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Characterization of Streptomyces promoter sequences using the Escherichia coli galactokinase gene.

A gene fusion system that uses the Escherichia coli galK gene has been developed to characterize Streptomyces transcriptional regulatory sequences. The system consists of galK-deficient Streptomyces lividans mutants and plasmids containing the E. coli galK gene with its natural ribosome-binding site and sites upstream of galK for insertion of transcription signals. Expression of the E. coli galK gene in S. lividans can be quantitated by either an enzymatic or immunoblot assay or detected by genetic complementation of an S. lividans galK- mutant. The utility of the plasmid to select, detect and assess promoter function was examined using the S. lividans XP55 and S. fradiae aph gene promoters. The potential use of the galK fusion system to isolate and characterize Streptomyces transcription signals is discussed.

Base Sequence↗

Comparison of different separation technologies for proteome analyses: isoform resolution as a prerequisite for the definition of protein biomarkers on the level of posttranslational modifications.

In this article we evaluate methods used to reveal the molecular complexity, which is generated in biological samples by posttranslational modifications (PTM) of proteins. We show how distinct molecular differences on the level of phosphorylation sites in a single protein (ovalbumin) can be resolved with different success using 1D and 2D gel-electrophoresis and reversed-phase liquid chromatography (LC) with monolithic polystyrol-divinylbenzol (PS-DVB) columns for protein separation, and matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) for protein identification. Phosphorylation site analysis was performed using enzymatic dephosphorylation in combination with differential peptide mass mapping. Liquid chromatography-MALDI-TOF MS coupling with subsequent on-target tryptic protein digestion turned out to be the fastest method tested but yielded low resolution for the analysis of PTM, whereas 2D gel-electrophoresis, due to its unique capability of resolving highly complex isoform pattern, turned out to be the most suitable method for this purpose. The evaluated methods complement one another and in connection with efficient technologies for differential and quantitative analysis, these approaches have the potential to reveal novel molecular details of protein biomarkers.

Amino Acid Sequence↗

Evaluation of the complexity of motor unit potentials in anal sphincter electromyography.

OBJECTIVE: Motor unit potential (MUP) morphology can be quantified using parameters describing electrophysiological size and shape (complexity). Traditionally, MUP complexity has been estimated using parameters number of phases and turns. Recently, 'irregularity coefficient' (IR), measuring the length of the MUP curve normalized with its amplitude, has been introduced. The aim of this study was to evaluate IR in the external anal sphincter muscle. METHODS: Sensitivity was examined in 61 patients with chronic cauda equina lesions, and specificity in 75 controls using a standard concentric EMG needle and EMG system with multi-MUP analysis. RESULTS: When evaluated separately the sensitivity of IR was 16% lower, and specificity 5% higher compared to number of turns, with both differences decreasing to only 2 and 1%, respectively, when each of these parameters was added to MUP area and duration. CONCLUSIONS: Our present results suggest that IR provides a similar diagnostic usefulness, but a more appropriate description of MUP complexity compared to traditional MUP parameters. SIGNIFICANCE: IR seems to be suitable to complement one of MUP parameters measuring electrophysiological MUP size in the future quantitative EMG.

Action Potentials↗

Development of local knowledge of environmental contamination in Sydney, Nova Scotia: environmental health practice from an environmental justice perspective.

In Sydney, Nova Scotia, from 1901 through 1988 a coke and steel factory operated with no pollution controls, depositing over a million tons of particulate matter and releasing several thousands of tons of coal tar into the estuary. Previously we documented the presence of lead, arsenic and PAHs, in soil above Canadian guidelines, and in house dust in the communities surrounding the site [Lambert, TW, Lane, S. Lead, arsenic, and polycyclic aromatic hydrocarbons in soil and house dust in the communities surrounding the Sydney, Nova Scotia, tar ponds. Environ Health Perspect 2004; 112:35-41.]. In this paper we further the research by documenting and developing community knowledge with a study of resident's observations and experiences of the industrial contamination. We conducted two surveys, a quantitative door-to-door survey and qualitative dust interview, designed to complement each other and bring together the observations and experiences in the different communities to develop the local knowledge. The combined methodology uses techniques from both social and physical science, and was developed with the cooperation of community members. The research supports the proposition that local knowledge adds contextual meaning that complements the physical measurement of environmental contaminants, in order to understand the complex environment in which people live, and the multiple exposure pathways through which they can be affected. Residents in all three communities provided vivid observations and detailed experiences of the industrial pollution in their community and homes. The local knowledge is consistent with our physical data and review of the historical scientific research in Sydney, and supports the inference that the community was adversely impacted by the coke and steel facility. From a justice perspective, the three communities should be equally considered for remediation as part of the 'tar pond remediation policy' rather than the current policy of including only a few streets and houses.

