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Results for “Equilibrium optimization”

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At least 181 records · Page 10Linked to original sources
PubMed1990

Binding of [125I]iodipine to parathyroid cell membranes: evidence of a dihydropyridine-sensitive calcium channel.

The parathyroid cell is unusual, in that an increase in extracellular calcium concentrations inhibits PTH release. Calcium channels are glycoproteins that span cell membranes and allow entry of extracellular calcium into cells. We have demonstrated that the calcium channel agonist (+)202-791, which opens calcium channels, inhibits PTH release and that the antagonist (-)202-791, which closes calcium channels, stimulates PTH release. To identify the calcium channels responsible for these effects, we used a radioligand that specifically binds to calcium channels. Bovine parathyroid cell membranes were prepared and incubated under reduced lighting with [125I] iodipine (SA, 2000 Ci/mmol), which recognizes 1,4-dihydropyridine-sensitive calcium channels. Bound ligand was separated from free ligand by rapid filtration through Whatman GF/B filters. Nonspecific binding was measured by the inclusion of nifedipine at 10 microM. Specific binding represented approximately 40% of the total binding. The optimal temperature for [125I] iodipine binding was 4 C, and binding reached equilibrium by 30 min. The equilibrium dissociation constant (Kd) was approximately 550 pM, and the maximum number of binding sites was 780 fmol/mg protein. Both the calcium channel agonist (+)202-791 and antagonist (-)202-791 competitively inhibited [125I] iodipine binding, with 50% inhibition concentrations of 20 and 300 nM, respectively. These data indicate the presence of dihydropyridine-sensitive calcium channels on parathyroid cell membranes.

Animals↗
PubMed1993

Characterization and subtype identification of the Na(+)-H+ exchanger in bovine corneal epithelium.

Amiloride analogues with N5-alkyl substitutions are specific high-affinity ligands for the Na(+)-H+ exchanger in various tissues. As a means to characterize the Na(+)-H+ exchanger in the bovine corneal epithelium, we determined the binding properties of [3H] methylisobutylamiloride (MIA) to a fraction enriched in plasma membrane from this tissue. [3H]MIA bound to these membranes in a time, -a temperature-, and -a pH-dependent manner. The binding was optimal at 4 degrees C and at pH 8.5 and it reached equilibrium at 60 min. Under these conditions, specific binding, which was inhibitable by excess unlabeled MIA, was about 85%. Scatchard analysis of this specific binding revealed a single saturable binding component with a Kd of 61 nM and a Bmax of 271 pmoles/mg protein. Inhibition of [3H]MIA specific binding by amiloride analogues showed the following order of potency: MIA > dimethylamiloride (DMA) > benzamil > amiloride. Na+ did not compete with MIA for binding. The effectiveness of clonidine, an alpha 2 agonist, and cimetidine, an H2 receptor antagonist, as inhibitors of Na(+)-H+ exchange activity was also determined because these compounds are used to distinguish between the exchanger subtypes. At concentrations higher than those needed for receptor interaction, clonidine was more effective than cimetidine in decreasing MIA binding. The activity of Na(+)-H+ exchanger, which was measured as the uptake of 22Na+ in the presence of an outwardly directly H+ gradient, was also inhibited by DMA, benzamil and amiloride with the same order of potency as obtained in the binding studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed2000

Fundamental cryobiology of rat immature and mature oocytes: hydraulic conductivity in the presence of Me(2)SO, Me(2)SO permeability, and their activation energies.

The hydraulic conductivity in the presence of dimethyl sulfoxide Me(2)SO (L(p)(Me(2)SO)), Me(2)SO (P(Me(2)SO)) permeability and reflection coefficient (sigma) of immature (germinal vesicle; GV) and mature (metaphase II; MII) rat oocytes were determined at various temperatures. A temperature controlled micropipette perfusion technique was used to conduct experiments at five different temperatures (30, 20, 10, 4, and -3 degrees C). Kedem and Katchalsky membrane transport theory was used to describe the cell volume kinetics. The cell volumetric changes of oocytes were calculated from the measurement of two oocyte diameters, assuming a spherical shape. The activation energies (E(a)) of L(p)(Me(2)SO) and P(Me(2)SO) were calculated using the Arrhenius equation. Activation energies of L(p)(Me(2)SO) for GV and MII oocytes were 34.30 Kcal/mol and 16.29 Kcal/mol, respectively; while the corresponding E(a)s of P(Me(2)SO) were 19.87 Kcal/mol and 21.85 Kcal/mol, respectively. These permeability parameters were then used to calculate cell water loss in rat oocytes during cooling at subzero temperatures. Based on these values, the predicted optimal cooling rate required to maintain extra- and intracellular water in near equilibrium for rat GV stage oocytes was found to be between 0.05 degrees C/min and 0. 025; while for rat MII oocytes, the corresponding cooling rate was 1 degrees C/min. These data suggest that standard cooling rates used for mouse oocytes (e.g., 0.5-1 degrees C/min) can also be employed to cryopreserve rat MII oocytes. However, the corresponding cooling rate required to avoid damage must be significantly slower for the GV stage rat oocyte. J. Exp. Zool. 286:523-533, 2000.

