Quantitative analysis of receptor-mediated uptake and pro-apoptotic activity of mistletoe lectin-1 by high content imaging.

Ribosome inactivating proteins (RIPs) are highly potent cytotoxins that have potential as anticancer therapeutics. Mistletoe lectin 1 (ML1) is a heterodimeric cytotoxic protein isolated from European Mistletoe and belongs to RIP class II. The aim of this project was to systematically study ML1 cell binding, endocytosis pathway(s), subcellular processing and apoptosis activation. For this purpose, state of the art cell imaging equipment and automated image analysis algorithms were used. ML1 displayed very fast binding to sugar residues on the membrane and energy-dependent uptake in CT26 cells. The co-staining with specific antibodies and uptake blocking experiments revealed involvement of both clathrin-dependent and -independent pathways in ML1 endocytosis. Co-localization studies demonstrated the toxin transport from early endocytic vesicles to Golgi network; a retrograde road to the endoplasmic reticulum. The pro-apoptotic and antiproliferative activity of ML1 were shown in time lapse movies and subsequently quantified. ML1 cytotoxicity was less affected in multidrug resistant tumor cell line 4T1 in contrast to commonly used chemotherapeutic drug (ML1 resistance index 6.9 vs 13.4 for doxorubicin; IC50: ML1 1.4 ng/ml vs doxorubicin 24000 ng/ml). This opens new opportunities for the use of ML1 as an alternative treatment in multidrug resistant cancers.

Substitution of Yor1p NBD1 residues improves the thermal stability of Human Cystic Fibrosis Transmembrane Conductance Regulator.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a plasma membrane chloride channel protein that regulates vertebrate fluid homeostasis. The inefficiency of wild type human CFTR protein folding/trafficking is exacerbated by genetic mutations that can cause protein misfolding in the endoplasmic reticulum (ER) and subsequent degradation. This project investigates small changes in protein sequence that can alter the thermal stability of the large multi-domain CFTR protein. We target a conserved 70-residue α-subdomain located in the first nucleotide-binding domain that hosts the common misfolding mutation ∆F508. To investigate substitutions that can stabilize this domain, we constructed chimeras between human CFTR and its closest yeast homolog Yor1p. The α-subdomain of Yor1p was replaced with that of CFTR in Saccharomyces cerevisiae. Cellular localization of green fluorescence protein-tagged Yor1p-CFTR chimeras was analyzed by fluorescence microscopy and quantitative multispectral imaging flow cytometry, steady-state protein levels were compared by SDS-PAGE and protein function probed by a phenotypic oligomycin resistance assay. The chimeras exhibited ER retention in yeast characteristic of defective protein folding/processing. Substitution of seven CFTR α-subdomain residues that are highly conserved in Yor1p and other transporters but differ in CFTR (S495P/R516K/F533L/A534P/K536G/I539T/R553K) improved Yor1p-CFTR chimera localization to the yeast plasma membrane. When introduced into human CFTR expressed in mammalian cells, the same substitutions improve the purified protein thermal stability. This stabilized human CFTR protein will be directly useful for structural and biophysical studies that have been limited by the thermal sensitivity of wild type CFTR. The insights into critical structural residues within CFTR could facilitate development of effective therapeutics for CF-causing mutations.

Electron holography on HfO2/HfO2-x bilayer structures with multilevel resistive switching properties.

Unveiling the physical nature of the oxygen-deficient conductive filaments (CFs) that are responsible for the resistive switching of the HfO2-based resistive random access memory (RRAM) devices represents a challenging task due to the oxygen vacancy related defect nature and nanometer size of the CFs. As a first important step to this goal, we demonstrate in this work direct visualization and a study of physico-chemical properties of oxygen-deficient amorphous HfO2-x by carrying out transmission electron microscopy electron holography as well as energy dispersive x-ray spectroscopy on HfO2/HfO2-x bilayer heterostructures, which are realized by reactive molecular beam epitaxy. Furthermore, compared to single layer devices, Pt/HfO2/HfO2-x /TiN bilayer devices show enhanced resistive switching characteristics with multilevel behavior, indicating their potential as electronic synapses in future neuromorphic computing applications.

FIB based fabrication of an operative Pt/HfO2/TiN device for resistive switching inside a transmission electron microscope.