Air Pollution, Indoor↗

Surprising evolutionary predictions from enhanced ecological realism.

A focus on the eco-evolutionary feedback continually operating between a population's evolution and its environment helps to appreciate the generality of ESS theory. Here we illustrate, through a sequence of four examples, how respecting such feedback in the evolutionary dynamics of quantitative traits may result in qualitatively unexpected outcomes. Reviewing existing insights and complementing these with new results, we show (1) that evolutionary matrix games are fundamentally degenerate and allow a natural unfolding, (2) that selection-driven extinction may not be rare in nature, (3) that evolutionary epidemiology should not rely on R0 maximization, and (4) why the occurrence of Hardy-Weinberg proportions generically requires an evolutionary explanation.

Biological Evolution↗

A visual approach to proteomics.

Cryo-electron tomography is an emerging imaging technique that has unique potential for molecular cell biology. At the present resolution of 4-5 nm, large supramolecular structures can be studied in unperturbed cellular environments and, in the future, it will become possible to map molecular landscapes inside cells in a more comprehensive manner. 'Visual proteomics' aims to complement and extend mass-spectrometry-based inventories, and to provide a quantitative description of the macromolecular interactions that underlie cellular functions.

Cryoelectron Microscopy↗

Biological sciences in Project 2000: an exploration of status.

Through Project 2000 preregistration nursing courses it was intended to establish a broad knowledge base for nurse education. This is likely to affect the way biological sciences are viewed as subjects within the curriculum. The aim of this study was to investigate the status of biological sciences in the curriculum following educational reform. A small-scale, descriptive study was undertaken investigating one institution. Quantitative and qualitative data were gathered using a postal questionnaire; this was complemented by contextual information obtained from documents. Findings indicated that there was a lack of consensus amongst teachers regarding what emphasis should be given to different subjects within the curriculum. It was commented that 'nursing' may have been displaced by pure subject disciplines, and that application of biological theory to practice was inadequate. Biological sciences, social sciences and behavioural sciences were allotted equal hours in the curriculum; concern was expressed, however, that there was insufficient time available for some subjects. This appeared to foster competition and tension, in particular between proponents of biological and social sciences. Teachers demonstrated subject loyalty, and promoted their own discipline within the curriculum. In an attempt to explain findings a conceptual framework based upon the sociology of knowledge and conflicts and tensions between groups within a profession was employed. Issues identified in the study suggest that there is cause for concern with respect to the structuring, teaching and learning of biological concepts, and their application to nursing practice.

Biological Science Disciplines↗

Anatomy and neurophysiology of the taste system in aged animals.

In summary, the recent data from quantitative studies of taste buds in old humans, rhesus monkeys, and rats complement neurophysiological data on taste responses from aged rats and lead to the general conclusion that the peripheral taste system is maintained structurally and functionally across the life span. Although some statistically significant differences were observed, the magnitude of these differences would not lead one to predict altered taste preferences or feeding behavior in old animals. The robust nature of the peripheral sense of taste in old age appears to be in sharp contrast to other sensory systems. Not only do receptors alter with age, but accessory organs, such as the lens in the visual system, also alter structurally so that sensory function is compromised. One factor contributing to maintenance of the receptor organ of taste bud and papilla is the turnover and replacement of taste bud cells and surrounding epithelial cells. Recent studies in aged mice indicate no differences in turnover time in epithelial mucosa lining the mouth. There are no data on turnover time, however, for taste bud cells or for gustatory papilla epithelium in old animals. Alterations in taste bud cell or membrane receptor turnover could affect function. In other cell types and receptors, age-related differences in membrane lipids, proteins, and fluidity characteristics are reported. Membrane changes such as these are proposed to account for age-related differences in response properties of the system of cardiac muscarinic receptors, a system in which density of receptors does not alter. The small, but statistically significant, differences in neurophysiological responses to taste stimuli from the chorda tympani nerve might well relate to age-related differences in membrane receptors. Not only is there an absence of data on turnover time or membrane characteristics for taste bud cells in old age, but there are no observations on the ultrastructure of taste buds in old animals. The taste bud is a complex structure, composed of many cells and different cell types, with a specific orientation and access to the oral cavity via the taste pore, and with extensive associations with the innervating afferent fibers. Ultrastructural observations could provide information on changes in synapses and other nerve/cell contacts--in microvilli and taste pore structure, and in numbers of various cell types within the bud. Such data will be essential to understanding the nature and extent of age-related alterations in the taste system.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗

A murine skeletal muscle ischemia-reperfusion injury model: differential pathology in BALB/c and DBA/2N mice.