Animals↗
PubMed1992

[Model of therapeutic programming in NIDDM diabetes].

The usual choice of therapy in NIDDM diabetes, using oral anti-diabetic compounds, insulin or associated treatments, is based on the results of treatment evaluated empirically using glycemic profiles. As a contribution to the search for a method of choosing a treatment which is based on laboratory data and can restore metabolic equilibrium as quickly as possible using the most efficacious drug at an optimal dose, the paper reports a method of assessing the severity of diabetes according to mean daily glucose concentrations, the degree of instability in relation to the standard deviation, and proposes a sensitivity test to SU which indicates the choice of therapy, together with an insulin sensitivity test which is useful for evaluated the optimal dose. Sensitivity to SU evaluated using this method is not dependent on the degree of severity of diabetes. With regard to its practical use for prescribing treatment the test is highly predictive in positive cases since it is extremely sensitive, while its low specificity does not rule out its use in cases of resistance. The analysis of the results obtained after treatment which was not indicated by the sensitivity tests but based on personal experience and the comparison of the two methods shows that a high percentage of patients received inadequate therapy either due to the prescription of the wrong type of treatment or an incorrect dosage.

Blood Glucose↗
PubMed1991

Human postural dynamics.

The study of posture dynamics is important not only to understand disorders of impaired equilibrium and protective reactions to unexpected displacements of the human body, but also to the design of prosthesis and functional neuromuscular stimulation as aids to patients with impaired postural stability and locomotion. Coordinated control of the body segments is a complex aspect of motor behavior, owing to the multiple degrees of freedom of the controlled system. Several interacting subsystems are involved in the dynamics of human posture and locomotion, including the skeletal, neuromuscular, and sensory systems. Human posture control is maintained by somatosensory, vestibular, and visual feedback, integrated within the locomotor and central nervous systems. Studies of posture dynamics and stability therefore entail the study of mechanical aspects of the human body, its sensory systems, and the principles governing coordination in motion control. In this paper is reviewed some of the research done in the field of human posture dynamics, including such topics as biomechanics, equilibrium, stability, motion coordination, neural feedback, neural control systems modeling, motion strategies, optimality of motion, and adaptation. We consider experimental approaches and theoretical models, as well as the gap between them. Principles for the experimental investigation of control systems are considered.

Animals↗
PubMed1988

Changes in phosphoproteins during commitment of HL60 cells to monocyte differentiation: evidence for multiple protein kinase involvement.

The human promyeloid cell line HL60 differentiates toward monocytes when treated with TPA. We have analyzed, by two-dimensional gel electrophoresis, the phosphoprotein patterns within HL60 cells, labeled to equilibrium with [32P]orthophosphate when cells were treated with suboptimal (1 nM), optimal (5 and 10 nM), and supraoptimal (40 and 100 nM) concentrations of 12-O-tetradecanylphorbol-13-acetate (TPA) as regards the induction of differentiation. No change was detected in the phosphoprotein pattern at 1 nM TPA, whereas four phosphoproteins showed increased levels of phosphorylation at 5 and 10 nM TPA. When cells were treated with 40 and 100 nM TPA, in total eight and ten proteins, respectively, were phosphorylated, including the above four proteins. Two proteins were dephosphorylated when cells were treated with 40 and 100 nM TPA. A 15-kd protein, phosphorylated when HL60 cells were treated with 5 nM TPA, was observed as an intense spot in autoradiographs of total cellular phosphoproteins of two variant HL60 cell lines that are unable to differentiate toward monocytes and prior to treatment with TPA. In the case of three variant cell lines, which like HL60 differentiate toward monocytes, the phosphoprotein spot was almost absent. Thus, paradoxically, the 15-kd phosphoprotein is affected by TPA although its constitutive level of expression or increased phosphorylation state is inversely related to the potential for monocyte differentiation. This observation, together with the TPA dose-response effects on protein phosphorylation, is discussed in relation to multiple protein kinase involvement.

