Investigating non-specific binding to chemically engineered sensor surfaces using liposomes as models.

Nanoparticles are ubiquitously used for signal enhancement in (bio)sensors, but their true possible performance is typically hampered by non-specific binding. A better understanding of the nature and the prevention of non-specific binding through surface engineering of the particles and sensor surfaces is needed to intelligently design (bio)sensors and potentially avoid bulk blocking methods. Hence, two types of liposomes were used as model for signal-enhancing nanoparticles. Their surface was engineered to bear negative surface charge. One type was synthesized with additional 6 mol% -COOH groups. Their interaction with four typical chemically modified sensor surfaces was then mechanistically characterized by surface plasmon resonance (SPR) spectroscopy. It was shown that the non-specific binding can be described with Langmuir isotherms providing quantitative information of dissociation constants and surface loading with especially high correlation coefficients (>0.97) for all the studied sensor surfaces modified with hydrophilic alkane thiols. By tailoring the sensor surface chemistry, non-specific binding was significantly minimized. Here, carboxyl- or methyl-terminated surfaces performed best. In fact, the pairing of -COOH groups on the sensor surface with -COOH groups on the liposomes almost completely eliminated non-specific binding, resulting in a SPR signal change of only 1 mRIU (refractive index unit) at 100 µM phospholipid concentration. Surprisingly though, -OH groups on the surface, which are also commonly used in sensing applications, did not lead to decreased adsorption, but caused significant signal changes (4 mRIU at 100 µM phospholipid) due to non-specific binding. Overall, the mechanistic studies presented here demonstrate that by careful design of the nanoparticle surface and by choosing sensor surfaces with terminal -CH3 or -COOH groups, improved sensing (micro)systems with very low non-specific adsorption can be obtained.

Impacts of metal mining on river systems: a global assessment.

An estimated 23 million people live on floodplains affected by potentially dangerous concentrations of toxic waste derived from past and present metal mining activity. We analyzed the global dimensions of this hazard, particularly in regard to lead, zinc, copper, and arsenic, using a georeferenced global database detailing all known metal mining sites and intact and failed tailings storage facilities. We then used process-based and empirically tested modeling to produce a global assessment of metal mining contamination in river systems and the numbers of human populations and livestock exposed. Worldwide, metal mines affect 479,200 kilometers of river channels and 164,000 square kilometers of floodplains. The number of people exposed to contamination sourced from long-term discharge of mining waste into rivers is almost 50 times greater than the number directly affected by tailings dam failures.

Multiple forces contribute to cell sheet morphogenesis for dorsal closure in Drosophila.

The molecular and cellular bases of cell shape change and movement during morphogenesis and wound healing are of intense interest and are only beginning to be understood. Here, we investigate the forces responsible for morphogenesis during dorsal closure with three approaches. First, we use real-time and time-lapsed laser confocal microscopy to follow actin dynamics and document cell shape changes and tissue movements in living, unperturbed embryos. We label cells with a ubiquitously expressed transgene that encodes GFP fused to an autonomously folding actin binding fragment from fly moesin. Second, we use a biomechanical approach to examine the distribution of stiffness/tension during dorsal closure by following the response of the various tissues to cutting by an ultraviolet laser. We tested our previous model (Young, P.E., A.M. Richman, A.S. Ketchum, and D.P. Kiehart. 1993. Genes Dev. 7:29-41) that the leading edge of the lateral epidermis is a contractile purse-string that provides force for dorsal closure. We show that this structure is under tension and behaves as a supracellular purse-string, however, we provide evidence that it alone cannot account for the forces responsible for dorsal closure. In addition, we show that there is isotropic stiffness/tension in the amnioserosa and anisotropic stiffness/tension in the lateral epidermis. Tension in the amnioserosa may contribute force for dorsal closure, but tension in the lateral epidermis opposes it. Third, we examine the role of various tissues in dorsal closure by repeated ablation of cells in the amnioserosa and the leading edge of the lateral epidermis. Our data provide strong evidence that both tissues appear to contribute to normal dorsal closure in living embryos, but surprisingly, neither is absolutely required for dorsal closure. Finally, we establish that the Drosophila epidermis rapidly and reproducibly heals from both mechanical and ultraviolet laser wounds, even those delivered repeatedly. During healing, actin is rapidly recruited to the margins of the wound and a newly formed, supracellular purse-string contracts during wound healing. This result establishes the Drosophila embryo as an excellent system for the investigation of wound healing. Moreover, our observations demonstrate that wound healing in this insect epidermal system parallel wound healing in vertebrate tissues in situ and vertebrate cells in culture (for review see Kiehart, D.P. 1999. Curr. Biol. 9:R602-R605).

