The circadian Per1 and Per2 genes influence alcohol intake, reinforcement, and blood alcohol levels.

BACKGROUND: Perturbations in the function of core circadian clock components such as the Period (Per) family of genes are associated with alcohol use disorder, and disruptions in circadian cycles may contribute to alcohol abuse and relapse. This study tested ethanol consumption, reinforcement, and metabolism in mice containing functional mutations in Per1 and/or Per2 genes on an ethanol-preferring background, C57BL/6J mice. METHODS: Mice were tested in: (A) free-access intake with ascending concentrations of ethanol (2-16%, v/v), (B) conditioned place preference using ethanol (2g/kg for males; 2.5g/kg for females) vs. saline injections, (C) recovery of the righting reflex following a 4g/kg bolus of ethanol, and (D) blood ethanol levels 1h after a 2g/kg bolus of ethanol. RESULTS: All Per mutant (mPer) mice showed increased ethanol intake and condition place preference compared to controls. There were also genotypic differences in blood ethanol concentration: in males, only mPer1 mice showed a significantly higher blood ethanol concentration than WT mice, but in females, all mPer mice showed higher blood ethanol levels than WT mice. CONCLUSIONS: Mutation of either Per1 or Per2, as well as mutations of both genes, increases ethanol intake and reinforcement in an ethanol-preferring mouse model. In addition, this increase in ethanol seeking behavior seems to result both from a change in ethanol metabolism and a change in reward responding to ethanol, but not from any change in sensitivity to ethanol's sedating effects.

Who's listening? Experiences of women with breast cancer in communicating with physicians.

The purpose of this qualitative study was to describe communication behaviors and attitudes of physicians that were most important to women living with breast cancer. Two focus group sessions were conducted, 1 month apart, involving 15 women who were members of a community-based breast cancer support group in Vancouver, British Columbia, Canada. Group dialogue was audiotaped, and notes were taken at each session by the coinvestigators, also members of the support group. Audiotapes, coinvestigators' written notes from the two focus group sessions, and the written homework assignments were used in the qualitative data analysis. Conceptual themes were identified and grouped to discern patterns within the data. The women were asked the following: (a) What were the most helpful things your doctor said or did at the time of your diagnosis? (b) What does a good intervention feel or look like? They were then asked to describe behaviors and attitudes they would like to influence in medical students who might later be communicating with women facing a diagnosis of breast cancer and to indicate which behaviors and attitudes they felt were most important. Women's positive experiences with physicians were characterized by communication based on active listening, awareness of the women's knowledge of their illness, honesty, and partnership. Physicians who showed interest in their patients as persons and who used touch to communicate caring were perceived as supportive communicators. Not surprisingly, there were similarities between the participants' positive experiences with their own physicians and the behaviors and attitudes desired in future physicians. Once again, "listening" was ranked as most important, followed by willingness to discern the individual patient's knowledge level.

An adult education model for training third-year medical students in use of the Physician Data Query (PDQ) System.

BACKGROUND AND METHODS: An adult education model was developed to familiarize third-year medical students with the Physician Data Query (PDQ) system, a computerized, full-text database of state-of-the-art cancer information developed by the National Cancer Institute. The educational model was designed in collaboration with a medical librarian and was implemented within the context of a busy surgery clerkship using only modest resources that were readily available within the medical school. RESULTS: During three years, 275 medical students participated in the exercise and evaluated both the PDQ system and the educational model. Overall, 87% of the students considered the PDQ system to be a valuable source of information, and 84% anticipated using PDQ after completing their surgical rotations. Ninety-six percent of the students agreed that the objectives of the exercise were met. CONCLUSIONS: This article provides a description of the educational model and discusses the principles of adult education and andragogy on which it was developed. The importance of emphasizing the process of learning as well as the content is described relative to self-directed and life-long learning.

Metabolism and bioactivation of clozapine by human liver in vitro.

The metabolism of clozapine by human liver has been investigated in vitro. Irreversible protein-binding and conjunction with model nucleophiles have been used as markers for bioactivation of clozapine, while stable metabolite formation has been assessed using radiometric HPLC. In all nine liver microsomal preparations investigated, clozapine was extensively metabolized to the stable products desmethylclozapine (range 19%-27.2%), N-oxide (1.5-20.5%) and three polar metabolites (0-20.8%), and was bioactivated to a protein-reactive metabolite (0.6-2.1%). The CYP2D6 genotype did not influence the capacity of the livers to form these metabolites. All metabolic pathways were inhibited by ketoconazole, indicating the involvement of the cytochrome P450 enzymes. Isozyme-selective inhibitor studies demonstrated that whereas demethylation was performed by CYP1A2, N-oxidation and chemically reactive metabolite formation were dependent upon multiple forms of P450. The N-oxide was readily reduced back to clozapine in the presence of NADPH, this conversion being inhibited by ascorbic acid. Glutathione (1 mM) decreased covalent binding by 70%. The amount of putative adduct formed in the presence of glutathione (13.4 +/- 0.9%) was much greater than the covalent binding (mean 1.1 +/- 0.2%). The bioactivation of clozapine was, like the N-oxidation of clozapine, a reversible process. In summary, our results indicate clozapine undergoes extensive metabolism by human liver to both stable and chemically reactive metabolites, the formation of which is catalyzed by the cytochrome P450 enzymes. The role of the reactive metabolite, which may be a free radical, in the pathogenesis of clozapine agranulocytosis and hepatotoxicity requires further study.