Recent advances in microelectromechanical systems (MEMS) based chips for in situ transmission electron microscopy are opening exciting new avenues in nanoscale research. The capability to perform current-voltage measurements while simultaneously analyzing the corresponding structural, chemical or even electronic structure changes during device operation would be a major breakthrough in the field of nanoelectronics. In this work we demonstrate for the first time how to electrically contact and operate a lamella cut from a resistive random access memory (RRAM) device based on a Pt/HfO2/TiN metal-insulator-metal (MIM) structure. The device was fabricated using a focused ion beam (FIB) instrument and an in situ lift-out system. The electrical switching characteristics of the electron-transparent lamella were comparable to a conventional reference device. The lamella structure was initially found to be in a low resistance state and could be reset progressively to higher resistance states by increasing the positive bias applied to the Pt anode. This could be followed up with unipolar set/reset operations where the current compliance during set was limited to 400 µA. FIB structures allowing to operate and at the same time characterize electronic devices will be an important tool to improve RRAM device performance based on a microstructural understanding of the switching mechanism.

Justice and impoverished women: the ethical implications of work-based welfare.

Current U.S. welfare policy, Temporary Assistance for Needy Families, requires impoverished people to work in order to receive welfare, and it limits cash support to 5 years. Most of the people who have used this program are single-parent women, and a disturbing number have been terminated at 5 years, not having made a successful transition to work. The purpose of this longitudinal study was to explore the barriers to success and the social justice of the program from the perspective of single-parent women who were terminated. In all, 41 women were recruited through community-based purposive sampling, and the primary research methods were a qualitative, narrative interview approach and narrative analysis. Data from the semistructured interview guide are reported here. Findings describe health and socioeconomic burdens, and barriers that lie within the social policy. The study has ethical implications for nursing advocacy, and it informs nursing interventions for impoverished women and their families.

Cell cycle-dependent degradation of the Saccharomyces cerevisiae spindle motor Cin8p requires APC(Cdh1) and a bipartite destruction sequence.

Saccharomyces cerevisiae Cin8p belongs to the BimC family of kinesin-related motor proteins that are essential for spindle assembly. Cin8p levels were found to oscillate in the cell cycle due in part to a high rate of degradation imposed from the end of mitosis through the G1 phase. Cin8p degradation required the anaphase-promoting complex ubiquitin ligase and its late mitosis regulator Cdh1p but not the early mitosis regulator Cdc20p. Cin8p lacks a functional destruction box sequence that is found in the majority of anaphase-promoting complex substrates. We carried out an extensive mutagenesis study to define the cis-acting sequence required for Cin8p degradation in vivo. The C terminus of Cin8p contains two elements required for its degradation: 1) a bipartite destruction sequence composed of a KEN-box plus essential residues within the downstream 22 amino acids and 2) a nuclear localization signal. The bipartite destruction sequence appears in other BimC kinesins as well. Expression of nondegradable Cin8p showed very mild phenotypic effects, with an increase in the fraction of mitotic cells with broken spindles.

Preceptors: a perspective of what works.

This article provides a perspective of clinical preceptor contributions to advanced practice nurse education. It supports quality clinical education in the face of the proliferation of academic programs, and the economics of the resulting competition. The strategies of 2 universities are used as examples of how nurse practitioner programs respond to issues such as who precepts students, how preceptors are identified, what draws preceptors to the role, and the effects of students on clinic productivity.

Mitotic motors in Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae provides a unique opportunity for study of the microtubule-based motor proteins that participate in mitotic spindle function. The genome of Saccharomyces encodes a relatively small and genetically tractable set of microtubule-based motor proteins. The single cytoplasmic dynein and five of the six kinesin-related proteins encoded have been implicated in mitotic spindle function. Each motor protein is unique in amino acid sequence. On account of functional overlap, no single motor is uniquely required for cell viability, however. The ability to create and analyze multiple mutants has allowed experimental dissection of the roles performed by each mitotic motor. Some of the motors operate within the nucleus to assemble and elongate the bipolar spindle (kinesin-related Cin8p, Kip1p, Kip3p and Kar3p). Others operate on the cytoplasmic microtubules to effect spindle and nuclear positioning within the cell (dynein and kinesin-related Kip2p, Kip3p and Kar3p). The six motors apparently contribute three fundamental activities to spindle function: motility, microtubule cross-linking and regulation of microtubule dynamics.

Focus groups and vulnerable populations. Insight into client strengths and needs in complex community health care environments.

Focus groups are a useful qualitative research technique to assist in interpreting quantitative community assessment data. Data obtained from focus groups can provide sociological and psychological insights into the perceptions of population subgroups and suggest answers to the "why" questions raised by descriptive data about such issues as teen pregnancy, poverty, immunization levels, or lifestyle-related morbidity and mortality. Application of these insights can lead to the better use of community strengths and the creation of community-specific responses to barriers to health care. Focus groups work well for involving hard-to-reach members of a community in program development, planning, and evaluation. They may be more effective than face-to-face interviews and questionnaires because people often have not thought about how they feel and tend not to form opinions in isolation (1). The information sought through the use of focus groups is not randomly distributed in the population. Thus, groups are not randomly selected, and data are not gathered with the intent to generalize to all populations.