Drosophila wing melanin patterns form by vein-dependent elaboration of enzymatic prepatterns.

BACKGROUND: Animal melanin patterns are involved in diverse aspects of their ecology, from thermoregulation to mimicry. Many theoretical models have simulated pigment patterning, but little is known about the developmental mechanisms of color pattern formation. In Drosophila melanogaster, several genes are known to be necessary for cuticular melanization, but the involvement of these genes in melanin pattern evolution is unknown. We have taken a genetic approach to elucidate the developmental mechanisms underlying melanin pattern formation in various drosophilids. RESULTS: We show that, in D. melanogaster, tyrosine hydroxylase (TH) and dopa decarboxylase (DDC) are required for melanin synthesis. Ectopic expression of TH, but not DDC, alone was sufficient to cause ectopic melanin patterns in the wing. Thus, changes in the level of expression of a single gene can result in a new level of melanization. The ontogeny of this ectopic melanization resembled that found in Drosophila species bearing wing melanin patterns and in D. melanogaster ebony mutants. Importantly, we discovered that in D. melanogaster and three other Drosophila species these wing melanin patterns are dependent upon and shaped by the circulation patterns of hemolymph in the wing veins. CONCLUSIONS: Complex wing melanin patterns are determined by two distinct developmental mechanisms. Spatial prepatterns of enzymatic activity are established late in wing development. Then, in newly eclosed adults, melanin precursors gradually diffuse out from wing veins and are oxidized into dark brown or black melanin. Both the prepatterning and hemolymph-supplied components of this system can change during evolution to produce color pattern diversity.

Drosophila stretchin-MLCK is a novel member of the Titin/Myosin light chain kinase family.

Members of the titin/myosin light chain kinase family play an essential role in the organization of the actin/myosin cytoskeleton, especially in sarcomere assembly and function. In Drosophila melanogaster, projectin is so far the only member of this family for which a transcription unit has been characterized. The locus of another member of this family, a protein related to Myosin light chain kinase, was also identified. The cDNA and genomic sequences published explain only the shorter transcripts expressed by this locus. Here, we report the complete molecular characterization of this transcription unit, which spans 38 kb, includes 33 exons and accounts for transcripts up to 25 kb in length. This transcription unit contains both the largest exon (12,005 nt) and the largest coding region (25,213 nt) reported so far for Drosophila. This transcription unit features both internal promoters and internal polyadenylation signals, which enable it to express seven different transcripts, ranging from 3.3 to 25 kb in size. The latter encodes a huge, titin-like, 926 kDa kinase that features two large PEVK-rich repeats, 32 immunoglobulin and two fibronectin type-III domains, which we designate stretchin-MLCK. In addition, the 3' end of the stretchin-MLCK transcription unit expresses shorter transcripts that encode 86 to 165 kDa isoforms of stretchin-MLCK that are analogous to vertebrate Myosin light chain kinases. Similarly, the 5' end of the Stretchin-Mlck transcription unit can also express transcripts encoding kettin and Unc-89-like isoforms, which share no sequences with the MLCK-like transcripts. Thus, this locus can be viewed as a single transcription unit, Stretchin-Mlck (genetic abbreviation Strn-Mlck), that expresses large, composite transcripts and protein isoforms (sequences available at http://www.academicpress.com/jmb), as well as a complex of two independent transcription units, the Stretchin and Mlck transcription units (Strn and Mlck, respectively) the result of a "gene fission" event, that encode independent transcripts and proteins with distinct structural and enzymatic functions.

Transcriptional basis for hyporesponsiveness of the human inducible nitric oxide synthase gene to lipopolysaccharide/interferon-gamma.

