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PubMed1997

Optimized conditions for the production of recombinant amphotropic retroviral vector preparations.

The production and stability of recombinant retroviral vectors was examined at various temperatures. The two studied recombinant retroviral vectors, based on different packaging cell lines, exhibited a four-fold increased half-life at 32 degrees C as compared to 37 degrees C. Surprisingly, this increased stability at 32 degrees C was only observed within a very narrow temperature window. At 30 degrees C and 34 degrees C, retroviral vector half-lives were quite similar to that at 37 degrees C. Regardless of the vector half-life, retroviral vectors accumulated in the culture medium for a period of 48 h before an equilibrium was reached between retroviral vector production and decay. Maximal accumulated recombinant retroviral titers were five- to ten-fold increased after a medium incubation at 32 degrees C as compared to 37 degrees C. Furthermore, multiple cycles of freezing and thawing of retroviral vector supernatants hardly affected the recombinant retroviral vector titer, independent of the presence of serum. This knowledge on characteristics of recombinant retroviral vectors has practical implications for the manufacturing of these viruses for clinical gene therapy protocols.

Cell Line↗
PubMed2001

Methods for the determination of binding constants by capillary electrophoresis.

Apparent equilibrium constants for molecular association (e.g., association constants, binding constants, dissociation constants, partition coefficients) can be determined with a variety of different capillary electrophoresis (CE) approaches. In many cases, the investigated association behavior is between a smaller molecule or ion (i.e., the solute, drug, or analyte of interest) and a larger entity (e.g., proteins, micelles, polymers, chiral selectors such as cyclodextrins, etc.). Each experimental approach has advantages and disadvantages. Frequently, it is the nature of the system being evaluated that determines the optimal experimental approach. Six different CE-based techniques for evaluating binding constants are reviewed. Examples of each method, and recent references on its use are given.

Animals↗
PubMed1993

The binding of phosphorylase kinase to immobilized calmodulin.

The binding of phosphorylase kinase to calmodulin-Sepharose 4B was studied by column and batch methods. It was found that the Ca2+ dependence of the interaction strongly depended on the degree of substitution of agarose with calmodulin. Equilibrium adsorption isotherms (i.e., bulk ligand binding functions and lattice site binding functions) of phosphorylase kinase were measured on calmodulin-Sepharose. Sigmoidal bulk ligand binding functions (bulk adsorption coefficients: 1.5-5.8) were found which indicate intermolecular attraction during binding. Hyperbolic lattice site binding functions (lattice adsorption coefficients: 1.0) were obtained thus excluding the existence of a critical surface concentration of immobilized calmodulin and indicating single independent binding sites on the gel surface and on phosphorylase kinase. These findings were combined to optimize the adsorption of phosphorylase kinase on calmodulin-Sepharose, for purification procedures at low Ca2+ concentrations (5-10 microM) minimizing proteolysis by calpains. With this novel method phosphorylase kinase from rabbit and frog skeletal muscle could be purified ca 100- and 200-fold, respectively, in two steps.

Adsorption↗
PubMed1998

Structural plasticity in RNA and its role in the regulation of protein translation in coliphage Q beta.

We have analyzed both conformational and functional changes caused by two large cis-acting deletions (delta 159 and delta 549) located within the read-through domain, a 850 nucleotide hairpin, in coliphage Q beta genomic RNA. Studies in vivo show that co-translational regulation of the viral coat and replicase genes has been uncoupled in viral genomes carrying deletion delta 159. Translational regulation is restored in deletion delta 549, a naturally evolved pseudorevertant. Structural analysis by computer modeling shows that structural features within the read-through domain of delta 159 RNA are less well determined than they are in the read-through domain of wild-type RNA, whereas predicted structure in the read-through domain of evolved pseudorevertant delta 549 is unusually well determined. Structural analysis by electron microscopy of the genomic RNAs shows that several long range helices at the base of the read-through domain, that suppress translational initiation of the viral replicase gene in the wild-type genome, have been destabilized in delta 159 RNA. In addition, the structure of local hairpins within the read-through region is more variable in delta 159 RNA than in wild-type RNA. Stable RNA secondary structure is restored in the read-through domain of delta 549 RNA. Our analyses suggest that structure throughout the read-through domain affects the regulation of viral replicase expression by altering the likelihood that long-range interactions at the base of the domain will form. We discuss possible kinetic and equilibrium models that can explain this effect, and argue that observed changes in structural plasticity within the read-through domain of the mutant genomes are key in understanding the process. During the course of these studies, we became aware of the importance of the information contained in the energy dot plot produced by the RNA secondary structure prediction program mfold. As a result, we have improved the graphical representation of this information through the use of color annotation in the predicted optimal folding. The method is presented here for the first time.

