scholarly journals New Applications for the Kinetic Exclusion Assay (KinExA)

2021 ◽  
Author(s):  
Mark Harrison Smith
Keyword(s):  
Kinetic Parameters ◽  
Free Ligand ◽  
Analytical Techniques ◽  
Membrane System ◽  
Dna Aptamers ◽  
Artificial Membranes ◽  
Binding Model ◽  
Whole Cells ◽  
Analytical System ◽  
Membrane Components

This dissertation examines the fundamental principles and applicability of the kinetic exclusion assay (KinExA), developed and marketed by Sapidyne Instruments of Boise, Idaho, since 1995. Chapter One reviews and consolidates the manufacturer’s guidance and many early papers that delineate the practical and theoretical aspects of the technology. In brief, KinExA is a two stage analytical system. In stage one, a number of solutions are prepared, whereby one of the partners is kept constant (the constant binding partner, or CBP), while the other (the titrant) is varied, usually in serial dilution. As the titrant is increased, the free CBP decreases, and is analyzed by a sophisticated and precise microfluidic fluorometric device (stage two). The resulting signal can be related mathematically to the affinity (KD) of the two molecules for each other, and to the kinetic parameters of binding (kon) and dissociation (koff). A comparison of KinExA with other current technology available for quantification of interactions is provided. In Chapter Two, I investigate the use of KinExA technology with DNA aptamers. DNA aptamers are short nucleotide oligomers selected to bind a target ligand with affinity and specificity rivaling that of antibodies. These remarkable features make them promising alternatives for analytical and therapeutic applications that traditionally use antibodies as biorecognition elements. Numerous traditional and emerging analytical techniques have been proposed and successfully implemented to utilize aptamers for sensing purposes. In this work, I exploited the analytical capabilities offered by the KinExA technology to measure the affinity of fluorescent aptamers for their target molecule thrombin, and quantify the concentration of analyte in solution. Standard binding curves constructed by using equilibrated mixtures of aptamers titrated with thrombin were fitted with a 1:1 binding model and provided an effective KD of the binding in the sub-nanomolar range. However, the experimental results suggest that this simple model does not satisfactorily describe the binding process; therefore, the possibility that the aptamer is composed of a mixture of two or more distinct KD populations is discussed. The same standard curves, together with a four-parameter logistic equation, were used to determine “unknown” concentrations of thrombin in mock samples. The ability to identify and characterize complex binding stoichiometry, together with the determination of target analyte concentrations in the pM–nM range, supports the adoption of this technology for kinetics, equilibrium, and analytical purposes by employing aptamers as biorecognition elements. In Chapter Three, I explore complex capture agents in the KinExA system. Liposomes made from purified reagents, or from natural cellular membranes, are attached to the beads used in the KinExA process to capture the analyte. Model molecules representing lipophilic dyes, antibodies, and bacterial toxins were successfully captured by the beads for measurement. Residual free ligand captured after pre-equilibration with membrane components, presented as either liposomes or whole cells, could be quantified, and kinetic parameters determined. By this process the “bi-molecular” interaction of the B subunit of cholera toxin for the ganglioside GM1 incorporated into artificial membranes could be quantified, and shown to be dependent upon the presence of the ganglioside in the membrane. The diffusion into artificial membranes of the lipophilic dye DID could be quantified and shown to be dependent upon the amount of lipid available in the equilibration step. In addition, the bulk affinity of a commercial polyclonal antibody for the surface antigens of their target red blood cells could be evaluated. This membrane immobilization process appears to be generally applicable to any membrane system. Thus, it promises to be valuable for the study of signaling molecules for which purified soluble target cellular components may result in misleading information, or for which soluble fragments are unavailable. Likewise, this process should aid in the search for drugs which mimic or antagonize these signaling ligands.

scholarly journals Moraxella catarrhalis Bacterium without Endotoxin, a Potential Vaccine Candidate

2005 ◽  
Vol 73 (11) ◽  
pp. 7569-7577 ◽  
Author(s):  
Daxin Peng ◽  
Wenzhou Hong ◽  
Biswa P. Choudhury ◽  
Russell W. Carlson ◽  
Xin-Xing Gu
Keyword(s):  
Moraxella Catarrhalis ◽  
Lipid A ◽  
Vaccine Candidate ◽  
Gene Encoding ◽  
Potential Vaccine Candidate ◽  
Whole Cells ◽  
Potential Vaccine ◽  
Major Surface ◽  
Membrane Components ◽  
Human Infections