Ischemia-reperfusion injuries can occur with diseases such as myocardial infarction and stroke and during surgical procedures such as organ transplantation and correction of aortic aneurysms. We developed a murine model to mimic abdominal aortic aneurysm repair with cross-clamping of the aorta distal to the renal artery. After model development, we compared the normal complement BALB/c mouse with the C5-deficient DBA/2N mouse. To assess quantitative differences, we measured neuromuscular function up to 72 h after ischemia with a subjective clinical scoring system, as well as plasma chemistries, hematology, and histopathology. There were significant increases in clinical scores and creatine phosphokinase, lactate dehydrogenase, and muscle histopathology scores in BALB/c mice compared with those in DBA/2N mice and sham-surgery mice. Muscle histopathology scores of the cranial tibialis and quadriceps correlated well with clinical signs, creatine phosphokinase, and lactate dehydrogenase, and indicated the greatest pathology in these muscle groups. We developed a murine model of skeletal muscle ischemia-reperfusion injury that can utilize the benefits of murine genetic and transgenic models to assess therapeutic principles of this model. Additionally, we have shown a significant reduction in clinical signs, plasma muscle enzyme concentrations, and muscle pathology in the C5-deficient DBA/2N mouse in this model.

Animals↗

Human oligodendroglial cells express low levels of C1 inhibitor and membrane cofactor protein mRNAs.

BACKGROUND: Oligodendrocytes, neurons, astrocytes, microglia, and endothelial cells are capable of synthesizing complement inhibitor proteins. Oligodendrocytes are vulnerable to complement attack, which is particularly observed in multiple sclerosis. This vulnerability may be related to a deficiency in their ability to express complement regulatory proteins. METHODS: This study compared the expression level of complement inhibitor mRNAs by human oligodendrocytes, astrocytes and microglia using semi-quantitative RT-PCR. RESULTS: Semi-quantitative RT-PCR analysis showed that C1 inhibitor (C1-inh) mRNA expression was dramatically lower in oligodendroglial cells compared with astrocytes and microglia. The mRNA expression level of membrane cofactor protein (MCP) by oligodendrocytes was also significantly lower than for other cell types. CONCLUSION: The lower mRNA expression of C1-inh and MCP by oligodendrocytes could contribute to their vulnerability in several neurodegenerative and inflammatory diseases of the central nervous system.

Journal Article↗

Characterization of physical properties of supported phospholipid membranes using imaging ellipsometry at optical wavelengths.

Subnanometer-scale vertical z-resolution coupled with large lateral area imaging, label-free, noncontact, and in situ advantages make the technique of optical imaging ellipsometry (IE) highly suitable for quantitative characterization of lipid bilayers supported on oxide substrates and submerged in aqueous phases. This article demonstrates the versatility of IE in quantitative characterization of structural and functional properties of supported phospholipid membranes using previously well-characterized examples. These include 1), a single-step determination of bilayer thickness to 0.2 nm accuracy and large-area lateral uniformity using photochemically patterned single 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers; 2), hydration-induced spreading kinetics of single-fluid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers to illustrate the in situ capability and image acquisition speed; 3), a large-area morphological characterization of phase-separating binary mixtures of 1,2-dilauroyl-sn-glycero-3-phosphocholine and galactosylceramide; and 4), binding of cholera-toxin B subunits to GM1-incorporating bilayers. Additional insights derived from these ellipsometric measurements are also discussed for each of these applications. Agreement with previous studies confirms that IE provides a simple and convenient tool for a routine, quantitative characterization of these membrane properties. Our results also suggest that IE complements more widely used fluorescence and scanning probe microscopies by combining large-area measurements with high vertical resolution without the use of labeled lipids.

Cholera Toxin↗

Computational modeling of cardiac ventricular action potentials in rat and mouse: review.

