Structural and phylogenetic approaches to assess the significance of human Apolipoprotein E variation

Apolipoprotein E (APOE) is an important gene whose common polymorphism, and precisely the e *4 allele, has been reportedly associated with some disorders, including Alzheimer's disease (AD) and coronary artery disease. In the course of previous surveys on AD patients and healthy individuals some rare variants were detected by means of Isoelectric focusing and denaturing high-performance liquid chromatography techniques. After a mutation in a gene is identified, the problem arises to understand its effective significance. Structure modelling and phylogenetic analysis methods are widely used to establish the possible deleterious effect of mutations. In this study their usefulness in the analysis of APOE variants was evaluated. The two combined methods provided helpful indications for distinguishing between mutations possibly involved in AD susceptibility and not deleterious mutations.     

Apocynin derivatives interrupt intracellular signaling resulting in decreased migration in breast cancer cells

Cancer cells are defined by their ability to divide uncontrollably and metastasize to secondary sites in the body. Consequently, tumor cell migration represents a promising target for anticancer drug development. Using our high-throughput cell migration assay, we have screened several classes of compounds for noncytotoxic tumor cell migration inhibiting activity. One such compound, apocynin (4-acetovanillone), is oxidized by peroxidases to yield a variety of oligophenolic and quinone-type compounds that are recognized inhibitors of NADPH oxidase and may be inhibitors of the small G protein Rac1 that controls cell migration. We report here that while apocynin itself is not effective, apocynin derivatives inhibit migration of the breast cancer cell line MDA-MB-435 at subtoxic concentrations; the migration of nonmalignant MCF10A breast cells is unaffected. These compounds also cause a significant rearrangement of the actin cytoskeleton, cell rounding, and decreased levels of active Rac1 and its related G protein Cdc42. These results may suggest a promising new route to the development of novel anticancer therapeutics.     

Dielectrophoresis tweezers for single cell manipulation

Positioning single cells is of utmost importance in areas of biomedical research as diverse as in vitro fertilization, cell-cell interaction, cell adhesion, embryology, microbiology, stem cell research, and single cell transfection. Here we describe dielectrophoretic tweezers, a sharp glass tip with electrodes on either side, capable of trapping single cells with electric fields. Mounted on a micromanipulator, dielectrophoresis tweezers can position a single cell in three dimensions, holding the cell against fluid flow of hundreds of microns per second with more than 10 pN of force. We model the electric field produced by the tweezers and the field produced by coaxial microelectrodes. We show that cells are trapped without harm while they divide in the trap. In addition, dielectrophoretic tweezers offer the possibility for trapping, electroporating, and microinjecting a single cell with one probe.     

Redox stress and the contributions of BH3-only proteins to infarction

Ischemia followed by reperfusion is the primary cause of tissue injury and infarction during heart attack and stroke. The initiating stimulus is believed to involve reactive oxygen species that are produced during reperfusion when electron transport resumes in the mitochondria after suppression by ischemia. Programmed death has been shown to be a significant component of infarction, and evidence indicates that multiple pathways are initiated during both ischemia and reperfusion phases. Major infarction is preceded by severe ischemia that includes hypoxia, intracellular acidosis, glucose depletion, loss of ATP, and elevation of cytoplasmic calcium. The superimposition of a reactive oxygen surge on the latter condition provides the impetus for maximal damage. Compelling evidence implicates mitochondria not only as the source of initiating ROS but also as the focal sensors that translate the redox stress signal into a cellular-death response. Pivotal to this response are the BH3-only proteins that are activated by death signals and regulate mitochondrial communication with executioner proteins in the cytoplasm. The BH3-only proteins do this by controlling the activity of pores and channels in the outer mitochondrial membrane. To date at least six BH3-only proteins have been shown to contribute to ischemia-reperfusion death pathways in heart and/or brain; these include Bnip3, PUMA, Bid, Bad, HGTD-P, and Noxa. Here we review the evidence for these cell-death pathways and discuss their relevance to ischemic disease and infarction.     