Identification and characterization of an endo/exonuclease in Pneumocystis carinii that is inhibited by dicationic diarylfurans with efficacy against Pneumocystis pneumonia.

Dicationic diarylfurans and dicationic carbazoles are under development as therapeutic agents against opportunistic infections. While their ability to bind to the minor groove of DNA has been established, the complete mechanism of action has not. We demonstrate here that an effective diarylfuran, 2,5-bis[4-(N-isopropylguanyl)phenyl]furan, inhibits an endo/exonuclease activity present in Pneumocystis carinii, Cryptococcus neoformans, Candida albicans, and Saccharomyces cerevisiae. This activity was purified from the particulate fraction of P. carinii. The enzyme requires Mg++ or Mn++, and shows preferences for single-over double stranded DNA and for AT-rich over GC-rich domains. A panel of 12 dicationic diarylfurans and eight dicationic carbazoles, previously synthesized, were evaluated for inhibition of the purified nuclease and for efficacy against Pneumocystis pneumonia in rats. Among the diarylfurans, potency of nuclease inhibition, in vivo antimicrobial activity, and DNA binding strength were all strongly correlated (p < 0.001). These findings suggest that one target for antimicrobial action of the diarylfurans may be a nucleolytic or other event requiring unpairing of DNA strands. Dicationic carbazoles which were strong nuclease inhibitors all displayed anti-Pneumocystis activity in vivo, but there were also noninhibitory carbazoles with in vivo efficacy.

Survey data collection: operationalizing the research design.

This article describes how indigenous interviewers were used to collect data about the health needs and resources in a black South African township. The survey was done during the dismantling of the apartheid political system of South Africa. The political unrest, distrust, and tension were barriers to carrying out a survey and threatened the quality of the data collection. A vulnerability of survey research is the difficulty in controlling the variables of the community and the interviewer during the process of data collection. How this survey was carried out, in this unstable setting, influenced the quality of the data and the validity and reliability of the research. The data-collection requirements were carried out in a way that was functional in the real-world setting while maintaining research standards. The criteria used for hiring interviewers and the content and delivery of training were effective in this tense, educationally disadvantaged community setting. Methods that were used to motivate and supervise interviewers were successful and are recommended for use in similar survey research.

Building community participation in health care: a model and example from South Africa.

A conceptual model was developed for a community-based intervention study in a Black township in South Africa. The model shows a useful way to structure the complex role public health nurses play as they meet community health needs using a community's priorities and building toward community involvement in health and self-care. The model was applied over a 2-year period in an under-developed community of 100,00 people where the unemployment rate was over 50%, fewer than 10% of the homes had electricity, and only one-third had access to the sewage removal system. Over half of older adults interviewed were illiterate. The residents, in collaboration with the nurse researcher, gathered data, prioritized needs, and chose projects to produce solutions. The model guided activities for community empowerment through a deliberate transfer of information and expertise from the nurse to members of the community. Conceptual models or paradigms are useful to focus nursing strategies, to guide professional nursing practice, and to support interdisciplinary goals for cooperative efforts. The principles are also applicable in the United States and other developed countries as more effective ways to achieve health goals are sought.

Regulation of calcium influx and catecholamine secretion in chromaffin cells by a cytochrome P450 metabolite of arachidonic acid.

These studies were designed to determine the role of arachidonic acid metabolites in catecholamine secretion from adrenal chromaffin cells. Inhibitors of the cytochrome P450-dependent metabolism of arachidonic acid were shown to interfere with stimulus-secretion coupling in cultured chromaffin cells. Ketoconazole (10 microM), clotrimazole (20 microM), and piperonyl butoxide (50 microM) inhibited carbachol-dependent catecholamine secretion by 44%, 83%, and 100%, respectively; histamine-dependent secretion by 25%, 60%, and 81%, and secretion induced by 59 mM KCl depolarization by 25%, 55%, and 89%. Uptake of 45Ca2+ into the cells in response to carbachol was inhibited 63% by ketoconazole, 86% by clotrimazole, and 95% by piperonyl butoxide; KCl-dependent uptake was inhibited 7%, 56%, and 85%, respectively. However, cytochrome P450 inhibitors did not inhibit catecholamine secretion when cells were stimulated with the calcium ionophores ionomycin or lasalocid. These results indicated the involvement of a cytochrome P450 product in controlling Ca2+ influx in response to membrane depolarization. Cells prelabeled with [3H]arachidonic acid formed a 3H-labeled metabolite which comigrated with authentic 5,6-epoxyeicosatrienoic (5,6-EET) acid on reverse phase and normal phase HPLC. Pretreatment with clotrimazole inhibited the production of this 3H-labeled metabolite. Addition of synthetic 5,6-EET (1 nM) to cells pretreated with piperonyl butoxide resulted in catecholamine secretion. These data suggest a role for a cytochrome P450 metabolite of arachidonic acid in agonist-stimulated catecholamine secretion.