ABSTRACT Lipooligosaccharide (LOS) is a major surface component of Moraxella catarrhalis and a possible virulence factor in the pathogenesis of human infections caused by this organism. The presence of LOS on the bacterium is an obstacle to the development of vaccines derived from whole cells or outer membrane components of the bacterium. An lpxA gene encoding UDP-N-acetylglucosamine acyltransferase responsible for the first step of lipid A biosynthesis was identified by the construction and characterization of an isogenic M. catarrhalis lpxA mutant in strain O35E. The resulting mutant was viable despite the complete loss of LOS. The mutant strain showed significantly decreased toxicity by the Limulus amebocyte lysate assay, reduced resistance to normal human serum, reduced adherence to human epithelial cells, and enhanced clearance in lungs and nasopharynx in a mouse aerosol challenge model. Importantly, the mutant elicited high levels of antibodies with bactericidal activity and provided protection against a challenge with the wild-type strain. These data suggest that the null LOS mutant is attenuated and may be a potential vaccine candidate against M. catarrhalis.

Topological and regulatory aspects of dolichyl phosphate mediated glycosylation of proteins

1982 ◽  
Vol 300 (1099) ◽  
pp. 129-144 ◽  
Keyword(s):  
Secretory Pathway ◽  
Membrane System ◽  
Post Translational Modifications ◽  
Regulatory Aspects ◽  
Dolichyl Phosphate ◽  
Complex Membrane ◽  
Secretory Glycoproteins ◽  
Membrane Components ◽  
Polypeptide Chains

From the time of their synthesis in the rough endoplasmic reticulum until they are secreted, packaged in lysosomes, or appear as membrane components at the cell surface, the polypeptide chains of N - and O -linked glycoproteins remain associated with intracellular membranes that are components of the secretory pathway. The various co-translational and post-translational modifications of the carbohydrate moieties of glycoproteins have been shown to occur within morphologically and functionally distinct regions of this complex membrane system. However, the sugar nucleotides, which serve as precursors to the oligosaccharide moieties of these glycoproteins, are synthesized almost exclusively in the cytoplasm. These findings raise a number of questions about the mechanisms involved in the transmembrane assembly of membrane and secretory glycoproteins. In this paper these questions are reviewed and recent studies directed towards providing answers to them are summarized. In addition, information related to the possible role of dolichyl phosphate in regulating the glycosylation of proteins is presented.

scholarly journals Aromatic indolinic aminoxyls as antioxidants in cardiac sarcoplasmic reticulum lipid and protein oxidation.

2002 ◽  
Vol 49 (1) ◽  
pp. 43-49 ◽  
Author(s):  
Dorota Kulawiak-Gałaska ◽  
Michał Woźniak ◽  
Lucedio Greci
Keyword(s):  
Lipid Peroxidation ◽  
Sarcoplasmic Reticulum ◽  
Protein Oxidation ◽  
Acyl Chain ◽  
Thiobarbituric Acid ◽  
Membrane System ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Lipid And Protein Oxidation ◽  
Lipid Peroxidation Inhibition ◽  
Membrane Components

The results presented demonstrate the influence of aromatic indolinic aminoxyls: 1,2-dihydro-2-ethyl-2-phenyl-3H-indole-3-phenylimino-1-oxyl (IA-C2) and 1,2-dihydro-2-octadecyl-2-phenyl-3H-indole-3-phenylimino-1-oxyl (IA-C18) on oxidation of lipids and proteins of cardiac sarcoplasmic reticulum membranes. We have used doxorubicin and t-butyl hydroperoxide as agents inducing oxidative stress in isolated rat cardiac sarcoplasmic reticulum membrane system. Carbonyl groups were measured as the end product of membrane protein oxidation, and thiobarbituric acid reactive substances were assessed as a marker of lipid peroxidation. Inhibition of peroxidation of certain membrane components depends on the length of acyl chain. Aminoxyl IA-C2 inhibits the lipid peroxidation process while IA-C18 is an efficient protector against protein oxidation.

A Versatile XRF Analytical System for Geochemical Exploration and Other Applications

1983 ◽  
Vol 27 ◽  
pp. 481-486 ◽  
Author(s):  
T. K. Smith
Keyword(s):  
Large Scale ◽  
Mineral Exploration ◽  
Analytical Techniques ◽  
Geochemical Analysis ◽  
Geochemical Exploration ◽  
Fourth Period ◽  
Conventional Methods ◽  
Analytical System ◽  
Geological Sciences ◽  
And Tungsten

For more than a decade the Institute of Geological Sciences has carried out large-scale geochemical analysis in pursuit of mineral exploration and regional geochemical reconnaissance programmes for the Department of Industry. Over this period an XRF analytical system has been developed to meet part of this requirement for multielement data. The elements of interest range from the fourth period to uranium, and have become more numerous as exploration and analytical techniques have expanded. In particular, additions have been made of those elements such as arsenic, molybdenum and tungsten which are more difficult of determination by conventional methods.

