Mechanism-based inactivation of human liver cytochrome P450 2A6 by 8-methoxypsoralen

The P450 2A6 catalyzed 7-hydroxylation of coumarin proceeded with a mean Km of 0.40 (+/-0.13) microM and Vmax of 6.34 nmol/nmol P450/min (36-fold variation) in microsomal preparations from a panel of 12 human livers. Substrate depletion was avoided during the kinetic determinations. 8-Methoxypsoralen (8-MOP) is a potent mechanism-based inactivator of human liver P450 2A6 and reconstituted purified recombinant P450 2A6 based on the following evidence: 1) 8-MOP causes time, concentration, and NADPH-dependent loss of P450 2A6 activity that is not reversed by potassium ferricyanide or extensive dialysis, 2) loss of P450 2A6 activity is associated with a loss of spectrally observable P450, 3) addition of nucleophiles or reactive oxygen scavengers to the incubations does not prevent inactivation of P450 2A6, and 4) 8-MOP-dependent P450 2A6 inactivation is inhibited (concentration dependent) by the addition of a competitive inhibitor (pilocarpine). Inactivation is selective for P450 2A6 at low concentrations of 8-MOP (2.5 microM) after short incubation time periods (3 min) and was characterized by a KI of 0.8 and 1.9 microM in a reconstituted and microsomal system, respectively, and a kinact of 1 min-1 and 2 min-1 in a reconstituted and microsomal system, respectively. A substrate depletion partition ratio of 21 was calculated for the inactivation of recombinant P450 2A6. Potency and selectivity suggest that 8-MOP could be a useful tool in vitro for evaluating P450 2A6 activity in various enzyme preparations.     

Dynamic endocrine testing. The Mayo Clinic model

An endocrine testing center (ETC) is a universal requirement for the practice of endocrinology. Modifications of the Mayo Clinic model for an ETC should be applicable to most endocrine practices. Key components of an ETC include a centralized testing area, registered nurse-physician team, detailed testing protocols, and patient education programs.     

Cleavage of single strand RNA adjacent to RNA-DNA duplex regions by Escherichia coli RNase H1

RNase H1 from Escherichia coli cleaves single strand RNA extending 3' from an RNA-DNA duplex. Substrates consisting of a 25-mer RNA annealed to complementary DNA ranging in length from 9-17 nucleotides were designed to create overhanging single strand RNA regions extending 5' and 3' from the RNA-DNA duplex. Digestion of single strand RNA was observed exclusively within the 3' overhang region and not the 5' overhang region. RNase H digestion of the 3' overhang region resulted in digestion products with 5'-phosphate and 3'-hydroxyl termini. The number of single strand RNA residues cleaved by RNase H is influenced by the sequence of the single strand RNA immediately adjacent to the RNA-DNA duplex and appears to be a function of the stacking properties of the RNA residues adjacent to the RNA-DNA duplex. RNase H digestion of the 3' overhang region was not observed for a substrate that contained a 2'-methoxy antisense strand. The introduction of 3 deoxynucleotides at the 5' terminus of the 2'-methoxy antisense oligonucleotide resulted in cleavage. These results offer additional insights into the binding directionality of RNase H with respect to the heteroduplex substrate.     

CCD-3693: an orally bioavailable analog of the endogenous neuroactive steroid, pregnanolone, demonstrates potent sedative hypnotic actions in the rat

Signal detectability was measured in three temporal conditions as a function of the bandwidth and configuration of simultaneous maskers that either did or did not spectrally overlap the signal. The 20-ms signal was 250 Hz wide and was centered at 2500 Hz (fs). Although there were marked individual differences, performance was typically poorer when signal onset came 1 ms rather than 250 ms after the onset of a 420-ms masker, and poorest when signal onset came 1 ms after the onset of a 23-ms masker. The results support the idea that two separate across-channel processes contribute to temporal changes in signal detectability. One process contributes to the improvement observed as signal onset is delayed from masker onset, and its influence is reduced by the presence of masking components at fs only when the masker extends exclusively below fs. The other process is associated with the improvement observed as masker offset is delayed from signal offset, and its influence is reduced by the presence of masking components at fs when the masker extends exclusively above, or both below and above fs. Both of these processes are primarily activated by frequencies ranging from 0.6 to 0.8fs and 1.2 to 1.4fs. The data also demonstrate that the measured critical bandwidth narrows as signal onset is delayed from masker onset.     