An investigation of the formation of cytotoxic, genotoxic, protein-reactive and stable metabolites from naphthalene by human liver microsomes.

Chemically reactive epoxide metabolites have been implicated in various forms of drug and chemical toxicity. Naphthalene, which is metabolized to a 1,2-epoxide, has been used as a model compound in this study in order to investigate the effects of perturbation of detoxication mechanisms on the in vitro toxicity of epoxides in the presence of human liver microsomes. Naphthalene (100 microM) was metabolized to cytotoxic, protein-reactive and stable, but not genotoxic, metabolites by human liver microsomes. The metabolism-dependent cytotoxicity and covalent binding to protein of naphthalene were significantly higher in the presence of phenobarbitone-induced mouse liver microsomes than with human liver microsomes. The ratio of trans-1,2-dihydrodiol to 1-naphthol was 8.6 and 0.4 with the human and the induced mouse microsomes, respectively. The metabolism-dependent toxicity of naphthalene toward human peripheral mononuclear leucocytes was not affected by the glutathione transferase mu status of the co-incubated cells. Trichloropropene oxide (TCPO; 30 microM), an epoxide hydrolase inhibitor, increased the human liver microsomal-dependent cytotoxicity (19.6 +/- 0.9% vs 28.7 +/- 1.0%; P = 0.02) and covalent binding to protein (1.4 +/- 0.3% vs 2.8 +/- 0.2%; P = 0.03) of naphthalene (100 microM), and reversed the 1,2-dihydrodiol to 1-naphthol ratio from 6.6 (without TCPO) to 2.6, 0.6 and 0.1 at TCPO concentrations of 30, 100 and 500 microM, respectively. Increasing the human liver microsomal protein concentration reduced the cytotoxicity of naphthalene, while increasing its covalent binding to protein and the formation of the 1,2-dihydrodiol metabolite. Co-incubation with glutathione (5 mM) reduced the cytotoxicity and covalent binding to protein of naphthalene by 68 and 64%, respectively. Covalent binding to protein was also inhibited by gestodene, while stable metabolite formation was reduced by gestodene (250 microM) and enoxacin (250 microM). The study demonstrates that human liver cytochrome P450 enzymes metabolize naphthalene to a cytotoxic and protein-reactive, but not genotoxic, metabolite which is probably an epoxide. This is rapidly detoxified by microsomal epoxide hydrolase, the efficiency of which can be readily determined by measurement of the ratio of the stable metabolites, naphthalene 1,2-dihydrodiol and 1-naphthol.

Generation and transit pathway of H+ is critical for inhibition of palmar sweating by iontophoresis in water.

Passing galvanic current across the skin (known as "tap water iontophoresis" or TWI) inhibits sweating; however, its mechanism of action is unclear. Using improved methods, we confirmed that anodal current has more of an inhibitory effect than cathodal current, water is superior to saline, and the inhibitory effect is a function of the amperage used. To address the importance of current flowing through the pores, a layer of silicone grease was placed on the skin to reduce the shunt pathway across the epidermis. With silicone, total skin conductance decreased 60% without the sweat pores being occluded, swelling of the stratum corneum and collapse of the poral lumen was prevented, and current-induced inhibition of sweating was enhanced, most likely because of an increase in current density in the pores. The pH of anodal water, but not of saline, dropped to 3, whereas that of cathodal water increased to 10 during passage of current through the skin. Acidified anodal water was superior to alkaline water. Sweat glands isolated from TWI-induced anhidrotic palmar skin responded to methacholine in vitro, but the sweat rate and pharmacological sensitivity were slightly lowered. Thus the strong acidity generated by hydrolysis of water in the anodal bath and the further accumulation of H+ in the sweat duct by anodal current may be responsible for TWI-induced inhibition of sweating due to an unknown lesion(s) in the duct or sweat pore. The secretory coil function may also be altered because of exposure to intense acidity during TWI. The importance of H+ movement into the sweat pore for inhibition of sweating could be further exploited to develop new strategies for the control of sweating.

Alkyl- and aryl-substituted salicyl phosphates as detection reagents in enzyme-amplified fluorescence DNA hybridization assays on solid support.

