Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis.

Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.

A modified hTERT promoter-directed oncolytic adenovirus replication with concurrent inhibition of TGFbeta signaling for breast cancer therapy.

We were interested in developing oncolytic adenoviral vectors that can be administered systemically for the treatment of breast cancer. To restrict viral replication in breast tumor cells, we constructed mhTERTAd.sTbetaRFc, a 01/07-based adenoviral vector expressing the soluble form of transforming growth factor-beta (TGFbeta) receptor II fused with the human Fc IgG1 (sTGFbetaRIIFc) gene, in which viral replication is under the control of a modified human telomerase reverse transcriptase (mhTERT) promoter. In addition, mhTERTAd.sTbetaRFc-mediated sTGFbetaRIIFc production targets the TGFbeta pathway known to contribute to the tumor progression of breast cancer metastasis. We chose to use the mhTERT promoter because it was found to be relatively more active (approximately 20 times) in breast cancer cells compared with normal human cells. We showed that infection of MDA-MB-231 and MCF-7 breast cancer cells for 48 h with mhTERTAd.sTbetaRFc produced high levels of sTGFbetaRIIFc (greater than 1 microg ml(-1)) in the medium. Breast cancer cells produced nearly a 6000-fold increase in viral titers during the 48 h infection period. However, mhTERTAd.sTbetaRFc replication was attenuated in normal cells. Infection of breast cancer cells with a replication-deficient virus Ad(E1(-)).sTbetaRFc also produced high levels of sTGFbetaRIIFc, but under these conditions, no detectable viral replication was observed. Adenoviral-mediated production of sTGFbetaRIIFc was shown to bind with TGFbeta-1, and to abolish the effects of TGFbeta-1 on downstream SMAD-3 phosphorylation. The administration of mhTERTAd.sTbetaRFc intravenously into MDA-MB-231 human xenograft-bearing mice resulted in a significant inhibition of tumor growth and production of sTGFbetaRIIFc in the blood. Conversely, intravenous injection of Ad(E1(-)).sTbetaRFc did not show a significant inhibition of tumor growth, but resulted in sTGFbetaRIIFc in the blood, suggesting that viral replication along with sTGFbetaRIIFc protein production is critical in inducing the inhibition of tumor growth. These results warrant future investigation of mhTERTAd.sTbetaRFc as an antitumor agent in vivo.

Mechanisms of chiral discrimination by topoisomerase IV.

Topoisomerase IV (Topo IV), an essential ATP-dependent bacterial type II topoisomerase, transports one segment of DNA through a transient double-strand break in a second segment of DNA. In vivo, Topo IV unlinks catenated chromosomes before cell division and relaxes positive supercoils generated during DNA replication. In vitro, Topo IV relaxes positive supercoils at least 20-fold faster than negative supercoils. The mechanisms underlying this chiral discrimination by Topo IV and other type II topoisomerases remain speculative. We used magnetic tweezers to measure the relaxation rates of single and multiple DNA crossings by Topo IV. These measurements allowed us to determine unambiguously the relative importance of DNA crossing geometry and enzymatic processivity in chiral discrimination by Topo IV. Our results indicate that Topo IV binds and passes DNA strands juxtaposed in a nearly perpendicular orientation and that relaxation of negative supercoiled DNA is perfectly distributive. Together, these results suggest that chiral discrimination arises primarily from dramatic differences in the processivity of relaxing positive and negative supercoiled DNA: Topo IV is highly processive on positively supercoiled DNA, whereas it is perfectly distributive on negatively supercoiled DNA. These results provide fresh insight into topoisomerase mechanisms and lead to a model that reconciles contradictory aspects of previous findings while providing a framework to interpret future results.

Statistical determination of the step size of molecular motors.

Molecular motors are enzymatic proteins that couple the consumption of chemical energy to mechanical displacement. In order to elucidate the translocation mechanisms of these enzymes, it is of fundamental importance to measure the physical step size. The step size can, in certain instances, be directly measured with single-molecule techniques; however, in the majority of cases individual steps are masked by noise. The step size can nevertheless be obtained from noisy single-molecule records through statistical methods. This analysis is analogous to determining the charge of the electron from current shot noise. We review methods for obtaining the step size based on analysing, in both the time and frequency domains, the variance in position from noisy single-molecule records of motor displacement. Additionally, we demonstrate how similar methods may be applied to measure the step size in bulk kinetic experiments.

Screening for substance use in pregnancy: a practical approach for the primary care physician.

Our goal was to identify risk factors for substance use during pregnancy for primary care physicians so that we could assess a woman's risk of alcohol or illicit drug use. Participants were 2002 Medicaid-eligible pregnant women with < or =2 visits to prenatal care clinics in South Carolina and Washington State. Structured interviews were used to collect data. Logistic regressions and classification and regression trees identified predictors for pregnant women at high risk for substance use. Approximately 9% of the sample reported current use of either drugs or alcohol or both. Past use of alcohol or cigarettes, including during the month before pregnancy, most differentiated current drug or alcohol users from current nonusers. Our analysis suggests that primary care physicians can ask 3 questions in the context of a prenatal health evaluation to target women for referral to a full clinical assessment for drug and alcohol use.