Cell Differentiation↗
PubMed1984

Spectrophotometric assay of bisphosphoglycerate mutase: a reexamination of Rapoport-Luebering's method.

The saturation by substrates and cofactors, the effects of pH and the influence of salts and auxiliary enzymes have been studied. The linear NAD+ reduction observed before addition of haemolysate to the assay system was proportional to pH, being higher with phosphate than with Tris-HCl buffer. In the presence of bisphosphoglycerate mutase, an optimal pH (7.8-8.1) was obtained and the inhibition by sulfate ions could be confirmed. It can then be suggested that the absence of an equilibrium, the pH used by several authors and sulfate inhibition could be sources of error in the spectrophotometric assay of bisphosphoglycerate mutase activity. Once optimal conditions have been established, activities found in both human and rat erythrocytes are similar to those given by other accurate methods.

Animals↗
PubMed1993

Regulation of inositol 1,4,5-trisphosphate receptors in rat basophilic leukemia cells. I. Multiple conformational states of the receptor in a microsomal preparation.

A detailed characterization of the inositol 1,4,5-trisphosphate (IP3) receptor in rat basophilic leukemia (RBL) cells, a neoplastic mast cell line, has been possible through the growth of solid RBL cell tumors which provide a rich source of IP3 receptor. Equilibrium binding studies show a 1.6 +/- 0.1 pmol/mg of protein maximal binding capacity for [3H]IP3 at optimal Ca2+ (10 microM). The specificity of the RBL cell IP3 receptor towards phosphoinositides, ATP and heparin parallels those previously described with excitable and nonexcitable tissues. [3H]IP3 binding is slightly enhanced from < 1 nM to 10 microM Ca2+ and inhibited by > 10 microM Ca2+. Kinetic and equilibrium studies provide evidence for at least two classes or conformational states of binding sites with pico- and nanomolar affinities. At nM concentrations of IP3, neither binding to the IP3 receptor nor IP3-induced Ca2+ efflux from permeabilized cells demonstrates cooperativity. In contrast, at pM concentrations, IP3 binding kinetics deviate from simple mass action suggesting a complex interaction among binding sites for IP3 on the receptor-channel oligomer. The mechanisms that regulate [3H]IP3 binding in RBL cells are unique when compared to what has been reported in other cells.

Animals↗
PubMed1996

Characterization of topoisomerase II-DNA interaction and identification of a DNA-binding domain by ultraviolet laser crosslinking.

We have used ultraviolet laser crosslinking to characterize the DNA-binding properties of highly purified yeast topoisomerase II in the absence of ATP. A single 5 ns, 20 mJ pulse of 266 nm light produced optimal crosslinking to a short DNA duplex, with an efficiency of 0.25%. An equilibrium binding constant (Keq) of 1.2 +/- 0.5 x 10(8) M(-1) was determined from kinetic analysis. Topoisomerase II showed highest affinity for supercoiled DNA. Limited proteolysis of crosslinked topoisomerase II-DNA complexes showed a site of crosslinking to be within a 29-kDa fragment with Leu-681 at its amino-terminal end. This region contains the active Tyr-783 and is homologous to the amino-terminal region of the DNA-binding bacterial gyrase GyrA subunit, suggesting a conserved DNA-binding mechanism.

Base Sequence↗
PubMed1988

Angiotensin II receptors in the fowl aorta.

In the domestic fowl, angiotensin II (ANG II) causes an in vivo depressor response and in vitro relaxation of aortic rings which appear to be a direct action of ANG II on the blood vessels. Thus, we determined whether binding sites specific to ANG II exist in the membrane fraction of the fowl aorta. The particulate fraction of aortas from adult female fowl, Gallus gallus, exhibits high specific binding to ANG II ligand. 125I-[Ile5]ANG II (0.5 nM) binding to the above fraction (30 micrograms protein) in 50 mM Tris (pH 7.2), 10 mM MgCl2, and 0.2% bovine serum albumin at 12 degrees (1) is rapid, saturable, and reversible; (2) increases as a function of ligand or membrane concentration, time, and temperature; and (3) optimally fits to a two-site (high and low affinity) model. The equilibrium dissociation constant (0.15 +/- 0.03 nM) and binding site concentration (28.7 +/- 8.1 fmol/mg protein) of the high affinity site as well as association (0.055 nM-1.min-1) and dissociation (0.0122 min-1) rate constants are similar to those of mammalian vascular ANG II receptors. Both 125I-[Ile5]ANG II and 125I-[Val5]ANG II are competitively displaced by unlabeled ANG II. These results suggest that specific ANG II receptors exist in the fowl aorta.