Nine salicyl phosphate esters with hydrophobic substituents (5-phenyl, 5-(2,4-difluorophenyl), 5-tert-octyl, 5-cumyl, 5-(4-tert-butylphenyl, 5-(1-adamantyl), 5-(n-dodecyl), 5-(1,1-diphenylethyl, and 5-trityl) were synthesized and found to be good substrates for calf intestinal alkaline phosphatase. The enzymatic hydrolysis produced the corresponding salicylates, which were strongly fluorescent when excited by ultraviolet light around 300 nm with maximum emission at 420-435 nm. The salicylates were less soluble and/or more adhesive than the nonfluorescent salicyl phosphate substrates, resulting in localization of fluorescence signal, which is a requirement for membrane-based assays. The salicyl phosphates bearing 8-14 carbon substitutents were found to be suitable detection reagents for dot-blot DNA hybridization assays on nylon membrane using a biotinylated probe, allowing the detection of 125 pg of target pBR322 plasmid DNA using a simple apparatus consisting of a transilluminator, a camera. and a 455-nm cutoff optical filter.

Time-resolved fluorescence detection of enzyme-amplified lanthanide luminescence for nucleic acid hybridization assays.

A new nonisotopic detection method based on time-resolved fluorescence for nucleic acid hybridization assays with alkaline phosphatase labels has been developed: enzyme-amplified lanthanide luminescence (EALL). EALL combines the amplification of an enzyme label with the sensitivity and background elimination of time-resolved fluorescence detection of lanthanide ion luminescence. The detection system for alkaline phosphatase makes use of a phosphorylated salicylic acid derivative that, upon dephosphorylation, gives a product capable of forming a luminescent terbium chelate. We demonstrate DNA hybridization assays by using two substrates, one for membrane and one for solution-based formats. Using the substrate that produces a more adhesive product allows performance of dot-blot and Southern blot assays on nylon membranes; results can be recorded with a time-resolved photographic camera system, or with an ultraviolet transilluminator-based system. Less than 4 pg of target sequence can be detected in a dot-blot assay after incubation with substrate for 2-4 h. DNA microwell-plate hybridization assays with the more soluble substrate/product pair can be quantified with time-resolved fluorescence plate readers, giving a similar detection sensitivity. EALL is thus a practical time-resolved fluorescence-based alternative to other detection systems for DNA hybridization assays.

Enzyme-amplified lanthanide luminescence for enzyme detection in bioanalytical assays.

Enzyme-amplified lanthanide luminescence (EALL) is a new method which has been developed for enzymatically amplified signal detection in ultrasensitive bioanalytical assays where an enzyme is used as label or is itself the analyte of interest. Signal generation is performed by enzymatically transforming a substrate into a product which forms a luminescent lanthanide chelate; the product chelate can then be detected using time-resolved or normal fluorescence methods. Alkaline phosphatase substrates have been developed and demonstrated in a model immunoassay in microwell format. The method has also been demonstrated for detection of a variety of other hydrolytic and oxidative enzymes. Thus the EALL method shows promise for use in a wide variety of bioanalytical applications.

A new europium chelate for protein labelling and time-resolved fluorometric applications.

Synthetic procedures are presented for a new chelator that forms stable and highly fluorescent complexes with Eu3+. This chelator, 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) is synthesized in a high-yield three-step procedure. BCPDA can be covalently incorporated into proteins under relatively mild conditions, and when complexed with Eu3+ forms a fluorescent product that has a lifetime in the range of 0.4 to 0.7 ms. Thus, it is useful for time-resolved fluorescence immunoassay applications.

MEDLINE on CD-ROM: end user searching in a medical school library.

End users at the Medical College of Pennsylvania enjoyed searching CD-ROM versions of MEDLINE and judged such searches "extremely useful." To assess the effectiveness of their searches, a sample of 500 search statements from Compact Cambridge (CC) MEDLINE was examined. A high proportion of search statements (224, or 45 percent) retrieved no documents. Over one-third of search statements (185, or 37 percent) contained at least one error, which usually resulted in zero retrieval for that search statement. Searchers attempting to enter Medical Subject Headings were frequently defeated by the elaborate punctuation requirement of CC MEDLINE. "Missed opportunities" were evident in over three-fourths of search statements. One-on-one instruction and library classes may increase search success, but these measures reach a limited audience. Improvement in the search software itself is needed to help searchers improve the effectiveness of their searches.

Charge transfer between fluorescein and tryptophan as a possible interaction in the binding of fluorescein to anti-fluorescein antibody.

Steady-state and time-resolved fluorescence studies of fluorescein (Fl) and 9-hydroxyphenylfluoron (HPF) bound to high-affinity rabbit anti-Fl IgG antibody (anti-Fl IgG) have been performed. The heterogeneity in the fluorescence properties observed for Fl bound to anti-Fl IgG is reduced for HPF bound to anti-Fl IgG. A charge transfer between a tryptophyl residue in the binding site and the hapten was considered as a possible binding interaction. Fl was observed to form complexes in solution with the amino acids tryptophan, tyrosine and methionine, probably due to charge transfer. Also, the fluorescence of tryptophyl residues of the protein is quenched on binding. While such charge transfer complexes may be present, there is no direct evidence that charge transfer complexes between Fl and tryptophan are necessarily present for Fl bound to all high-affinity anti-Fl IgG molecules.