts formed from livers of DBC-treated mouse (adduct 3) using identical conditions.

Administration, Topical↗

PubMed2006

Comparison of p53 mutations induced by PAH o-quinones with those caused by anti-benzo[a]pyrene diol epoxide in vitro: role of reactive oxygen and biological selection.

Polycyclic aromatic hydrocarbons (PAH) are one of the major carcinogens in tobacco smoke. They are metabolically activated through different routes to form either diol-epoxides, PAH o-quinones, or radical cations, each of which has been proposed to be an ultimate carcinogen. To study how PAH metabolites mutate p53, we used a yeast reporter gene assay based on p53 transcriptional activity. Colonies expressing wt p53 turn white (ADE +) and those expressing mutant p53 turn red (ADE -). We examined the mutagenicity of three o-quinones, benzo[a]pyrene-7,8-dione, benz[a]anthracene-3,4-dione, and dimethylbenz[a]anthracene-3,4-dione, and compared them with (+/-)-anti-benzo[a]pyrene diol epoxide ((+/-)-anti-BPDE) within the same system. The PAH o-quinones tested gave a dose-dependent increase in mutation frequency in the range of 0.160-0.375 microM quinone, provided redox-cycling conditions were used. The dominant mutations were G to T transversions (>42%), and the incidence of hotspot mutations in the DNA-binding domain was more than twice than that expected by a random distribution. The dependence of G to T transversions on redox cycling implicates 8-oxo-dGuo as the lesion responsible, which is produced under identical conditions (Chem. Res. Toxicol. (2005) 18, 1027). A dose-dependent mutation frequency was also observed with (+/-)-anti-BPDE but at micromolar concentrations (0-20 microM). The mutation pattern observed was G to C (63%) > G to A (18%) > G to T (15%) in umethylated p53 and was G to A (39%) > G to C (34%) > G to T (16%) in methylated p53. The preponderance of G mutations is consistent with the formation of anti-BPDE-N2-dGuo as the major adduct. The frequency of hotspots mutated by (+/-)-anti-BPDE was essentially random in umethylated and methylated p53, suggesting that 5'-CpG-3' islands did not direct mutations in the assay. These data suggest that smoking may cause mutations in p53 by formation of PAH o-quinones, which produce reactive oxygen species. The resultant 8-oxo-dGuo yields a pattern of mutations but not a spectrum consistent with that seen in lung cancer; we suggest that the emergence of the spectrum requires biological selection.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed2004

Genetic vaccination of mice with plasmids encoding the NS1 non-structural protein from tick-borne encephalitis virus and dengue 2 virus.

Although there is a safe, inexpensive and efficacious vaccine against yellow fever, vaccination against other flavivirus diseases is less successful. There is no licensed vaccine against dengue fever and current vaccines against tick-borne encephalitis (TBE) and Japanese encephalitis are expensive and require several injections. Furthermore novel vaccines containing only virus envelope proteins may raise fears over antibody mediated enhancement (ADE) of disease. Here we report the successful use of genetic vaccination against TBE in an experimental animal model using a plasmid containing the coding sequence of a non-structural protein (NS1). Such vaccines would provide inexpensive protection against disease, without raising concerns over inducing ADE on subsequent exposure to heterotypic infectious virus. Attempts to generate chaemeric plasmids to protect against both TBE and dengue fever were less successful. Although these chaemeric plasmids directed the synthesis and secretion of the virus NS1 protein normally, no protection was observed against either TBE or dengue fever.

Amino Acid Sequence↗

PubMed2004

mGluR5 antagonist MPEP reduces ethanol-seeking and relapse behavior.

The glutamatergic system plays an important role in mediating neurobehavioral effects of ethanol. Metabotropic glutamate receptors subtype 5 (mGluR5) are modulators of glutamatergic neurotransmission and are abundant in brain regions known to be involved in ethanol self-administration. Here, we studied the effects of 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a highly potent, noncompetitive mGlu5 receptor antagonist, on voluntary ethanol consumption and relapse behavior. For this purpose, we used two models for the measurement of relapse behavior: (i) reinstatement of ethanol-seeking behavior by drug-associated cues and (ii) the alcohol deprivation effect in long-term ethanol-consuming rats. In the first set of experiments, rats were trained to lever press for ethanol in the presence of a distinct set of cues. After extinction, the animals were exposed to the respective cues that initiated reinstatement of responding. A response-contingent ethanol prime further enhanced responding compared to the conditioned cues alone. Under these conditions, MPEP (0, 1, 3, and 10 mg/kg) attenuated ethanol seeking significantly and in a dose-related manner. However, at the highest dose, MPEP also decreased the number of inactive lever responses. In the second set of experiments, rats with 1 year of ethanol experience and repeated deprivation phases were used. A subchronic treatment with MPEP (twice daily; 0, 3, and 10 mg/kg) resulted in a significant and dose-dependent reduction of the alcohol deprivation effect (ADE). Although the same MPEP treatment regimen decreased baseline drinking, this effect was not as pronounced as on the ADE. These results show in two commonly used models of relapse to ethanol that pharmacological targeting of mGlu5 receptors may be a promising approach for the treatment of alcoholism.

Alcohol Drinking↗

PubMed2005

Structure, reactivity and solution behaviour of [Re(ser)(7-MeG)(CO)(3)] and [Re(ser)(3-pic)(CO)(3)]: "nucleoside-mimicking" complexes based on the fac-[Re(CO)(3)](+) moiety.

The fac-[Re(CO)(3)](+) moiety was reacted with the amino acid serine (D- and L-ser) and with 7-methylguanine (7-MeG), 3-methylpyridine (3-pic) or adenine (ade) to yield novel complexes intended as nucleoside-mimicking compounds. Reaction of [Re(H(2)O)(3)(CO)(3)](+)(1) with L-ser yields the complex [Re(L-ser)(2)(CO)(3)](L-2). X-Ray structure analysis of L-2 reveals that one of the two amino acids is bound to the metal centre in a bidentate fashion while the other amino acid is bound as a zwitterion via the carboxylate oxygen only. Reaction of L-2 and of [Re(D-ser)(2)(CO)(3)](D-2) with 7-MeG yields complexes [Re(L-ser)(7-MeG)(CO)(3)](L-3) and [Re(D-ser)(7-MeG)(CO)(3)](D-3) respectively. Complexes L-3 and D-3 are received as a mixture of diastereomers. If 3-pic is used instead of 7-MeG complex [Re(L-ser)(3-pic)(CO)(3)](L-4) is obtained in good yield, while interaction of L-2 with ade gives a mixture of five distinct species. Crystallization gave one single diastereomer for L-3 and D-3 and the two forms for 4 respectively. X-Ray structure analyses reveal that in all cases the amino acid is bound in a chelate fashion with the base occupying the sixth co-ordination site. When crystals of either 2 or 3 are dissolved in a CD(3)OD/D(2)O mixture (1:1, 293 K) rapid transformation to the diastereomeric mixture is observed. While for L-2 this reorganisation is fast on the NMR time scale even at 193 K, the rate constant for the rearrangement of L-3 and D-3 is 1.36 +/- 0.24 x 10(-2) s(-1) at 293 K.

Journal Article↗