ED2-positive perivascular phagocytes produce interleukin-1beta during delayed neuronal loss in the facial nucleus of the rat

An adeno-associated virus (AAV) vector, expressing genes for human tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC), demonstrated significantly increased production of dopamine in 293 (human embryonic kidney) cells. This bicistronic vector was used to transduce striatal cells of six asymptomatic but dopamine-depleted monkeys which had been treated with the neurotoxin MPTP. Striatal cells were immunoreactive for the vector-encoded TH after stereotactic injection for periods up to 134 days, with biochemical effects consistent with dopamine biosynthetic enzyme expression. A subsequent experiment was carried out in six more severely depleted and parkinsonian monkeys. Several TH/aadc-treated monkeys showed elevated levels of dopamine near injection tracts after 2.5 months. Two monkeys that received a beta-galactosidase expressing vector showed no change in striatal dopamine. Behavioral changes could not be statistically related to the vector treatment groups. Toxicity was limited to transient fever in several animals and severe hyperactivity in one animal in the first days after injection with no associated histological evidence of inflammation. This study shows the successful transfection of primate neurons over a period up to 2.5 months with suggestive evidence of biochemical phenotypic effects and without significant toxicity. While supporting the idea of an in vivo gene therapy for Parkinson's disease, more consistent and longer lasting biochemical and behavioral effects will be necessary to establish the feasibility of this appraoch in a primate model of parkinsonism.     

Comparison of fatty acid tracers FTHA and BMIPP during myocardial ischemia and hypoxia

To study the sensitivity of two fatty acid tracers to changes in beta-oxidation, the myocardial retention kinetics of 125I-iodine-15-(p-iodophenyl)-3(R,S)-methylpentadecanoic acid (BMIPP) and 14-18F-fluoro-6-thia-heptadecanoic acid (FTHA) were compared in states of oxygen deprivation due to ischemia and hypoxia. METHODS: Nineteen swine were studied by extracorporeal perfusion of the three coronary arteries. Fatty acid beta-oxidation rates were determined by infusion of tritiated palmitate into the left anterior descending artery (LAD) and by measurement of labeled water production in the LAD perfusion bed. After a baseline period of 30 min, animals were divided into three groups and subjected to a 50-min intervention period. For the control group, there was no change in perfusion; for the ischemia group, there was a 60% decrease in LAD perfusion; and for the hypoxia group, the perfusion rate was unchanged, but venous blood was used as the LAD perfusate. Continuous infusion of FTHA and BMIPP into the LAD started 10 min into the intervention period and continued until the end of the intervention period. Retention rates of the two tracers were compared between the LAD and circumflex perfusion beds. RESULTS: No difference in beta-oxidation rate occurred from the baseline to the intervention period in the control group. A 50% reduction in beta-oxidation occurred in the ischemia group, and an 80% reduction occurred in the hypoxia group. No difference in retention of BMIPP or FTHA occurred in the control group. In the ischemia group, reduction in retention of both tracers occurred. However, in the hypoxia group, FTHA uptake was unchanged, whereas BMIPP retention increased compared to the circumflex arterial bed. CONCLUSION: Decreased retention of both BMIPP and FTHA occurred with ischemia, despite the known differences in metabolism of the two tracers. This difference in metabolism was further highlighted in the setting of hypoxia with increased BMIPP uptake. Thus, these results suggest that uptake of both FTHA and BMIPP tracks reduction of fatty acid utilization in myocardial ischemia but fails in tracking reduction of fatty acid oxidation during hypoxia.     

Computer-based smoking cessation interventions in adolescents: description, feasibility, and six-month follow-up findings

The impact of adolescent smoking cessation clinics has been disappointing due to low participation rates, high attrition, and low quit rates. This paper describes two computerized self-help adolescent smoking cessation intervention programs: 1) a program utilizing the expert system which is based on the transtheoretical model of change and 2) a popular action-oriented smoking cessation clinic program for teens which was modified for computer presentation. High participation rates in the program among 132 smokers demonstrate the high feasibility and acceptability of the programs. Quit rates of up to 20% were observed during the intervention, and an additional 30% made unsuccessful quit attempt(s). The 6-month follow-up findings indicated that adolescents were poorly prepared to maintain abstinence.