The work reported here resolves, at the level of gene regulation, the controversy as to whether or not human monocytes/macrophages can produce nitric oxide (NO) when stimulated with lipopolysaccharide (LPS), with or without co-stimulation by interferon-gamma (IFN-gamma). Studies included structural comparison of the promoters for human and mouse inducible NO synthase (iNOS) genes, transfection and assay of human and mouse iNOS promoter regions in response to LPS +/- IFN-gamma, and electrophoretic mobility shift assays of kappa B response elements. Two explanations for hyporesponsiveness of the human iNOS promoter to LPS +/- IFN-gamma were found: (1) multiple inactivating nucleotide substitutions in the human counterpart of the enhancer element that has been shown to regulate LPS/IFN-gamma induced expression of the mouse iNOS gene; and (2) and absence of one or more nuclear factors in human macrophages (e.g., an LPS-inducible nuclear factor-kappa B/Rel complex), that is (are) required for maximal expression of the gene. The importance of resolution of this controversy is that future research in this area should be directed toward the understanding of alternative mechanisms that can result in the successful production of NO.

Heavy metal contamination of water, soil and produce within riverine communities of the Río Pilcomayo basin, Bolivia.

The Río Pilcomayo heads on the Cerro Rico de Potosí precious metal-polymetallic tin deposits of Southern Bolivia. Mining of the Potosí deposits began in 1545 and has led to the severe contamination of the Pilcomayo's water and sediments for at least 200 km downstream of the mines. This investigation addresses the potential human health affects of metal and As contamination on four communities located along the upper Río Pilcomayo by examining the potential significance of human exposure pathways associated with soils, crops and water (including river, irrigation and drinking water supplies). The most significantly contaminated agricultural soils occur upstream at Mondragón where Cd, Pb and Zn concentrations exceed recommended guideline values for agricultural use. Further downstream the degree of contamination decreases, and metal concentrations are below Dutch, German and Canadian guideline values. Metal and As concentrations in agricultural products from the four communities were generally below existing guidelines for heavy metal content in commercially-sold vegetables. Thus, the consumption of contaminated produce does not appear to represent a significant exposure pathway. A possible exception is Pb in carrots, lettuce and beetroots from Sotomayor and Tuero Chico; 37% and 55% of the samples, respectively, exceeded recommended guidelines. Most communities obtain drinking water from sources other than the Río Pilcomayo. In general, dissolved concentrations of metals and As in drinking water from the four studied communities are below the WHO guideline values with the exception of Sb, which was high at Tasapampa. The inadvertent ingestion of contaminated water from irrigation canals and the Río Pilcomayo represents a potential exposure pathway, but its significance is thought to be minimal. Given the degree of soil contamination in the area, perhaps the most significant exposure pathway is the ingestion of contaminated soil particles, particularly particles attached to, and consumed with vegetables. The risks associated with this pathway can be reduced by thoroughly washing or peeling the vegetables prior to consumption. Other exposure pathways that are currently under investigation include the consumption of contaminated meat from livestock and poultry, which drink polluted waters and the ingestion of contaminated wind-blown dust.

An index for assessing weight change in children: weight/height ratios.

Weight and height data from two studies were recomputed, and original data were computed using a Weight Index formula that accounts for the interaction of actual weight/height changes in growing children and compares this ratio with that of normed weight/height ratios for equal-aged children. Recomputing the data of one study showed that the Weight Index is a more sensitive procedure for assessing long-term behavioral treatment of obesity for girls than weight alone. Recomputing the data of a second study showed that the Weight Index formula is consistent for boys and girls and is a more sensitive procedure for assessing long-term effects of a variety of medical treatments for obesity than the Ponderal Index or weight alone. Computation of the data for 17 "normal" children in a preschool class showed a zero Weight Index score before and after a six-month interval elapsed without treatment. The procedure may be useful in assessing ponderosity or anorexia over intervals of six months or more with growing children or difference between actual and normed weight over shorter intervals.

Research methodology. Part I: Understanding correlational analysis.

This paper is part one of a four part series on basic statistical analytical methods. Using a case method presentation, the basic essentials of correlational analysis is described.

Research methodology. Part IV: Understanding canonical correlation analysis.

Canonical correlation is presented as a technique to determine how sets of dependent variables are related with sets of independent variables. Canonical correlation reveals the strength of the relationship between the clusters using case data as illustration, three pairs of clusters (factors or profiles) emerged. Interpretation of the clusters are presented. As indicated in the case presentation, Canonical Correlation (CA) is the fourth in a series of methodologies selected for illustration as precursors to advanced statistics and modeling. In this paper, background will be given, a schematic example presented, sample size and CA, SPSS procedure to perform CA, and interpretation of CA and possible uses of CA in nursing research.