Allolevivirus↗
PubMed1989

Rabbit distal colon epithelium: II. Characterization of (Na+,K+,Cl-)-cotransport and [3H]-bumetanide binding.

Loop diuretic-sensitive (Na+,K+,Cl-)-cotransport activity was found to be present in basolateral membrane vesicles of surface and crypt cells of rabbit distal colon epithelium. The presence of gradients of all three ions was essential for optimal transport activity. (Na+,K+) gradient-driven 36Cl fluxes were half-maximally inhibited by 0.14 microM bumetanide and 44 microM furosemide. While 86Rb uptake rates showed hyperbolic dependencies on Na+ and K+ concentrations with Hill coefficients of 0.8 and 0.9, respectively, uptakes were sigmoidally related to the Cl concentration, Hill coefficient 1.8, indicating a 1 Na+:1 K+:2 Cl stoichiometry of ion transport. The interaction of putative (Na+,K+,Cl-)-cotransport proteins with loop diuretics was studied from equilibrium-binding experiments using [3H]-bumetanide. The requirement for the simultaneous presence of Na+,K+, and Cl-, saturability, reversibility, and specificity for diuretics suggest specific binding to the (Na+,K+,Cl-)-cotransporter. [3H]-bumetanide recognizes a minimum of two classes of diuretic receptor sites, high-affinity (KD1 = 0.13 microM; Bmax1 = 6.4 pmol/mg of protein) and low-affinity (KD2 = 34 microM; Bmax2 = 153 pmol/mg of protein) sites. The specific binding to the high-affinity receptor was found to be linearly competitive with Cl- (Ki = 60 mM), whereas low-affinity sites seem to be unaffected by Cl-. We have shown that only high-affinity [3H]-bumetanide binding correlates with transport inhibition raising questions on the physiological significance of diuretic receptor site heterogeneity observed in rabbit distal colon epithelium.

Animals↗
PubMed2005

Ion association with alkylammonium cations for separation of anions by capillary electrophoresis.

A background electrolyte (BGE) containing a 100 mM concentration of an alkylammonium cation with ethyl, propyl or butyl groups provides an excellent medium for separation of anions by capillary electrophoresis (CE). Two major effects were noted. Use of one of a series of alkylammonium cations in the BGE at a selected pH provides a simple and effective way to vary and control electroosmotic flow (EOF) over a broad range. It is believed that the alkylammonium cations are coated onto the capillary surface through a reversible dynamic equilibrium. Secondly, alkylammonium cations modify the electrophoretic migration of sample anions and the electroosmotic migration of neutral organic analytes by association interaction. This selective interaction results in improved anion separations and permits the simultaneous separation of neutral analytes. The degree of association interaction varies with the bulk and hydrophobicity of the alkylammonium cations. Incorporation of an aliphatic amine salt of moderate molecular weight in the running electrolyte provides a valuable new way to vary the migration times of sample anions and to optimize their resolution. The interactions between alkylammonium cations and sample anions or neutral organics appear to take place entirely within the liquid phase and do not require a polymeric or micellar pseudo phase.

Anions↗
PubMed1997

DNA-lipid complexes: stability of honeycomb-like and spaghetti-like structures.