[Lipid island (xanthelasma) of the duodenal mucosa--a rarely found diagnosis and differential diagnosis]. / Die Lipidinsel (Xanthelasma) der Duodenalschleimhaut--ein seltener Befund Diagnose und Differentialdiagnose.

A case report ist given on a 54-year-old patient with the so called lipid island (xanthelasma) in the duodenal mucosa. Xanthelasmas are very rarely located in the duodenal mucosa. The diagnosis and differential diagnosis of this entity are reported. The relevance of histochemistry and immunohistochemistry for differentiation of signet ring carcinoma cells in the mucosa is discussed.

Comparison of recombination in vitro and in E. coli cells: measure of the effective concentration of DNA in vivo.

Despite the extremely high concentration of DNA in nucleoid/nuclear regions, chromosomal dimerization and entanglement are avoided. To help understand this, we measured the effective concentration of DNA in E. coli, a value that reflects the functional impact of the cellular milieu on DNA site reactivity. We used as probes plasmid fusion reactions by two site-specific recombinases. The normalized extents and rates of fusion in these systems were much lower in vivo than in analogous in vitro reactions. We calculate that the effective concentration of plasmid DNA is about one order of magnitude lower than the chemical concentration. We suggest that in bacterial cells DNA accessibility is highly restricted and that this dominates the forces that increase DNA activity, such as macromolecular crowding.

A model for community involvement in health (CIH) program development.

Community involvement in health (CIH), a central concept in health development, is a participatory approach to health care that is organized from the perspective of the recipient. Putting CIH into practice represents a learning experience for the community, the health professionals involved and those responsible for the national climate in which this change takes place. The CIH process was operationalized over a two-year period in a black township in South Africa. A community survey identified the health needs and capacities related to the elderly, their families and their support system. Community groups and individuals, in partnership with the researcher, prioritized the needs that had been identified and then implemented four programs related to those needs. A process model was developed that provided the structure for initiating and maintaining these programs. The model helped people who were new to the community organizing to focus on general principles. It was flexible so that programs could be interpreted and implemented in the context of local culture and resources. The model was functional in guiding community nurses, lay community members and employees in health-related programs through the process of starting new programs. This approach empowered participants to move beyond only hoping for change or being puzzled by its elusiveness.

Prevention of atherosclerosis and end-organ damage: a basis for antihypertensive interventional strategies.

AIM: To review the nature of the complex relationships between essential hypertension and cardiovascular end-organ damage, with a particular focus on the pathogenesis and prevention of coronary heart disease, the major complication of untreated hypertension. RISK FACTORS FOR CORONARY HEART DISEASE: Both atherosclerosis and hypertension have their origins in childhood; in the second and third decades of life development of the more advanced fibrous plaques accelerates, emphasizing the need for early diagnosis and intervention. Perplexing and complex relationships have been found among the principal risk factors for coronary heart disease, hyperinsulinemia, insulin resistance, dyslipidemia and hypertension. In the pathogenesis of atherosclerosis at the cellular and molecular level, the important features are the effects of monocyte-macrophages, oxidant stress, lipoprotein modification, inflammatory mediators and the focal hemodynamic environment. Even brief periods of experimental hypertension can accentuate atherogenesis, the effects of which are greatest but not limited to the cervical and cerebral arteries. Further, acute hypertension lasting for even a few minutes causes a 'leakage' of plasma proteins and particulate probes into the artery wall, which has far-reaching implications for antihypertensive therapy. Recent work has shown that 24-h blood pressure variability is correlated with target-organ damage in hypertensive patients. THERAPY: Antihypertensive therapy should not only lower blood pressure but also prevent significant short-term blood pressure fluctuations. The trough: peak ratio has been used to assess the effect of antihypertensive treatment on blood pressure variability. CONCLUSION: More intensive research is required to clarify the nature of the interface between hypertension and atherogenesis.