Little is known about the ionic mechanisms underlying the action potential heterogeneity in ventricle-associated healthy and disease conditions, even though five decades of histological, electrophysiological, pharmacological, and biochemical investigations exist. The computational modeling in murine ventricular myocytes can complement our knowledge of the experimental data and provide us with more quantitative descriptions in understanding different conditions related to normal and disease conditions. This paper initially reviews the theoretical modeling for cardiac ventricular action potentials of various species and the related experimental work. It then presents the progress of the computational modeling of cardiac ventricular cells for normal, diabetic, and spontaneously hypertensive rats. The paper also introduces recent modeling efforts for the action potential heterogeneity in mouse ventricular cells. The computational insights gained into the ionic mechanisms in rodents will continue to enhance our understanding of the heart and provide us with new knowledge for future studies to treat cardiac diseases in children and adults. Because the dissemination of computational models is very important, we continue to disseminate these models by iCell, the interactive cell modeling resource. iCell (http://ssd1.bme.memphis.edu/icell/) has been developed as a simulation-based teaching and learning tool for electrophysiology and contains JAVA applets that present models of various cardiac cells and neurons and simulation data of their bioelectric activities at cellular level.

Action Potentials↗

Endogenous glutathione adducts.

This review provides an overview of the formation, pharmacology, and toxicology of endogenous glutathione (GSH)-adducts with particular emphasis on GSH-adducts that arise from lipid peroxidation. GSH is the major low-molecular-weight thiol in mammalian cells. It is involved in the formation of endogenous bioactive eicosanoids and is a source of reducing equivalents in a number of biosynthetic reactions. GSH has long been recognized to act as a co-factor in the reduction of reactive oxygen species and lipid hydroperoxides by glutathione peroxidases and glutathione-S-transferases (GSTs). It also plays an important role in the reduction of reactive intermediates derived from arylamines and in the conjugation of reactive intermediates to form S-substituted endogenous GSH-adducts through its nucleophilic cysteine sulfhydryl group. Although some reactive intermediates can form adducts directly, GST-mediated reactions generally predominate. This results in the formation of bioactive endogenous GSH-adducts derived from eicosanoids, isoprostanes, estrogens, catecholamines, and 4-hydroxy-2(E)-nonenal (HNE). Cellular oxidative stress causes increased lipid peroxidation with the concomitant formation of DNA- and protein-reactive bifunctional electrophiles. It has generally been considered that HNE is the most abundant bifunctional electrophile that is formed. Several years ago we discovered that 4-oxo-2(E)-nonenal (ONE) was also a major lipid hydroperoxide-derived bifunctional electrophile. From in vitro studies, we showed that ONE and HNE arose from the common intermediate, 4-hydroperoxy-2(E)-nonenal and also showed that ONE was formed in greater amounts than HNE. We have recently made the unexpected discovery that GSH addition to ONE leads to the formation of an unusual thiadiazabicyclo-ONE-GSH-adduct (TOG), which was characterized as (2S,7R) - 7 - [N - (carboxymethyl)carbamoyl] - 5 - oxo - 12 - pentyl - 9 - thia - 1,6 - diazabicyclo[8.2.1]trideca - 10(13), 11-diene-2-carboxylic acid. TOG is one of the most abundant GSH-adducts formed during peroxide/Fe(II)- or Fe(II)-mediated oxidative stress in EA.hy 926 endothelial cells. As TOG is formed from ONE, these experiments have confirmed that ONE is a major lipid hydroperoxide-derived bifunctional electrophile formed during intracellular oxidative stress. TOG represents the first member of a new class of endogenous GSH-adduct biomarkers that can be used to quantify intracellular oxidative stress. Two other members of the TOG family arise from GST-mediated GSH-adduct formation with dioxododecenoic acid and dioxooctenoic acid, bifunctional electrophiles derived from the carboxy terminus of lipid hydroperoxides. The formation of TOG and TOG-related endogenous GSH-adducts can result from free radical- as well as cyclooxygenase- and lipoxygenase-mediated pathways. Analysis of the GSH-adducts by stable isotope dilution mass spectrometry-based methodology will provide a quantitative measure of enzymatic and non-enzymatic cellular oxidative stress to complement isoprostane measurements. In future studies, it will also be important to establish the biological activity of TOG and its analogs in view of the potent activity of many other endogenous GSH-adducts such as the leukotrienes.

Aldehydes↗

Use of interactive online histopathology modules at different stages of a veterinary program.