Angiotensin II↗
PubMed1982

The manpower situation in pathology: implications for the future.

With the exception of academic pathology and specific subspecialty areas, manpower in pathology is currently in a state of apparent equilibrium between supply and demand. Exogenous influences, and especially an influx of Americans who have attended foreign-chartered medical schools, could disturb this apparent equilibrium. Other key manpower issues with respect to pathology include mechanisms to determine optimal numbers of pathologists, the future productivity of pathologists, current and prospective needs in academic pathology and subspecialty areas, and the role of the federal government in the area of medical manpower. Recommendations to deal with these and related issues are presented.

Foreign Medical Graduates↗
PubMed1998

Length changes of the cat soleus muscle under frequency-modulated distributed stimulation of efferents in isotony.

The length changes of the cat soleus muscle have been examined in isotony within closed cycles of the stimulation rate change. Successive stimuli were applied in a cycle to five filaments of the preliminary dissected L7-S1 ventral roots (method of distributed stimulation), maximal rate did not exceed 80-90/s (16-18/s per single filament). At the beginning of a slow linear increase in the rate a muscle began shortening rather quickly, the rate changes without muscle reaction consisted of 5.91 +/- 0.28/s (mean +/- S.E.M.). A substantially linear movement was observed during increase of the input rate up to 40-50/s (i.e. 8-10/s per filament), a further rate increment could evoke a somewhat slowing of the shortening velocity with a distinctive infection of the rate-length curve. During a significant part of the rate decrease phase no movement was seen, the rate range with the absence of muscle lengthening was 29.93 +/- 1.57/s. After such a pronounced period of length fixation, a muscle began to elongate, the steady-state velocity at this part of the movement trajectory was invariably higher as compared with shortening velocity at the leading edge of the cycle. The striking feature of a powerful length clamping seen in active muscle at the phase of rate decrease preceded by previous rate increment allows us to suppose that this strongly non-linear behaviour of muscle might be a main reason for the existence of powerful dynamic components in efferent activity every time when muscle should shorten against external load in a ramp-and-hold fashion. We used the following experimental paradigm to check the assumption. Stimulation began with regular rate of 15/s; then, after transition from isometry to isotony and cessation of movement transients, the rate was raised linearly; after reaching a peak value of 50-90/s, it decreased linearly or exponentially, being afterwards fixed at several different levels between the maximal and initial values of the rate. It was demonstrated that such pattern of stimulation could be effective for a linear transition between two equilibrium lengths, provided that corresponding parameters in modulation signal were chosen in an optimal way. Duration and amplitude of the leading edge of the dynamic component seemed to completely define amplitude and velocity of the ramp phase of movement, parameters of decay to the final steady rate were, on the other hand, important for efficacy of the length clamping at hold phase. It was concluded that hysteresis effects of muscle contraction seemed to be an extremely important nonlinear property of muscle dynamics in producing any transition movements between two steady-states. Thus, oversimplified muscle models, like the spring model based on the isometric length-tension dependencies, seem to be incorrect. Possible mechanisms for the muscle hysteresis and its role in motor control are discussed.

Animals↗
PubMed1999

Efficient discrimination between different densities of target antigen by tetracycline-regulatable T bodies.

Engineered T cells expressing chimeric T cell receptors (chTCRs) are of interest for cancer gene therapy but many "cancer antigens" are thought to be unsuitable targets because they are expressed at low levels on normal tissues. We therefore sought to determine whether engineered T cells expressing variable surface densities of a high-affinity chTCR could discriminate different concentrations of the targeted antigen. We plotted the relationship between chTCR density and the concentration of target antigen using Jurkat T cells expressing a hapten-binding chTCR whose expression could be modulated by tetracycline. Our analysis reveals that there is a dynamic equilibrium between cell surface density of the chTCR and the antigen density that optimally triggers T cell activation. At a fixed density of target antigen, optimal T cell activation can be achieved only within a certain range of chTCR densities, while excessive TCR signaling triggers apoptosis of the engineered T cells. Our results show that T cells can be engineered to discriminate different antigen densities and that the T cell response to a fixed concentration of antigen can be optimized by tuning the cell surface density of TCRs.

Antigens, Neoplasm↗
PubMed1976

Creatine kinase in serum: 1. Determination of optimum reaction conditions.