A molecular level theory is presented for the thermodynamic stability of two (similar) types of structural complexes formed by (either single strand or supercoiled) DNA and cationic liposomes, both involving a monolayer-coated DNA as the central structural unit. In the "spaghetti" complex the central unit is surrounded by another, oppositely curved, monolayer, thus forming a bilayer mantle. The "honeycomb" complex is a bundle of hexagonally packed DNA-monolayer units. The formation free energy of these complexes, starting from a planar cationic/neutral lipid bilayer and bare DNA, is expressed as a sum of electrostatic, bending, mixing, and (for the honeycomb) chain frustration contributions. The electrostatic free energy is calculated using the Poisson-Boltzmann equation. The bending energy of the mixed lipid layers is treated in the quadratic curvature approximation with composition-dependent bending rigidity and spontaneous curvature. Ideal lipid mixing is assumed within each lipid monolayer. We found that the most stable monolayer-coated DNA units are formed when the charged/neutral lipid composition corresponds (nearly) to charge neutralization; the optimal monolayer radius corresponds to close DNA-monolayer contact. These conclusions are also valid for the honeycomb complex, as the chain frustration energy is found to be negligible. Typically, the stabilization energies for these structures are on the order of 1 k(B)T/A of DNA length, reflecting mainly the balance between the electrostatic and bending energies. The spaghetti complexes are less stable due to the additional bending energy of the external monolayer. A thermodynamic analysis is presented for calculating the equilibrium lipid compositions when the complexes coexist with excess bilayer.

Computer Simulation↗
PubMed2002

Study of the adsorption equilibria of the enantiomers of 1-phenyl-1-propanol on cellulose tribenzoate using a microbore column.

Using competitive frontal analysis, the binary adsorption isotherms of the enantiomers of 1-phenyl-l-propanol were measured on a microbore column packed with a chiral stationary phase based on cellulose tribenzoate. These measurements were carried out using only the racemic mixture. The experimental data were fitted to four different isotherm models: Langmuir, BiLangmuir, Langmuir-Freundlich and Tóth. The BiLangmuir and the Langmuir-Freundlich models accounted best for the competitive adsorption data. An excellent agreement between the experimental and the calculated overloaded band profiles for various samples of racemic mixture was obtained when the equilibrium dispersive model of chromatography was used together with the BiLangmuir competitive isotherm. The isotherm parameters measured under competitive conditions were used to calculate the overloaded band profiles of large samples of the pure S- and R-enantiomers, too. A satisfactory agreement between the experimental and calculated band profiles was observed when using in the computation the corresponding single component BiLangmuir isotherm derived from the binary isotherm previously determined. Thus oniy data derived from the racemic mixture are required for computer optimization of the preparative chromatography separation of the enantiomers.

Adsorption↗
PubMed2001

Reactivity of (eta(6)-arene)tricarbonylchromium complexes toward additions of anions, cations, and radicals.

A computational and experimental study of additions of electrophiles, nucleophiles, and radicals to tricarbonylchromium-complexed arenes is reported. Competition between addition to a complexed arene and addition to a noncomplexed arene was tested using 1,1-dideuterio-1-iodo-2-((phenyl)tricarbonylchromium)-2-phenylethane. Reactions under anionic and cationic conditions give exclusive formation of 1,1-dideuterio-1-((phenyl)tricarbonylchromium)-2-phenylethane arising from addition to the complexed arene. Radical conditions (SmI(2)) afford two isomeric products, reflecting a 2:1 preference for radical addition to the noncomplexed arene. In contrast, intermolecular radical addition competition experiments employing ketyl radical addition to benzene and (benzene)tricarbonylchromium show that addition to the complexed aromatic ring is faster than attack on the noncomplexed species by a factor of at least 100,000. Density functional theory calculations using the B3LYP method, employing a LANL2DZ basis set for geometry optimizations and a DZVP2+ basis set for energy calculations, for all three reactive intermediates showed that tricarbonylchromium stabilizes all three types of intermediates. The computational results for anionic addition agree well with established chemistry and provide structural and energetic details as reference points for comparison with the other reactive intermediates. Intermolecular radical addition leads to exclusive reaction on the complexed arene ring as predicted by the computations. The intramolecular radical reaction involves initial addition to the complexed arene ring followed by an equilibrium leading to the observed product distribution due to a high-energy barrier for homolytic cleavage of an exo bond in the intermediate cyclohexadienyl radical complex. Mechanisms are explored for electrophilic addition to complexed arenes. The calculations strongly favor a pathway in which the cation initially adds to the metal center rather than to the arene ring.

Anions↗
PubMed2006

One-dimensional free-energy profiles of complex systems: progress variables that preserve the barriers.