A mixed-method educational research study was conducted to evaluate the use of a series of online histopathology modules by students at two levels in a professional veterinary medical program. The materials were hypothesized to support student learning of histopathology concepts by facilitating "dual coding" of the content, since they provide both visual and verbal information. Second- and fourth-year professional veterinary students were surveyed about their perceptions of the effectiveness, efficiency, and appeal of the online modules. A historical comparison of final grades in the second-year Pathology course was conducted to evaluate the impact of the materials on academic outcomes. Think-aloud interviews were conducted with the fourth-year students in which the researcher observed the students interacting with the materials and asked them to "tell me what you're thinking as you use the modules." Survey results show that the only area in which the two student groups quantitatively rated the materials differently was their perception of how the materials complemented their learning. In contrast, the qualitative data indicate that advanced students used and perceived the materials differently, perhaps because of their greater metacognitive skills and pre-existing knowledge. We concluded that these findings can be attributed to the manner in which students approached the materials: the fourth-year students felt the materials were less relevant because they did not directly correspond to cases they were seeing, while the second-year students appreciated their relevance to topics in the Pathology course. There was no significant difference in final exam performance for the group of students that used the supplemental online modules and the prior four years' classes, who did not have access to the materials. Overall, this study demonstrated that it is possible to create a set of materials to be used by multiple audiences, provided the needs of each group are taken into consideration during the design process.

Animals↗

[Hyperacute kidney graft rejection with and without demonstration of antibodies].

Hyperacute rejection was studied in presensitized recipients of renal homografts and pig-to-dog heterografts. In these experiments antidonor antibodies can be observed but their participation in the rejection process cannot be assessed quantitatively. The importance of antibodies lies in their capacity to activate the complement system, which in turn destroys the graft. Intrarenal complement activation was also demonstrated in two clinical cases of hyperacute rejection, in one of which activation was by the alternate pathway.

Adult↗

[The complement-fixation test in rabies. I. Antibody titer of vaccinated dogs (author's transl)].

Complement-fixation test based in 50% hemolytic end point was applied to investigate the immune status to rabies of dogs vaccinated with heigh egg-passage Flury vaccine. The complement-fixation titer was compared with serum neutralization results. Twenty-five sera was employed and the complement fixation titer varied of 0 to 256. Three sera was anticomplementary. The results indicated a lack of quantitative correlation, but was found a qualitative correlation between the two methods.

Animals↗

Optimized simultaneous determination of several elements in human intestinal Caco-2 TC7 cells by inductively coupled plasma-mass spectrometry after closed vessel microwave digestion.

A rapid procedure, based on closed vessels microwave digestion and inductively coupled plasma-mass spectrometry (ICP-MS), was evaluated to ascertain the effect of chronic exposure to cadmium on intracellular accumulation of minor and essential trace elements in cultured epithelial cells (Caco-2 TC7). For all measurements, the method of external calibration was used and 3 elements (Be, Sc, In) were selected as internal standards. Optimization procedures are discussed and results are presented for the total determination of 9 key analytes (Na, Mg, Ca, Cr, Mn, Cu, Zn, Mo, and Cd) in certified reference materials (CRMs) and 20 samples of Caco-2 TC7 cells long-term exposed to Cd. The performance characteristics of the analytical system were evaluated by calibration and linearity, limits of detection and quantitation, accuracy with spiking, trueness and repeatability with available CRMs. As a complement to the ICP-MS determinations, both available CRMs and cell samples were analyzed either by electro thermal- or flame atomic absorption spectrometry. The results were in good agreement with the ICP-MS results.

Caco-2 Cells↗

Enhancement of neutrophil function by granulocyte-macrophage colony-stimulating factor involves recruitment of a less responsive subpopulation.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances numerous functions of mature neutrophils (PMN) including phagocytosis, superoxide responses to chemotaxins, antibody-dependent cellular cytotoxicity, and expression of complement receptors. A central question concerns whether the mechanism of enhancement involves quantitative increases in the response of all cells v subpopulation recruitment. The effects of GM-CSF on individual cell light scatter changes, membrane potential, and oxidant responses induced by the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP) were assessed by flow cytometry and by scoring individual cells for nitroblue tetrazolium dye (NBT) reduction. GM-CSF produced a dose- and time-dependent shift in forward light scatter that was very similar in character to that seen with FMLP or leukotriene B4 stimulation. Although not capable of depolarizing the cells directly, GM-CSF primed PMNs for enhanced membrane potential responses to FMLP by significantly increasing the proportion of depolarizing cells when compared with diluent-treated controls after a 60-minute incubation at 37 degrees C (79.4% +/- 3.4% v 29.5% +/- 4.7% GM-CSF v diluent, mean +/- SE, P less than .005, n = 11). Subpopulation recruitment by GM-CSF treatment was also demonstrated by the FMLP-elicited NBT test. Taken together, these results indicate that GM-CSF can modulate the function of mature PMN by enhancing the proportion of responsive cells.

Colony-Stimulating Factors↗