Concepts et techniques d'analyse des phénomènes administratifs

1968 ◽  
Vol 1 (3) ◽  
pp. 310-335 ◽  
Author(s):  
Lionel Ouellet
Keyword(s):  
Comparative Analysis ◽  
General Theory ◽  
Information Exchange ◽  
Analytical Techniques ◽  
Functional Theory ◽  
Administrative Roles ◽  
Administrative Process ◽  
Analytical System

Concepts and Analytical Techniques of Administrative PhenomenaAmong the phenomena inherent in administrative activities, four key categories may be distinguished: (1) the “duties” of the administration which give rise to whole families of activities and operations; (2) administrative “roles” which facilitate execution of these duties and provide the functional means for their execution; (3) administrative “organs” or institutions, charged with exercising the executive powers and which organize the agents assuming various administrative roles; and (4) the administrative “process” or information exchange circuits which inter-connect administrative duties, roles, and organs.It is argued that the roles emerge from the type of operation, that the organs are, in several forms, postulated by the types of roles, and that the administrative processes are brought into being by the differing outcomes of the work of the administrative organs. We have here the embryo of a structural-functional theory of administration.The thesis is presented throughout as a method of analysing the organization and operation of administrative institutions and as a means which may be used for reforming them. Such an analytical system could serve as a bridge facilitating comparative analysis of different administrative institutions and as the administrative element of a general theory of politics.

Relationship of cytochrome content to the sensitivity of bacteria to NaCl on freezing and thawing

1977 ◽  
Vol 23 (4) ◽  
pp. 413-419 ◽  
Author(s):  
Sai K. Lee ◽  
Peter H. Calcott ◽  
Robert A. MacLeod
Keyword(s):  
Sodium Chloride ◽  
Respiratory Activity ◽  
Membrane Integrity ◽  
Model Organism ◽  
Salt Sensitivity ◽  
Freezing And Thawing ◽  
Whole Cells ◽  
E Coli ◽  
Membrane Components ◽  
Cytochrome Content

Eight species of bacteria representing rod, coccus, gram-positive, and gram-negative forms were tested for their sensitivity to sodium chloride during freezing and thawing. Six of the eight species tested were salt-sensitive, though to different degrees, while Lactobacillus casei and Streptococcus faecalis were resistant. Escherichia coli grown anaerobically exhibited only 38% of the salt sensitivity of aerobically grown cells. Analysis of cytochrome pigments in the organisms revealed that the six sensitive organisms all contained these pigments but in varying amounts, while the two resistant ones were devoid of them. Anaerobically grown E. coli contained 50% of the cytochromes of aerobically grown cells. A relationship between cytochrome content of the organisms and salt sensitivity during freezing and thawing was demonstrated with a correlation coefficient of 0.76 (P < 0.05); the higher the cytochrome content, the more salt-sensitive the organism. This indicated that 58% of the salt sensitivity was due to the cytochrome content.Using a model organism, E. coli, the effect of salt during freezing and thawing on the respiratory activity was examined. Freezing and thawing in water or saline decreased the respiration by whole cells of substrates expected to be NAD-linked while NADH-stimulated respiration was increased. In cell-free extracts derived from unfrozen cells or those frozen and thawed in water or saline, the respiration of ascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) was constant. The respiration of NADH, succinate, and lactate in cell-free extracts derived from cells frozen and thawed in saline was reduced compared with those extracts derived from unfrozen cells or cells frozen and thawed in water. Studies with E. coli showed that the decreased respiratory activity caused by disruptions in the electron-transport chain could not account for the salt sensitivity on freezing and thawing. More likely, salt sensitivity is related to the presence of bonds between cytochromes and other membrane components which are disrupted by sodium chloride on freezing and thawing. This would then result in loss of membrane integrity and function.

scholarly journals Role of Periplasmic Trehalase in Uptake of Trehalose by the Thermophilic Bacterium Rhodothermus marinus

2008 ◽  
Vol 190 (6) ◽  
pp. 1871-1878
Author(s):  
Carla D. Jorge ◽  
Luís L. Fonseca ◽  
Winfried Boos ◽  
Helena Santos
Keyword(s):  
Kinetic Parameters ◽  
Glucose Transport ◽  
Transport System ◽  
Glucose Transporter ◽  
Thermophilic Bacterium ◽  
Transport Systems ◽  
Uptake System ◽  
Rhodothermus Marinus ◽  
Whole Cells ◽  
Physiological Relevance

