Biologically active Rep proteins of adeno-associated virus type 2 produced as fusion proteins in Escherichia coli

Four Rep proteins are encoded by the human parvovirus adeno-associated virus type 2 (AAV). The two largest proteins, Rep68 and Rep78, have been shown in vitro to perform several activities related to AAV DNA replication. The Rep78 and Rep68 proteins are likely to be involved in the targeted integration of the AAV DNA into human chromosome 19, and the full characterization of these proteins is important for exploiting this phenomenon for the use of AAV as a vector for gene therapy. To obtain sufficient quantities for facilitating the characterization of the biochemical properties of the Rep proteins, the AAV rep open reading frame was cloned and expressed in Escherichia coli as a fusion protein with maltose-binding protein (MBP). Recombinant MBP-Rep68 and MBP-Rep78 proteins displayed the following activities reported for wild-type Rep proteins when assayed in vitro: (i) binding to the AAV inverted terminal repeat (ITR), (ii) helicase activity, (iii) site-specific (terminal resolution site) endonuclease activity, (iv) binding to a sequence within the integration locus for AAV DNA on human chromosome 19, and (v) stimulation of radiolabeling of DNA containing the AAV ITR in a cell extract. These five activities have been described for wild-type Rep produced from mammalian cell extracts. Furthermore, we recharacterized the sequence requirements for Rep binding to the ITR and found that only the A and A' regions are necessary, not the hairpin form of the ITR.     

Amphetamines in hair by enzyme-linked immunosorbent assay

Human hair was collected from the occipital crown region of the head from several subjects; these hair samples were presumptively positive for amphetamines by a previously evaluated immunoassay. Hair was washed briefly with methanol to remove external contamination, then extracted with hot methanol for 2 h to recover the drugs. The extracts were evaporated to dryness, reconstituted in buffer, and analyzed using a new enzyme-linked immunosorbent assay (ELISA) technique adapted for the detection of amphetamines in hair. Gas chromatography-mass spectrometry was used as the reference technique. Cross-reactivity of several related compounds was evaluated by equating the inverse of the ligand concentration at 50% antibody binding to the affinity constant for each compound. The ratio of a compound's affinity constant to that for d-methamphetamine was used to derive percent crossreactivity. These experiments yielded values of 30.8% for d-amphetamine, 7.4% for I-methamphetamine, 4.3% for phentermine, 2.9% for I-amphetamine, and <1% for ephedrine, methylenedioxyamphetamine, and methylenedioxymethamphetamine. Cross-reactivity of unrelated compounds was found to be non-existent. The optimum cutoff concentration was determined by receiver operating characteristic curve analysis to be 300 pg/mg and the observed limit of detection was 60 pg/mg. Intra-assay precision at 300 pg/mg was 3.3% (coefficient of variation, CV), and the interassay CV was 10.5%. The sensitivity and specificity of the method were 83% and 92%, respectively.     

Cost effectiveness of clinical training in a community mental health center.

The future of professional psychology may well rest on psychology training programs successfully defending their existence in purely monetary terms. This article examines the effectiveness of the training program of an urban mental health center that includes an American Psychological Association-accredited psychology internship. The training program was found to produce net revenues for the facility and to meet the training needs of its participants. Variables related to producing a cost effective psychology training program are discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Patient choice in diabetes education curriculum. Nutritional versus standard content for type 2 diabetes

The purpose of this study was to determine whether aging alters neuronal uptake of norepinephrine (NE) in the rat heart and if dietary restriction influenced the effect of age on this system. Cardiac synaptosomes were prepared from 6-, 12- and 24-month-old male F344 rats fed ad libitum (AL) or a diet restricted (DR) to 60% of AL intake. Cardiac synaptosomes were incubated with 50, 100, 200, or 400 nM [3H]NE for 10 min at 37 degrees C with and without desmethylimipramine (DMI), a selective neuronal-uptake blocker. DMI-sensitive [3H]NE uptake was calculated as the difference between samples with and without DMI. NE uptake was adjusted for the number of cardiac synaptosomes in each sample by dividing by the endogenous NE content in each sample. The Vmax for uptake ([3H]NE/min/ng NE) declined significantly between 6 and 12 months in AL rats and between 12 and 24 months in DR rats. Km was not significantly different between age or diet groups. The change in Vmax with age suggests that the number of NE transporters per synaptosome may decline with age and that DR delays this effect of age. There were no differences in the sensitivity to DMI between age or diet groups.     