We show that the balanced minimum-cut procedure introduced in PNAS 2004, 101, 14766 can be reinterpreted as a method for solving the constrained optimization problem of finding the minimum cut among the cuts with a particular value of an additive function of the nodes on either side of the cut. Such an additive function (e.g., the partition function of the reactant region) can be used as a progress coordinate to determine a one-dimensional profile (FEP) of the free-energy surface of the protein-folding reaction as well as other complex reactions. The algorithm is based on the network (obtained from an equilibrium molecular dynamics simulation) that represents the calculated reaction behavior. The resulting FEP gives the exact values of the free energy as a function of the progress coordinate; i.e., at each value of the progress coordinate, the profile is obtained from the surface with the minimal partition function among the surfaces that divide the full free-energy surface between two chosen end points. In many cases, the balanced minimum-cut procedure gives results for only a limited set of points. An approximate method based on p(fold) is shown to provide the profile for a more complete set of values of the progress coordinate. Applications of the approach to model problems and to realistic systems (beta-hairpin of protein G, LJ38 cluster) are presented.

Algorithms↗
PubMed2001

Predicting rates of inbreeding for livestock improvement schemes.

This article presents a deterministic method to predict rates of inbreeding (deltaF) for typical livestock improvement schemes. The method is based on a recently developed general theory to predict rates of inbreeding, which uses the concept of long-term genetic contributions. A typical livestock breeding population was modeled, with overlapping generations, BLUP selection, and progeny testing of male selection candidates. Two types of selection were practiced: animals were either selected by truncation on estimated breeding values (EBV) across age classes, or the number of parents selected from each age class was set to a fixed value and truncation selection was practiced within age classes. Bulmer's equilibrium genetic parameters were obtained by iterating on a pseudo-BLUP selection index and deltaF was predicted for the equilibrium situation. Predictions were substantially more accurate than predictions from other available methods, which ignore the effect of selection on deltaF. Predictions were accurate for schemes with up to 20 sires. Predicted deltaF was somewhat too low for schemes with more than 20 sires, which was due to the use of simple linear models to predict genetic contributions. The present method provides a computationally feasible (i.e., deterministic) tool to consider both the rate of inbreeding and the rate of genetic gain when optimizing livestock improvement schemes.

Animals↗
PubMed2006

[Comparison on desorptive behavior of cadmium in phaeozem and burozem].

Desorptive behavior of Cd2+ was comparatively studied by the batch method on the two typical soils in northeast China. The results indicated that the two soils exhibit statistically significant sorption-desorption hysteresis. Phaeozem has a longer hysteresis period than the Burozem. The desorptive behavior of Cd2+ in the two tested soils was a rapid reactions, which could account for 90% of the Cd2+ equilibrium desorption within the first 30 min. Compared with the total adsorption, the desorptive percentage of the adsorped Cd2+ in the phaeozem and burozem was in a low extent, which was ranged within rate of 9.0% and 15.1% respectively. The desorptive rates of Cd2+ in the soils were increased with the higher concentrations of Cd2+ in the treatments, and decreased with the increasing desorptive amount and time. Soil properties had no significant effect on the desorptive rate decreasing. Freundlich equation was the optimal model to describe the desorptive isotherm of Cd2+, while the Elovich equation was the best model to describe Cd2+ desorption kinetics in the two soils.

Adsorption↗
PubMed1989

Superiority of visual versus computerized echocardiographic estimation of radionuclide left ventricular ejection fraction.

An optimal method for determining left ventricular ejection fraction (LVEF) by echocardiography should be rapid, reliable, and widely applicable in order to be utilized routinely in a busy clinical laboratory. Most methods reported in the literature are reliable in selected, high-quality echocardiograms. Most require off-line computer analysis and are time-consuming and poorly suited to the routine of a busy laboratory. We compared in a blinded manner several echocardiographic methods of LVEF determination with the ejection fraction obtained by equilibrium radionuclide angiography (ERNA) in 44 patients unselected for image quality. Echocardiographic methods included: [1] cubed M-mode formula; [2] Teichholz M-mode formula; [3] subjective estimation of LVEF from two-dimensional echocardiographic videotape; [4] area-length method in one four-chamber view; [5] average of area-length method in three four-chamber views; [6] average of area-length method in four-chamber and two-chamber views (one beat each); [7] subjective estimation from stored videoloop of four-chamber and two-chamber view. In 30 cases M-mode tracings were available for analysis. In only 23 of the 44 patients were the apical views suitable for quantitative analysis. The ERNA ejection fraction was 44 +/- 17% (mean +/- 1 SD). The best echocardiographic correlation with ERNA ejection fraction in each patient subgroup studied was obtained by method 3. We concluded that subjective analysis of the videotaped study by an experienced cardiologist/echocardiographer provided the best estimation of ERNA ejection fraction. More time-consuming and costly computer techniques yielded a worse estimate.