Understanding organizational reengineering in health care: strategies for social work's survival.

Confronted with decreasing reimbursement and managed care, hospitals across the country have been "reengineering" in an effort to streamline processes and enhance revenue. Because of the financial implications of discharge planning, hospital social workers and their departments are typically examined and realigned in some way during a reengineering process. To survive in this environment, social workers must understand the principles of reengineering; its hoped for and actual impact; be able to identify opportunities to contribute; and have the capacity to cope with an evolving professional role. This paper presents a general overview of reengineering and its adaptation to the health care environment. Specific challenges for medical social workers are described along with strategies for surviving in a reengineered health care system.

Characterization of photodamage to Escherichia coli in optical traps.

Optical tweezers (infrared laser-based optical traps) have emerged as a powerful tool in molecular and cell biology. However, their usefulness has been limited, particularly in vivo, by the potential for damage to specimens resulting from the trapping laser. Relatively little is known about the origin of this phenomenon. Here we employed a wavelength-tunable optical trap in which the microscope objective transmission was fully characterized throughout the near infrared, in conjunction with a sensitive, rotating bacterial cell assay. Single cells of Escherichia coli were tethered to a glass coverslip by means of a single flagellum: such cells rotate at rates proportional to their transmembrane proton potential (Manson et al.,1980. J. Mol. Biol. 138:541-561). Monitoring the rotation rates of cells subjected to laser illumination permits a rapid and quantitative measure of their metabolic state. Employing this assay, we characterized photodamage throughout the near-infrared region favored for optical trapping (790-1064 nm). The action spectrum for photodamage exhibits minima at 830 and 970 nm, and maxima at 870 and 930 nm. Damage was reduced to background levels under anaerobic conditions, implicating oxygen in the photodamage pathway. The intensity dependence for photodamage was linear, supporting a single-photon process. These findings may help guide the selection of lasers and experimental protocols best suited for optical trapping work.

Do advanced practice nurses and physician assistants benefit small rural hospitals?

Advanced practice nurses and physician assistants have offered small, rural hospitals an alternative to scarce primary care physicians for 30 years. This paper uses survey data from 285 small rural hospitals and case studies of 36 of these hospitals to answer questions about the extent to which advanced practice nurses and physician assistants provide primary care in small, rural hospitals, the benefits that might bring to the hospitals as well as the reactions of the public. The study used survey data collected as part of an evaluation of 285 hospitals, which received a Rural Health Care Transition grant from the Health Care Financing Administration in 1993 and 1994. Most of the hospitals used the practitioners; 70 percent used nurse practitioners; 30 percent used physician assistants; and 20 percent used both. There were some negative reactions to the use of the practitioners, but, overall, there was acceptance and benefits to the hospitals in the form of reduced recruitment costs, increased revenues and increased service offerings. These practitioners are beneficial to rural hospitals, and mechanisms to encourage their acceptance should be developed and implemented.

Advance directives: the experience of health care professionals across the continuum of care.

This study explored the perceptions of health care providers across the continuum of care as to how effective advance directive arrangements were in assuring compliance with the patients' wishes, as well as their overall satisfaction levels with the process. The health care providers who responded to the survey indicated high levels of overall satisfaction with advance directives, despite low patient completion rates and, most significantly, low confidence levels that surrogate decision making accurately reflected the patient's wishes. A secondary analysis compared the perceptions of health care providers with consumers' reasons for failing to complete advance directives. The need for further education was perceived as a primary need by the health care professionals but not by the consumers. Recommendations for policy initiatives and further research are then presented.

Reengineering outcomes management: an integrated approach to managing data, systems, and processes.

The integration of outcomes management into organizational reengineering projects is often overlooked or marginalized in proportion to the entire project. Incorporation of an integrated outcomes management program strengthens the overall quality of reengineering projects and enhances their sustainability. This article presents a case study in which data, systems, and processes were reengineered to form an effective Outcomes Management program as a component of the organization's overall project. The authors describe eight steps to develop and monitor an integrated outcomes management program. An example of an integrated report format is included.

Transmission of vancomycin-resistant enterococcus among family members: a case study.

Enterococci are persistent organisms naturally occurring in the digestive tract, able to persist on environmental surfaces for days or weeks, surviving heat and desiccation. They are the second most common bacterial cause of nosocomial infection. Recent strains of enterococci resistant to vancomycin pose a serious health hazard to hospitalized, institutionalized, and immunocompromised patients. This article presents a case study of community-acquired vancomycin-resistant enterococcus in an otherwise healthy child and suggests strategies for management and containment in the community.

Nonlamellar phases induced by the interaction of gramicidin S with lipid bilayers. A possible relationship to membrane-disrupting activity.