Cardiovascular risks: among Black and White rural-urban low income women.

The purpose of this descriptive comparative pilot study was to determine if there are rural-urban and racial differences in the prevalence of four modifiable risk factors (blood pressure, serum cholesterol, diabetes and smoking) for cardiovascular disease among low income Black and White women. Of the 163 low income women who participated in the study, it was found that Black women had a significantly higher prevalence of elevated blood pressure and serum cholesterol levels than White women. The observed rates for smoking and diabetes were highest among rural White women. Implications for health education and continued research are presented.

Research methodology. Part III: understanding multiple correlation analysis and multiple regression.

This is part III of a case series on research methodology with additional case demographic information. The relationship of a dependent variable with two or more independent variables is explained and used to illustrate multiple correlation and multiple regression.

Calorie use and obesity among diabetic and non-diabetic Mvskoke Indians.

Type 2 diabetes is a major cause of death among American Indians and obesity and physical inactivity are modifiable risk factors in the development of type 2 diabetes. Physical activity may have both a preventive effect and a secondary preventive effect of lessening insulin resistance in persons with type 2 diabetes. Moreover, for some individuals participation in daily regimens of physical activity is very difficult. Culture-specific physical activity may be an intervention to reduce obesity and prevent diabetes-related complications. Therefore, the research questions for this study were: (1) What is the body fat percentage of diabetic and non-diabetic Mskoke Indians?, (2) How does body fat percentage of Mvskoke Indians compare with recommended percentages for age and gender?, (3) What is the caloric use of Mvskoke Indians during daily and traditional cultural activities of dancing and stickball?, and (4) Are there relationships among body fat percentage, calorie use, gender, and diabetic status? The findings revealed that female diabetics' percentage of body fat in three age cohorts (40-49, 50-59, and 70+) was higher than that of non-diabetic females. By contrast, male diabetics had lower percentage of body fat than non-diabetic males. The actual mean percentage of body fat of the female participants ranged from 38.5% to 44%, roughly twice the standard recommended percentages of body fat of 20-24%. No significant difference was found in calorie use by gender or diabetic status. Analysis of calories during traditional activities revealed that 30 minutes of stickball used an average of 135.7 calories. Dancing for 33 minutes used an average of 149 calories. No significant relationship was found among percentage of body fat, caloric use, gender, and diabetic status.

Identification of environmental lead sources and pathways in a mining and smelting town: Mount Isa, Australia.

Lead (Pb) concentrations and isotopic compositions from soils, dusts and aerosols from public land and residential lots adjacent to the copper and Pb mine and smelter at Mount Isa, Australia, were examined to understand the sources and risks of environmental Pb exposure. Urban soil samples contain elevated Pb concentrations (mean 1560 mg/kg), of which 45-85% of the Pb is bioaccessible. The Pb isotopic composition of surface soils (0-2 cm), aerosols and dusts ((206)Pb/(207)Pb, (208)Pb/(207)Pb range: 1.049, 2.322-1.069, 2.345) are dominated by Pb derived from the Mount Isa Pb-zinc ore bodies. Underlying soil horizons (10-20 cm) have distinctly different Pb isotopic compositions ((206)Pb/(207)Pb, (208)Pb/(207)Pb range: 1.093, 2.354-1.212, 2.495). Surface soil-, dust- and aerosol-Pb are derived predominantly from smelter emissions and fugitive mining sources and not from in situ weathered bedrock. Remediation strategies should target legacy and ongoing sources of environmental Pb to mitigate the problem of Pb exposure at Mount Isa.

Inverse effects of ethidium bromide on superoxide dismutase and lactate dehydrogenase of Artemia salina embryos.

Development of Artemia salina embryos in the presence of ethidium bromide, an inhibitor of mitochondrial transcription, results in a dose dependent increase in the specific activity of lactate dehydrogenase, and a concomitant decrease in the specific activity of a cyanide-resistant superoxide dismutase. The inhibition of mitochondrial function by ethidium bromide appears to exert opposite effects on the nuclear cistrons encoding lactate dehydrogenase and superoxide dismutase, and suggests that a common mitochondrial signal may exert diametric effects on nuclear cistrons whose products are characteristic of alternate states of respiration.

