Reassessment of cytochrome P450 2B2: catalytic specificity and identification of four active site residues

Cytochromes P450 2B metabolize a variety of compounds and have provided an excellent framework for identifying determinants of substrate specificity. Among the rat 2B enzymes, a puzzling difference has emerged between the reported substrate specificity of purified hepatic 2B2 and that of certain 2B1 mutants containing 2B1 --> 2B2 substitutions. To address these discrepancies, we have characterized two 2B2 variants. A cDNA clone designated 2B2FF was obtained from phenobarbital-induced Lewis rats and, like some previously isolated variants, was found to contain phenylalanine at positions 58 and 114. A second 2B2 clone was generated by restoring Leu and Ile, respectively, at these positions. These enzymes were expressed in Escherichia coli and analyzed with androstenedione, testosterone, progesterone, ethoxycoumarin, benzyloxyresorufin, and pentoxyresorufin. The expressed 2B2 variants metabolized most substrates at rates that were 1-9% of those of 2B1. When steroid regio- and stereospecificity was examined, the metabolite profiles of expressed 2B2 and 2B2FF conflicted with the 16beta- and 16alpha-hydroxylation observed for purified hepatic 2B2 from Sprague-Dawley rats. These and other results suggested that the purified hepatic 2B2 contained a small percent of the 2B1 enzyme. Masses of tryptic peptides were consistent with identity between purified hepatic 2B2 and 2B2FF. On the basis of a three-dimensional homology model and the construction and analysis of 2B2 mutants, residues 114, 363, 367, and 478 were identified as determinants of substrate specificity. In addition, 2B1 and the expressed 2B2 variants showed differential susceptibility to the mechanism-based inactivators chloramphenicol and N-(2-p-nitrophenethyl)chlorofluoroacetamide.     

Qualitative approach: a contribution to nursing

This paper focus on some characteristics of the qualitative methodology. Some of these methods are explored such as: participatory research, phenomenology, grounded theory and ethnography critical theory Perspectives of their utilization in nursing research are examined.     

Pertussis in Massachusetts, 1981-1991: incidence, serologic diagnosis, and vaccine effectiveness

Massachusetts provides diphtheria-tetanus toxoid-pertussis (DTP) vaccine, and since 1980 has monitored pertussis with a statewide diagnostic service. The incidence of bacteriologically confirmed pertussis was 104.5 per 100,000 person-years in 1-month-old infants and declined progressively thereafter. Infants < 6 months old experienced disproportionate morbidity: 44% of bacteriologically confirmed pertussis, 64% of hospitalizations, and 71% of hospital days. Most children with pertussis had received < 3 DTP doses during childhood, whereas 87% of adolescents with pertussis had received > or = 4 doses. Serodiagnosis by single serum anti-pertussis toxin antibody ELISA increased the incidence of confirmed pertussis in persons 11-19 years old from 3.0 to 12.9 per 100,000 and in persons > or = 20 years old from 0.16 to 0.56 per 100,000. Bacteriologic methods underestimate pertussis incidence, but a single serum anti-pertussis toxin antibody ELISA is a practical method for population-based diagnosis in adolescents and adults.     

Dexamethasone inhibition of interferon-alpha 2-induced stimulation of cortisol and growth hormone secretion in chronic myeloproliferative syndrome