To establish optimum conditions for creatine kinase (EC 2.7.3.2) activity measurement with the creatine phosphate in equilibrium creatine reaction, we re-examined all kinetics factors relevant to an optimal and standardized enzyme assay at 30 and 25 degrees C. We determined the pH optimum in vaious buffers, considering the effect of the type and concentration of the buffer, as well as the influence of various buffer anions on the activity. The relation between activity and substrate concentration was shown and the apparent Michaelis constants of creatine kinase for creatine phosphate and ADP were evaluated. We tested the effect on creatine kinase measurement of the concentration of substrates (glucose and NADP+) in the auxillary and indicator reactions, especially the influence of the added auxiliary (hexokinase) and indicator (glucose-6-phosphate dehydrogenase) enzymes on the lag phase, at different temperatures. The NADP+ concentration proved to be the factor limiting the duration of constant reaction rate. We studied the inhibition of creatine kinase and adenylate kinase by AMP and established a convenient AMP concentration. For reactivation of creatine kinase, N-acetyl cysteine as sulfhydryl compound was introduced. Finally, we examined the relationship between activity and temperature.

Blood Glucose↗
PubMed1992

Evolutionary optimization of the catalytic efficiency of enzymes.

1. The rate equation for a generalized Michaelian type of enzymic reaction mechanism has been analyzed in order to establish how the mechanism should be kinetically designed in order to optimize the catalytic efficiency of the enzyme for a given average magnitude of true and apparent first-order rate constants in the mechanism at given concentrations of enzyme, substrate and product. 2. As long as on-velocity constants for substrate and product binding to the enzyme have not reached the limiting value for a diffusion-controlled association process, the optimal state of enzyme operation will be characterized by forward (true and apparent) first-order rate constants of equal magnitude and reverse rate constants of equal magnitude. The drop in free energy driving the catalysed reaction will occur to an equal extent for each reaction step in the mechanism. All internal equilibrium constants will be of equal magnitude and reflect only the closeness of the catalysed reaction to equilibrium conditions. 3. When magnitudes of on-velocity constants for substrate and product binding have reached their upper limits, the optimal kinetic design of the reaction mechanism becomes more complex and has to be established by numerical methods. Numerical solutions, calculated for triosephosphate isomerase, indicate that this particular enzyme may or may not be considered to exhibit close to maximal efficiency, depending on what value is assigned to the upper limit for a ligand association rate constant. 4. Arguments are presented to show that no useful information on the evolutionary optimization of the catalytic efficiency of enzymes can be obtained by previously taken approaches that are based on the application of linear free-energy relationships for rate and equilibrium constants in the reaction mechanism.

Biological Evolution↗
PubMed1978

Experimental optimization of the detection limit of two-step solid-phase radioimmunoassay.

For two-step inhibition radioimmunoassay (sequential saturation or delayed addition of labeled antigen) generally a higher sensitivity than for one-step inhibition radioimmunoassay (equilibrium assay) is expected. The detection limit of a two-step solid phase inhibition radioimmunoassay for human serum immunoglobulin A was minimized by statistical methods of experimental optimization. Under optimal conditions the detection limit was 2.3 ng IgA. This is about 1.8 times lower than the minimal detection limit of the one-step assay under similar conditions of the qualitative variables such as the origin of the antibody. This increase in sensitivity was associated with a decrease in the precision of the assay. The results are discussed with respect to the comparison of the one-step and the two-step assay and the usefulness of a sensitive radioimmunoassay in practice.

Evaluation Studies as Topic↗
PubMed1999

A procedure to determine equilibrium postural configurations for arbitrary locations of the feet.

This research was directed toward predicting postural equilibrium configurations in normal humans for asymmetric locations of the feet. The objective of the study was to identify trends in the variation of the location of ground center of pressure (COP) with increasing levels of asymmetry in the foot placement. The procedure developed here minimized the muscular effort (active torques) in the lower extremities while maximizing postural stability margins for given foot locations. Minimizing muscular effort led to fully extended knees, and maximal stability margin led to the COP moving toward the rear foot in asymmetric stance. A combined analytical-numerical optimization scheme was used to avoid singularities that can arise due to the fact that at equilibrium postural configurations, the torso lies at or near the workspace boundary of the lower extremities. Experiments were conducted and the results obtained were in keeping with the model predictions. This basic understanding of asymmetric stance is important for studying asymmetric postural mechanics in the presence of external disturbances, and for extending the results from normal subjects to humans at both ends of the life span.

Algorithms↗