Adult↗
PubMed1989

Purification and properties of ornithine carbamoyl transferase from liver of Squalus acanthias.

Citrulline synthesis from ammonia by hepatic mitochondria in elasmobranchs involves intermediate formation of glutamine as the result of the presence of high levels of glutamine synthetase and a unique glutamine- and N-acetyl-glutamate-dependent carbamoyl phosphate synthetase, both of which have properties unique to the function of glutamine-dependent synthesis of urea, which is retained in the tissues of elasmobranchs at high concentrations for the purpose of osmoregulation [P.M. Anderson and C.A. Casey (1984) J. Biol. Chem. 259, 456-462; R.A. Shankar and P.M. Anderson (1985) Arch. Biochem. Biophys. 239, 248-259]. The objective of this study was to determine if ornithine carbamoyl transferase, which catalyzes the last step of mitochondrial citrulline synthesis and which has not been previously isolated from any species of fish, also has properties uniquely related to this function. Ornithine carbamoyl transferase was highly purified from isolated liver mitochondria of Squalus acanthias, a representative elasmobranch. The purified enzyme is a trimer with a subunit molecular weight of 38,000 and a native molecular weight of about 114,000. The effect of pH is significantly influenced by ornithine concentration; optimal activity is at pH 7.8 when ornithine is saturating. The apparent Km values for ornithine and carbamoyl phosphate at pH 7.8 are 0.71 and 0.05 mM, respectively. Ornithine displays considerable substrate inhibition above pH 7.8. The activity is not significantly affected by physiological concentrations of the osmolyte urea or trimethylamine-N-oxide or by a number of other metabolites. The results of kinetic studies are consistent with a steady-state ordered addition of substrates (carbamoyl phosphate binding first) and rapid equilibrium random release of products. Except for an unusually low specific activity, the properties of the purified elasmobranch enzyme are similar to the properties of ornithine carbamoyl transferase from mammalian ureotelic and other species and do not appear to be unique to its role in glutamine-dependent synthesis of urea for the purpose of osmoregulation.

Animals↗
PubMed2005

Biosorption of lead(II), cadmium(II), copper(II) and nickel(II) by anaerobic granular biomass.

Biosorption is potentially an attractive technology for treatment of wastewater for retaining heavy metals from dilute solutions. This study investigated the feasibility of anaerobic granules as a novel type of biosorbent, for lead, copper, cadmium, and nickel removal from aqueous solutions. Anaerobic sludge supplied from a wastewater treatment plant in the province of Quebec was used. Anaerobic granules are microbial aggregates with a strong, compact and porous structure and excellent settling ability. After treatment of the biomass with Ca ions, the cation exchange capacity of the biomass was approximately 111 meq/100 g of biomass dry weight which is comparable to the metal binding capacities of commercial ion exchange resins. This work investigated the equilibrium, batch dynamics for the biosorption process. Binding capacity experiments using viable biomass revealed a higher value than those for nonviable biomass. Binding capacity experiments using non-viable biomass treated with Ca revealed a high value of metals uptake. The solution initial pH value affected metal sorption. Over the pH range of 4.0-5.5, pH-related effects were not significant. Meanwhile, at lower pH values the uptake capacity decreased. Time dependency experiments for the metal ions uptake showed that adsorption equilibrium was reached almost 30 min after metal addition. It was found that the q(max) for Pb2+, Cd2+, Cu2+, and Ni2+ ions, were 255, 60, 55, and 26 mg/g respectively (1.23, 0.53, 0.87, and 0.44 mmol/g respectively). The data pertaining to the sorption dependence upon metal ion concentration could be fitted to a Langmiur isotherm model. Based on the results, the anaerobic granules treated with Ca appear to be a promising biosorbent for removal of heavy metals from wastewater due to its optimal uptake of heavy metals, its particulate shape, compact porous structure, excellent settling ability, and its high mechanical strength.