This study investigated the acute effects of interferon-alpha 2 (IFN-alpha 2) on hormonal secretion in adult patients affected by a chronic myeloproliferative syndrome and tried to shed some light on the mechanism by which IFN-alpha 2 stimulates cortisol and GH secretion in humans. We compared the pattern of IFN-alpha 2-induced cortisol and GH release with that elicited after the same challenge given subsequent to pretreatment with dexamethasone (Dex). We studied eight patients affected by a chronic myeloproliferative syndrome (thrombocythemia) who had been selected for treatment with IFN-alpha 2. Four sets of experiments were performed: 1) 2 mL iv saline was given at 0800 h in eight cases; 2) 3 x 10(6) IU iv IFN-alpha 2 was given at 0800 h in eight cases; 3) 3 x 10(6) IU iv IFN-alpha 2 was given at 0800 h after pretreatment with 1.5 mg Dex (1 mg at midnight the previous night and 0.5 mg at 0700 h on the day of the test) in six cases; and 4) 2 mL iv saline was given at 0800 h after the same Dex pretreatment in four cases. Cortisol and GH were measured in plasma samples drawn at 30-min intervals between 0800 and 1300 h. Acute iv administration of IFN-alpha 2 stimulated the release of both cortisol and GH in each patient with a significant increment vs. control values, as assessed by areas under the curve. The administration of Dex significantly decreased basal plasma cortisol secretion and abolished cortisol response to IFN-alpha 2 administration. These data suggest that the stimulatory action of IFN-alpha 2 on cortisol release is mediated via a modulation of the activity of the hypothalamic-pituitary axis rather than through a direct effect at the level of the adrenal cortex. After Dex plus saline administration, no significant effect was observed on plasma GH levels, which remained low. Dex administration significantly decreased GH response to IFN-alpha 2. These data suggest that a hypothalamic or pituitary stimulation (or both) is involved in the mechanism of IFN-alpha 2-induced GH secretion. It remains to be established whether IFN-alpha 2 directly stimulates pituitary somatotropic cells or whether the cytokine exerts a stimulatory action on GH secretion by indirectly modulating the hypothalamic or pituitary activity. In conclusion, acute iv administration of IFN-alpha 2 represents a potent stimulus for cortisol and GH secretion in adult human subjects.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neonatal androgen and estrogen treatments masculinize the size of motoneurons in the rat spinal nucleus of the bulbocavernosus

Electrically-assisted transdermal delivery (EATDD) is the facilitated transport of compounds across the skin using an electromotive force. It has been extensively explored as a potential means for delivering peptides and other hydrophilic, acid-labile or orally unstable products of biotechnology. The predominant mechanism for delivery is iontophoresis, although electroosmosis and electroporation have also been investigated. The focus of this review is to put these different mechanisms in perspective and relate them to the drug and skin model system being investigated.     

The posterior operative approach for cervical radiculopathy

Cervical disc disease includes acute herniation and chronic disc degeneration with secondary changes in the associated bone. The latter may lead to the spectrum of cervical spondylitic stenosis, which is considered to be multilevel and may be more of a bony disease. Clinically, cervical disc disorders can be divided into several disorders. The disorder of true cervical radiculopathy is associated with lateral compression of the nerve root. When this condition is due to a lateral soft disc herniation or lateral bony stenosis, the posterior cervical laminoforaminotomy is commonly used. It is a procedure that works extremely well in the vast majority of patients and there is no risk of spinal instability; therefore, no fusion is required. The details of operative care have been described. In patients who have persistent radicular problems after a failed anterior cervical interspace procedure, the posterior cervical laminoforaminotomy with posterior wiring and fusion is a simple and effective operative option.     

Beyond the interpersonal circumplex in group psychotherapy: the structure and relationship to outcome of the Individual Group Member Interpersonal Process Scale

Development of a new scale to measure patient behavior in group psychotherapy, the Individual Group Member Interpersonal Process Scale (IGIPS), is described. This scale captures clinically important aspects of how patients act in group, including how they respond to the other group members. The IGIPS was applied to videotapes of the first four sessions of seven 15-session outpatient therapy groups (52 patients) in the Mental Health Department of a health maintenance organization. The scale was found to have five factors (Activity, Interpersonal Sensitivity, Comfort with Self, Self-Focus, and Psychological Mindedness). Patient behavior that was moderate on two IGIPS dimensions was connected with better outcome than was that of patients with more extreme behavior in either direction.     

ACAT inhibitors derived from hetero-Diels-Alder cycloadducts of thioaldehydes

Acyl-CoA:cholesterol acyltransferase (ACAT) is the enzyme largely responsible for intracellular cholesterol esterification. A systemic inhibitor of ACAT is believed to be able to slow or even reverse the atherosclerotic process. Towards that goal, a series of cyclic sulfides, derived from the hetero-Diels-Alder reaction of thioaldehydes with 1,3-dienes, and bearing carboxamide substituents, were prepared and evaluated for in vitro (in several tissues and species) and ex vivo ACAT inhibition. Minor changes in subsequent structure were found to have a significant effect in optimization of the biological activity of this series of compounds.     

Adopting a policy of reuse

In many firms' experience, reusing elements of proven systems designs and software is best done through a companywide program. Reuse means savings in design, testing, manufacturing and support. The author gives the basic information a company needs to get started. The author discusses the context for reuse, planning and strategy involved in a reuse program