ABSTRACT Trehalose uptake at 65°C in Rhodothermus marinus was characterized. The profile of trehalose uptake as a function of concentration showed two distinct types of saturation kinetics, and the analysis of the data was complicated by the activity of a periplasmic trehalase. The kinetic parameters of this enzyme determined in whole cells were as follows: Km = 156 ± 11 μM and V max = 21.2 ± 0.4 nmol/min/mg of total protein. Therefore, trehalose could be acted upon by this periplasmic activity, yielding glucose that subsequently entered the cell via the glucose uptake system, which was also characterized. To distinguish the several contributions in this intricate system, a mathematical model was developed that took into account the experimental kinetic parameters for trehalase, trehalose transport, glucose transport, competition data with trehalose, glucose, and palatinose, and measurements of glucose diffusion out of the periplasm. It was concluded that R. marinus has distinct transport systems for trehalose and glucose; moreover, the experimental data fit perfectly with a model considering a high-affinity, low-capacity transport system for trehalose (Km = 0.11 ± 0.03 μM and V max = 0.39 ± 0.02 nmol/min/mg of protein) and a glucose transporter with moderate affinity and capacity (Km = 46 ± 3 μM and V max = 48 ± 1 nmol/min/mg of protein). The contribution of the trehalose transporter is important only in trehalose-poor environments (trehalose concentrations up to 6 μM); at higher concentrations trehalose is assimilated primarily via trehalase and the glucose transport system. Trehalose uptake was constitutive, but the activity decreased 60% in response to osmotic stress. The nature of the trehalose transporter and the physiological relevance of these findings are discussed.

scholarly journals A method for determining kinetic parameters at high enzyme concentrations

1982 ◽  
Vol 203 (1) ◽  
pp. 339-342 ◽  
Author(s):  
C J Halfman ◽  
F Marcus
Keyword(s):  
Enzyme Activity ◽  
Kinetic Parameters ◽  
Graphical Method ◽  
Free Ligand ◽  
Enzyme Concentration ◽  
Fructose 1,6 Bisphosphatase ◽  
High Enzyme ◽  
Substrate Inhibitor ◽  
The Relationship

A graphical method is described which allows determination of kinetic parameters when substrate, inhibitor or activator concentrations must be in the vicinity of the enzyme concentration and a significant fraction of ligand is bound. Velocity is measured at several ligand: enzyme ratios at two or more enzyme concentrations. Results are obtained in terms of free and bound ligand corresponding to particular velocities. The relationship between velocity and bound and free ligand may then be analysed by any desired plotting technique. Preknowledge of the reaction mechanism or experimental determination of Vmax. is not required. The relationship between ligand bound and enzyme activity need not be linear and the method is equally suitable for analysing co-operative as well as simple kinetics. Application of the method is demonstrated by analysis of the inhibition of fructose, 1,6-bisphosphatase by AMP.

scholarly journals Characterization of Early Steps in the Poliovirus Infection Process: Receptor-Decorated Liposomes Induce Conversion of the Virus to Membrane-Anchored Entry-Intermediate Particles

2006 ◽  
Vol 80 (1) ◽  
pp. 172-180 ◽  
Author(s):  
Tobias J. Tuthill ◽  
Doryen Bubeck ◽  
David J. Rowlands ◽  
James M. Hogle
Keyword(s):  
Conformational Change ◽  
Membrane System ◽  
Nitrilotriacetic Acid ◽  
Infection Process ◽  
Membrane Pore ◽  
Whole Cells ◽  
Poliovirus Infection ◽  
Poliovirus Receptor ◽  
N Terminus ◽  
Liposome System

ABSTRACT The mechanism by which poliovirus infects the cell has been characterized by a combination of biochemical and structural studies, leading to a working model for cell entry. Upon receptor binding at physiological temperature, native virus (160S) undergoes a conformational change to a 135S particle from which VP4 and the N terminus of VP1 are externalized. These components interact with the membrane and are proposed to form a membrane pore. An additional conformational change in the particle is accompanied by release of the infectious viral RNA genome from the particle and its delivery, presumably through the membrane pore into the cytoplasm, leaving behind an empty 80S particle. In this report, we describe the generation of a receptor-decorated liposome system, comprising nickel-chelating nitrilotriacetic acid (NTA) liposomes and His-tagged poliovirus receptor, and its use in characterizing the early events in poliovirus infection. Receptor-decorated liposomes were able to capture virus and induce a temperature-dependent virus conversion to the 135S particle. Upon conversion, 135S particles became tethered to the liposome independently of receptor by a membrane interaction with the N terminus of VP1. Converted particles had lost VP4, which partitioned with the membrane. The development of a simple model membrane system provides a novel tool for studying poliovirus entry. The liposome system bridges the gap between previous studies using either soluble receptor or whole cells and offers a flexible template which can be extrapolated to electron microscopy experiments that analyze the structural biology of nonenveloped virus entry.

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