Detailed analysis of structures and formulations of cationic lipids for efficient gene transfer to the lung.

Cationic lipid-mediated gene transfer of cystic fibrosis transmembrane conductance regulator (CFTR) cDNA represents a promising approach for treatment of cystic fibrosis (CF). Here, we report on the structures of several novel cationic lipids that are effective for gene delivery to the lungs of mice. An amphiphile (#67) consisting of a cholesterol anchor linked to a spermine headgroup in a "T-shape" configuration was shown to be particularly efficacious. An optimized formulation of #67 and plasmid vector encoding chloramphenicol acetyl-transferase (CAT) was capable of generating up to 1 microgram of CAT enzyme/lung following intranasal instillation into BALB/c mice. This represents a 1,000-fold increase in expression above that obtained in animals instilled with naked pDNA alone and is greater than 100-fold more active than cationic lipids used previously for CFTR gene expression. When directly compared with adenovirus-based vectors containing similar transcription units, the number of molecules of gene product expressed using lipid-mediated transfer was equivalent to vector administration at multiplicities of infection ranging from 1 to 20. The level of transgene expression in the lungs of BALB/c mice peaked between days 1 and 4 post-instillation, followed by a rapid decline to approximately 20% of the maximal value by day 7. Undiminished levels of transgene expression in the lung could be obtained following repeated intranasal administration of #67:DOPE:pCF1-CAT in nude mice. Transfection of cells with formulations of #67:DOPE:pCF1-CFTR generated cAMP-stimulated CFTR chloride channel and fluid transport activities, two well-characterized defects associated with CF cells. Taken together, the data demonstrate that cationic lipid-mediated gene delivery and expression of CFTR in CF lungs is a viable and promising approach for treatment of the disease.

Senior-led freshman groups: a strategy for professional development.

Faculty members are challenged to promote professional nurse development in undergraduate students. The authors discuss an experimental teaching/learning strategy for senior nursing students and groups of freshman students in a baccalaureate nursing program. This strategy provided a unique opportunity for professional role socialization and school integration in first-year students and increased competence in teaching skills and confidence as professional role models in the seniors. Based on evaluations by the participants, the experimental teaching strategy was a positive experience for both freshman and senior students.

Paternal perspectives of the childbirth experience.

This study examined the responses given by first-time fathers who were asked to describe their feelings about their childbirth experience. The fathers answered three open-ended questions about their feelings concerning labor and childbirth, and the paternal behaviors believed to be most useful to their wives during labor and delivery. Data from fathers who attended prenatal childbirth education classes were examined separately from fathers who did not attend. The findings indicated that for all fathers, regardless of prenatal preparation, the labor experience evoked generally positive responses in addition to a significant number of negative responses, while perceptions of the birth experience were primarily characterized by positive or very positive feelings. The results also indicated that the fathers perceived that they were most helpful to their partner during labor.

Adjustment to new parenthood: attenders versus nonattenders at prenatal education classes.

This longitudinal descriptive study compared the adjustment to new parenthood in two groups of first-time mothers and fathers. Participants included 106 married couples, 58 (55%) who attended prenatal childbirth education classes and 48 (45%) who did not. The study variables included prenatal, intrapartal, and new parent experiences. All mothers and fathers completed questionnaires during the last trimester of pregnancy and one month after delivery of a healthy newborn. Fathers were present during labor and birth regardless of prenatal class attendance. The groups differed in maternal age and in maternal and paternal education levels, but did not differ in measures of prenatal attachment, paternal childbirth involvement, childbirth satisfaction, parenting sense of competence, and ease of transition to parenthood. The results suggest the need for further study of the influence of prenatal classes on becoming a new parent, and of the effects of the father's presence during childbirth on birth and new parent experiences.

The use of bupropion hydrochloride for smoking cessation therapy.

All healthcare providers have an obligation to promote smoking cessation to their patients who smoke. While patients are advised to stop smoking, the use of specific smoking cessation strategies is rarely addressed by primary care providers. This article discusses smoking cessation for two specifically vulnerable groups: women and African-Americans. Both groups have been identified as having increased rates of nicotine metabolism and may be less likely to respond effectively to nicotine replacement for smoking cessation. Increased nicotine metabolism results in greatest difficulty with successful smoking cessation and creates an increased vulnerability to a wide range of cardiovascular and respiratory health problems associated with smoking. Nicotine replacement therapy for smoking cessation has been the most common treatment available; however, many patients have not been successful with that method, and a majority (75%) of smokers who attempt to stop smoking return to smoking. The use of bupropion hydrochloride (Zyban) as a nonnicotine replacement therapy for smoking cessation is discussed. Relapses in smoking cessation are due to nicotine craving and the attempt to alleviate symptoms of nicotine withdrawal. Bupropion hydrochloride, an oral antidepressant, is believed to work by elimination of nicotine cravings and to decrease the physiologic and psychologic symptoms associated with nicotine withdrawal. Patients who have not been successful in smoking cessation may benefit from bupropion hydrochloride therapy in conjunction with counseling and behavior modification.