Absorption↗
PubMed2004

Developmental changes of static standing balance in children.

The purpose of the present experiment was to investigate the time course by which children aged 6-, 8- and 10-year-old adapt and maintain their static balance. Participants (N=30) were required to stand on a force platform with their eyes closed. Ten adult subjects served as a reference group. We analyzed moment-to-moment modifications of quiet stance equilibrium by measuring the range and speed of the center of foot pressure (COP) displacements over time (i.e., periods of 2 s). Results showed that: (1) with age, the range of the COP decreased non-monotonically, with a maximum at 8 years of age, whereas the speed of the COP decreased linearly from 6 to 10 years of age, and (2) over time, both parameters decreased and stabilized, similarly for all age groups, suggesting that the processes underlying the maintenance of an optimal postural stability are mature at least as soon as 6 years of age.

Adult↗
PubMed2006

Optimization of extraction of phenolic compounds from flax shives by pressurized low-polarity water.

Pressurized low-polarity water (PLPW) extraction of phenolic compounds from flax shive was investigated using statistically based optimization and the "one-factor-at-a-time" method. Extraction variables examined using central composite design (CCD) included temperature, flow rate, and NaOH concentration of the extracting water. Extraction of phenolic compounds including p-hydroxybenzaldehyde, vanillic acid, syringic acid, vanillin, acetovanillone, and feruric acid was affected by temperature and NaOH concentration; and extraction of all phenolic compounds, except ferulic acid, increased with temperature and NaOH concentration of the extracting water. Flow rate had little effect on concentration of phenolic compounds at equilibrium, but the extraction rate at the early phase was higher for higher flow rates. The mechanism of PLPW extraction of flax shive phenolics was also investigated using a two-site kinetic model and a thermodynamic model. To determine the extraction mechanism, flow rate was varied from 0.3 to 4.0 mL/min while temperature and NaOH concentration were fixed at 180 degrees C and 0.47 M, respectively. The flow rate tests showed the extraction rates of total phenolic (TP) compounds increased with flow rate and can be described by a thermodynamic model. The results from the thermodynamic model demonstrated that a K(D) value of 30 agreed with the experimental data in the flow rate range of 0.3-4.0 mL/min. When the effect of the three independent variables was evaluated simultaneously using CCD, a maximum TP concentration of 5.8 g/kg of dry flax shive (DFS) was predicted from the combination of a high temperature (230.5 degrees C), a high initial concentration of NaOH (0.63 M), and a low flow rate (0.7 mL/min). Maximum TP concentration of 5.7 g/kg of DFS was obtained from extraction conditions of 180 degrees C, 0.3 or 0.5 mL/min, and 0.47 M NaOH at equilibrium. A second-order regression model generated by CCD predicted a maximum TP concentration of 5.8 g/kg of DFS under the same extraction conditions, which is well matched with the results from experimental data.

Flax↗
PubMed2006

A C-helix residue, Arg-123, has important roles in both the active and inactive forms of the cAMP receptor protein.

The cAMP receptor protein (CRP) of Escherichia coli exists in an equilibrium between active and inactive forms, and the effector, cAMP, shifts that equilibrium to the active form, thereby allowing DNA binding. For this equilibrium shift, a C-helix repositioning around the C-helix residues Thr-127 and Ser-128 has been reported as a critical local event along with proper beta4/beta5 positioning. Here we show that another C-helix residue, Arg-123, has a unique role in cAMP-dependent CRP activation in two different ways. First, Arg-123 is important for proper cAMP affinity, although it is not critical for the conformational change with saturating amounts of cAMP. Second, Arg-123 is optimal for stabilizing the inactive conformation of CRP when cAMP is absent, thereby allowing a maximal range of regulation by cAMP. However, Arg-123 does not appear to be critical for a functional response to cAMP, as has been proposed previously (Berman, H. M., Ten Eyck, L. F., Goodsell, D. S., Haste, N. M., Korney, A., and Taylor, S. S. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 45-50). Based on mutagenic evidence, we also propose the basis for the stabilization of the inactive form to be through a salt interaction between Asp-68 and Arg-123.

Arginine↗