Reported changes in sexual behavior in response to AIDS in the United States

BACKGROUND: The AIDS epidemic has profoundly affected public health policy and everyday choices of individuals. Understanding the self-protective strategies adopted by the general population is crucial to designing appropriate and more effective primary AIDS prevention strategies. METHODS: A probability sample of the U.S. adult population in 1992, the National Health and Social Life Survey (n = 3,159), addressed detailed aspects of sexual behavior. How respondents changed their sexual behavior due to AIDS was recorded verbatim and categorized for analysis. RESULTS: Twenty-nine percent of U.S. adults reported some sexual behavior change, primarily, reducing numbers of partners (12%), using condoms more frequently (9%), selecting partners more carefully (7%), changing attitude toward sex (4%), and abstinence (3%). Individuals most at risk show the greatest propensity to change although the actual risk-reduction strategies and the meanings assigned to those strategies vary considerably. Strategies are not all compatible with each other nor equally likely to be adopted by individuals with different lifestyles. CONCLUSIONS: Self-protective strategies to avoid AIDS are widespread, but primary prevention efforts should be targeted to particular circumstances and the self-protective propensities of each individual.     

Comparison of measured sleeping metabolic rate and predicted basal metabolic rate in the first year of life

In infants, sleeping metabolic rate (SMR) is used as a proxy for basal metabolic rate (BMR). BMR can be predicted from anthropometry using published equations. Our study was intended to evaluate the ability of these equations to predict measured SMR in infants aged 6 weeks to 12 months. SMR was measured in a mixed longitudinal study using the Douglas bag technique (n = 105). Measured SMR values were compared with BMR predicted from weight (BMR-1) or weight and length (BMR-2). These equations were not successful in predicting SMR in this age group. Percentage error of predicted BMR was related to infant weight (BMR-1: r = 0.26; p < 0.005; BMR-2: r = 0.18; p < 0.06). Alternative logarithmic equations were derived from this study (R = 0.84-0.87; SEE = 0.159-0.168). We conclude that the new equations, relating to contemporary infants, are more suitable but actual measurements remain preferred.     

Molecular cloning, sequencing, and heterologous expression of the vaoA gene from Penicillium simplicissimum CBS 170.90 encoding vanillyl-alcohol oxidase

The cDNA encoding vanillyl-alcohol oxidase (EC 1.1.3.7) was selected from a cDNA library constructed from mRNA isolated from Penicillium simplicissimum CBS 170.90 grown on veratryl alcohol by immunochemical screening. The vaoA-cDNA nucleotide sequence revealed an open reading frame of 1680 base pairs encoding a 560-amino acid protein with a deduced mass of 62,915 Da excluding the covalently bound FAD. The deduced primary structure shares 31% sequence identity with the 8alpha-(O-tyrosyl)-FAD containing subunit of the bacterial flavocytochrome p-cresol methyl hydroxylase. The vaoA gene was isolated from a P. simplicissimum genomic library constructed in lambdaEMBL3 using the vaoA-cDNA as a probe. Comparison of the nucleotide sequence of the vaoA gene with the cDNA nucleotide sequence demonstrated that the gene is interrupted by five short introns. Aspergillus niger NW156 prtF pyrA leuA cspA transformed with the pyrA containing plasmid and a plasmid harboring the complete vaoA gene including the promoter and terminator was able to produce vaoA mRNA and active vanillyl-alcohol oxidase when grown on veratryl alcohol and anisyl alcohol. A similar induction of the vaoA gene was found for P. simplicissimum, indicating that similar regulatory systems are involved in the induction of the vaoA gene in these fungi. Introduction of a consensus ribosome binding site, AGAAGGAG, in the vaoA-cDNA resulted in elevated expression levels of active vanillyl-alcohol oxidase from the lac promoter in Escherichia coli TG2. The catalytic and spectral properties of the purified recombinant enzyme were indistinguishable from the native enzyme.