A new technique for assessing hybrid layer interfacial micromorphology and integrity: two-photon laser microscopy

PURPOSE: This study describes a two-photon laser fluorescence microscopy technique developed to evaluate the interfacial micromorphology of the hybrid layer in bonded restorations. MATERIALS AND METHODS: Micropermeability of the hybrid layer was characterized by means of simultaneously contrasting a dye-containing adhesive with a differently colored dye placed into the pulp chamber and allowed to diffuse toward the different-colored hybrid layer. A fluorescent red dye (rhodamine B) was incorporated into a commercial dentin bonding agent. Class I preparations (margins in enamel) were made on extracted human third molars. The teeth were restored using conventional methods: bonding agent, composite, finishing, and polishing. An aqueous solution of a yellow/green dye (fluorescein) was then placed into the pulp chamber for 3 h, allowing time to diffuse toward the different-colored bonded interface. The teeth were then embedded, sectioned, and microscopically analyzed using two-photon laser microscopy at 40X magnification. RESULTS: Subsurface fluorescent imaging using this technique enabled interfacial micromorphology to be characterized at submicrometer resolution and provided high-contrast images. The quality of surrounding structures and potential presence of gaps were also precisely assessed. CONCLUSION: Two-photon laser microscopy provided high quality, high-resolution images of the bonded interface and surrounding areas, allowing accurate qualitative and quantitative analysis of the structure and integrity of the hybrid layer.     

Prosthetic intervention effects on activity of lower-extremity amputees

OBJECTIVE: To investigate the effect of prosthetic interventions on the functional mobility of lower-extremity amputees. DESIGN: Crossover with repeated measures. SETTING: Household and community environment. PARTICIPANTS: Twelve transtibial and 5 transfemoral amputees. INTERVENTIONS: For transtibial amputees, shock-absorbing versus rigid pylons. For transfemoral amputees, C-Leg versus Mauch SNS knees. MAIN OUTCOME MEASURES: Daily activity level (step count) and duration (minutes of activity). RESULTS: Pylon type had no effect on the daily activity level or duration of transtibial amputees. Knee type had no effect on the daily activity level or duration of transfemoral amputees. Transtibial amputees were more active on weekdays (3079+/-1515 steps/d) than weekends (2386+/-1225 steps/d) (P=.007). In general, lower-extremity amputees perform numerous short-duration bouts of activity (1-2 min) consisting of fewer than 17 steps/min, but activities of more than 15 minutes in duration were relatively rare (<1 per day). CONCLUSIONS: Intervention had no effect on amputee activity level and duration. Higher weekday activity levels of transtibial amputees suggest their vocational activities are more demanding than recreational activities. The fitting, alignment, and design of prosthetic components should be optimized for 1 to 2 minute bouts of activity consisting of only a few dozen steps.     

Immunoprivileged status of the liver is controlled by Toll-like receptor 3 signaling

The liver is known to be a classical immunoprivileged site with a relatively high resistance against immune responses. Here we demonstrate that highly activated liver-specific effector CD8+ T cells alone were not sufficient to trigger immune destruction of the liver in mice. Only additional innate immune signals orchestrated by TLR3 provoked liver damage. While TLR3 activation did not directly alter liver-specific CD8+ T cell function, it induced IFN-alpha and TNF-alpha release. These cytokines generated expression of the chemokine CXCL9 in the liver, thereby enhancing CD8+ T cell infiltration and liver disease in mice. Thus, nonspecific activation of innate immunity can drastically enhance susceptibility to immune destruction of a solid organ.     

Are patient characteristics helpful in recognizing mild COPD (GOLD I) in daily practice?

OBJECTIVE: To determine whether in a high-risk group of middle-aged male current smokers, patient characteristics are useful to recognize mild COPD (GOLD stage I). DESIGN: In a cross-sectional study spirometry was performed according to the American Thoracic Society criteria. COPD was defined according to the GOLD criteria for COPD. SETTING: Primary care. SUBJECTS: Male smokers, aged 40-65 years, without documented lung disease in the population at large. MAIN OUTCOME MEASURES: Medical records were scrutinized to collect patient characteristics. Multiple logistic regression analysis was used to identify independent determinants of mild COPD. RESULTS: A total of 567 subjects participated. COPD, defined by a FEV1/FVC ratio < 0.7, was detected in 170 subjects (30.0%, 95% CI 26.2-33.9%). In 149 subjects (26.3%; 22.7-30.1%) COPD was mild (GOLD stage I) and in 21 subjects (3.7%; 2.3-5.6%) moderate (GOLD stage II). Only age and cough were independently associated with the presence of mild COPD. The ability of these determinants to discriminate between subjects with or without mild COPD was relatively poor (ROC area 0.65). Therefore no prediction rule was developed. CONCLUSION: Our results indicate that patient characteristics are not helpful to recognize mild COPD (GOLD stage I) in middle-aged male smokers.