Differential inhibition of multiple cAMP phosphodiesterase isozymes by isoflavones and tyrphostins.

A series of isoflavone and tyrphostin compounds were found to inhibit the degradation of cAMP by several cyclic nucleotide phosphodiesterase (PDE) isozymes. Specific hydroxyl groups on the isoflavone structure were critical for PDE isozyme-selective inhibition. Replacement of the C-7 hydroxyl group of the isoflavone with a methoxy group raised the IC(50) for PDE1, PDE3, and PDE4. The absence of the C-5 hydroxyl group raised the IC(50) from 5 to >100 microM for PDE4, but actually lowered the IC(50) for PDE3 and PDE1. Replacement of the C-4' hydroxyl group with a methoxy group raised the IC(50) for PDE3 and PDE1, yet only slightly changed the IC(50) for PDE4. Various tyrphostins were also potent inhibitors of PDE1, PDE3, and PDE4. The four-carbon side chained tyrphostins were much less potent; however, a very interesting pattern was observed in which removal of phenolic hydroxyls on the tyrphostin structure increased the potency for PDE1 and PDE3, but not PDE4. These results may help to explain some of the therapeutic and intracellular signaling effects of isoflavones and tyrphostins. Moreover, the isozyme selectivity demonstrated by the isoflavones and tyrphostins can serve as a pharmacophore for the design of specific PDE inhibitors.

Tyrosine kinase-independent inhibition of cyclic-AMP phosphodiesterase by genistein and tyrphostin 51.

The phosphodiesterase activity in the HT4.7 neural cell line was pharmacologically characterized, and phosphodiesterase isozyme 4 (PDE4) was found to be the predominant isozyme. The Km for cAMP was 1-2 microM, indicative of a "low Km" phosphodiesterase, and the activity was inhibited by PDE4-selective inhibitors rolipram and Ro20-1724, but not PDE3- or PDE2-selective inhibitors. Calcium, calmodulin, and cGMP, regulators of PDE1, PDE2, and PDE3, had no effect on cAMP hydrolysis. The protein tyrosine kinase inhibitor, genistein, inhibited HT4.7 cAMP phosphodiesterase activity by 85-95% with an IC50 of 4 microM; whereas daidzein, an inactive structural analog of genistein, had little effect on phosphodiesterase activity. This is a common pharmacological criterion used to implicate the regulation by a tyrosine kinase. However, genistein still inhibited phosphodiesterase activity with a mixed pattern of inhibition even when ion-exchange chromatography was used to partially purify phosphodiesterase away from the tyrosine kinase activity. Moreover, tyrphostin 51, another tyrosine kinase inhibitor, was found to also inhibit partially purified phosphodiesterase activity noncompetitively. These data suggest that HT4.7 phosphodiesterase activity is dominated by PDE4 and can be regulated by genistein and tyrphostin 51 by a tyrosine kinase-independent mechanism.

Characterization of recombinant, soluble beta-secretase from an insect cell expression system.

The beta-site amyloid precursor protein-cleaving enzyme (BACE) cleaves the amyloid precursor protein to produce the N terminus of the amyloid beta peptide, a major component of the plaques found in the brains of Alzheimer's disease patients. Sequence analysis of BACE indicates that the protein contains the consensus sequences found in most known aspartyl proteases, but otherwise has only modest homology with aspartyl proteases of known three-dimensional structure (i.e., pepsin, renin, or cathepsin D). Because BACE has been shown to be one of the two proteolytic activities responsible for the production of the Abeta peptide, this enzyme is a prime target for the design of therapeutic agents aimed at reducing Abeta for the treatment of Alzheimer's disease. Toward this ultimate goal, we have expressed a recombinant, truncated human BACE in a Drosophila melanogaster S2 cell expression system to generate high levels of secreted BACE protein. The protein was convenient to purify and was enzymatically active and specific for cleaving the beta-secretase site of human APP, as demonstrated with soluble APP as the substrate in novel sandwich enzyme-linked immunosorbent assay and Western blot assays. Further kinetic analysis revealed no catalytic differences between this recombinant, secreted BACE, and brain BACE. Both showed a strong preference for substrates that contained the Swedish mutation, where NL is substituted for KM immediately upstream of the cleavage site, relative to the wild-type sequence, and both showed the same extent of inhibition by a peptide-based inhibitor. The capability to produce large quantities of BACE enzyme will facilitate protein structure determination and inhibitor development efforts that may lead to the evolution of useful Alzheimer's disease treatments.