Drosophila nonmuscle myosin II has multiple essential roles in imaginal disc and egg chamber morphogenesis.

Morphogenesis is characterized by orchestrated changes in the shape and position of individual cells. Many of these movements are thought to be powered by motor proteins. However, in metazoans, it is often difficult to match specific motors with the movements they drive. The nonmuscle myosin II heavy chain (MHC encoded by zipper is required for cell sheet movements in Drosophila embryos. To determine if myosin II is required for other processes, we examined the phenotypes of strong and weak larval lethal mutations in spaghetti squash (sqh), which encodes the nonmuscle myosin II regulatory light chain (RLC). sqh mutants can be rescued to adulthood by daily induction of a sqh cDNA transgene driven by the hsp70 promoter. By transiently ceasing induction of the cDNA, we depleted RLC at specific times during development. When RLC is transiently depleted in larvae, the resulting adult phenotypes demonstrate that RLC is required in a stage-specific fashion for proper development of eye and leg imaginal discs. When RLC is depleted in adult females, oogenesis is reversibly disrupted. Without RLC induction, developing egg chambers display a succession of phenotypes that demonstrate roles for myosin II in morphogenesis of the interfollicular stalks, three morphologically and mechanistically distinct types of follicle cell migration, and completion of nurse cell cytoplasm transport (dumping). Finally, we show that in sqh mutant tissues, MHC is abnormally localized in punctate structures that do not contain appreciable amounts of filamentous actin or the myosin tail-binding protein p127. This suggests that sqh mutant phenotypes are chiefly caused by sequestration of myosin into inactive aggregates. These results show that myosin II is responsible for a surprisingly diverse array of cell shape changes throughout development.

Purification and characterization of a type II DNA topoisomerase from bovine calf thymus.

We report here the large scale purification of DNA topoisomerase II from calf thymus glands, using the unknotting of naturally knotted P4 phage DNA as an assay for enzymatic activity. Topoisomerase II was purified more than 1300-fold as compared to the whole cell homogenate, with 22% yield. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands of apparent molecular masses of 125 and 140 kDa. Tryptic maps of the two bands indicated that they derive from the same protein. Using these fragments, specific polyclonal antisera to topoisomerase II were raised in rabbits. Immunoblotting of whole cell lysates from various species indicated that topoisomerase II is well conserved among mammals and has a native subunit molecular mass of 180 kDa. Analytical sedimentation and gel filtration were used to determine a sedimentation coefficient of 9.8 S and a Stokes radius of 68 A. The calculated solution molecular mass of 277 kDa implies a dimer structure in solution. The purified topoisomerase II unknots P4 DNA in an ATP-dependent manner and is highly stimulated in its relaxation activity by ATP. A DNA-stimulated ATPase activity, as has been found with other type II topoisomerases, is associated with the purified enzyme. Approximate kinetic parameters for the ATPase reaction were determined to be: a Vmax of 0.06 nmol of ATP/(micrograms of protein) (min) and Km of 0.2 mM in the absence of DNA, and a Vmax of 0.2 nmol of ATP/(micrograms of protein) (min) and Km of 0.4 mM ATP in the presence of supercoiled plasmid DNA.

Myosin heavy chain isoforms regulate muscle function but not myofibril assembly.

Myosin heavy chain (MHC) is the motor protein of muscle thick filaments. Most organisms produce many muscle MHC isoforms with temporally and spatially regulated expression patterns. This suggests that isoforms of MHC have different characteristics necessary for defining specific muscle properties. The single Drosophila muscle Mhc gene yields various isoforms as a result of alternative RNA splicing. To determine whether this multiplicity of MHC isoforms is critical to myofibril assembly and function, we introduced a gene encoding only an embryonic MHC into Drosophila melanogaster. The embryonic transgene acts in a dominant antimorphic manner to disrupt flight muscle function. The transgene was genetically crossed into an MHC null background. Unexpectedly, transformed flies expressing only the embryonic isoform are viable. Adult muscles containing embryonic MHC assemble normally, indicating that the isoform of MHC does not determine the dramatic ultrastructural variation among different muscle types. However, transformed flies are flightless and show reduced jumping and mating ability. Their indirect flight muscle myofibrils progressively deteriorate. Our data show that the proper MHC isoform is critical for specialized muscle function and myofibril stability.