The interactions of the cyclic peptide gramicidin S (GS) with a variety of single-component lipid bilayers, and with membrane polar lipid extracts of Acholeplasma laidlawii B and Escherichia coli, were examined by differential scanning calorimetry (DSC), 31P-nuclear magnetic resonance (NMR) spectroscopy, and X-ray diffraction. The DSC data indicate that the effects of GS on the thermotropic phase behavior of phosphatidylcholine and phosphatidylethanolamine dispersions are compatible with those expected of peptides interacting primarily with the polar headgroup and/or the polar/apolar interfaces of lipid bilayers. These DSC studies also suggest that GS exhibits stronger interactions with the more fluid bilayers. For mixtures of GS with lipids such as phosphatidylcholine, phosphatidylserine, cardiolipin, and sphingomyelin, axially symmetric 31P-NMR powder patterns are observed throughout the entire temperature range examined (0-90 degrees C), and there is little evidence for significant destabilization of the lipid bilayer with respect to nonlamellar phases. With mixtures of GS with either phosphatidylethanolamine, phosphatidylglycerol, or a nonlamellar phase-forming phosphatidylcholine, axially symmetric 31P-NMR powder patterns are also observed at low temperatures. However, at high temperatures, an isotropic component is observed in their 31P-NMR spectra, and the relative intensity of this component increases significantly with temperature and with GS concentration. Once formed at high temperatures, this isotropic component exhibits a marked cooling hysteresis and in most cases disappears only when the sample is recooled to temperatures well below the lipid hydrocarbon chain-melting phase transition temperature. We also show that GS induces the formation of isotropic components in the 31P-NMR spectra of heterogeneous lipid mixtures such as occur in A. laidlawii B and E. coli membranes. These observations suggest that GS induces the formation of cubic or other three dimensionally ordered inverted nonlamellar phases when it interacts with some types of lipid bilayers, a suggestion strongly supported by our X-ray diffraction studies. Our results also suggest that the capacity of GS to induce the formation of such phases increases with the intrinsic nonlamellar phase-preferring tendencies of the lipids with which it interacts probably by producing localized increases in membrane monolayer curvature stress. The latter effect could be part of the mechanism through which this peptide exhibits its antimicrobial and hemolytic activities.

Cell type specific regulation of COUP-TF II promoter activity.

COUP-TF family orphan receptors regulate activity of ligand-activated nuclear hormone receptors or function independently in the regulation of gene expression. COUP-TF II has a complex expression pattern suggesting that different mechanisms are involved in the regulation of its expression. We isolated the 5' regulatory region of the mouse COUP-TF II gene and demonstrated that the basal promoter is localized in a -200 bp region 5' from the transcription start site. All-trans retinoic acid and dibutyryl cyclic AMP have cell type specific effects on COUP-TF II promoter activity. The effect of cyclic AMP is mediated by the cyclic AMP response element that is localized 74 nucleotides upstream from the major transcriptional start. In vitro promoter analyses also demonstrated that the effect of all-trans RA is not directly mediated by the binding of RARs or RXRs to the promoter sequence.

Role of lipid polymorphism in pulmonary surfactant.

The development of artificial surfactants for the treatment of respiratory distress syndrome (RDS) requires lipid systems that can spread rapidly from solution to the air-water interface. Because hydration-repulsion forces stabilize liposomal bilayers and oppose spreading, liposome systems that undergo geometric rearrangement from the bilayer (lamellar) phase to the hexagonal II (HII) phase could hasten lipid transfer to the air-water interface through unstable transition intermediates. A liposome system containing dipalmitoylphosphatidylcholine was designed; the system is stable at 23 degrees C but undergoes transformation to the HII phase as the temperature increases to 37 degrees C. The spreading of lipid from this system to the air-water interface was rapid at 37 degrees C but slow at 23 degrees C. When tested in vivo in a neonatal rabbit model, such systems elicited an onset of action equal to that of native human surfactant. These findings suggest that lipid polymorphic phase behavior may have a crucial role in the effective functioning of pulmonary surfactant.

Do smog advisories work? Results of an evaluation of the Canadian Smog Advisory Program.

OBJECTIVE: To determine the effectiveness of the Canadian Smog Advisory Program. METHODS: Telephone interviews covering perceptions, knowledge, recall of and response to advisories, and general views on their usefulness and effectiveness were conducted with 1,474 randomly selected individuals in four geographic areas. Multiple logistic regression was used to model advisory recall as a function of explanatory variables. RESULTS: Recall of advisories was higher (72%; 95% CI 68-77%) when measured immediately following an advisory in southern New Brunswick. Recall was lower when measured at the end of the smog "season", and varied significantly between Toronto (46%; 42-51%), Haldimand-Norfolk (18%; 14-24%) and Vancouver (61%; 56-65%). Education and geographic area (urban versus rural) were the strongest explanatory variables in two final multiple logistic regression models. A minority of those who recalled an advisory reported taking action. CONCLUSIONS: Smog advisories were partially successful in generating awareness, but only marginally effective in promoting action.