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PubMed1994

Affinity purification and characterization of (2'-5')oligo(adenylate)-dependent RNase from mouse spleen.

Murine (2'-5')An-dependent RNase, a key enzyme of the interferon system, was purified from mouse spleen by affinity chromatography to immobilized (2'-5')An. Since the ribonuclease has high affinity to (2'-5')An, optimal non-denaturing conditions were obtained to disrupt the (2'-5')An-nuclease complex. Low-pH buffers in the presence of 0.1% Triton X-100 removed almost 80% of the enzyme from the (2'-5')An-agarose, preserving its (2'-5')An binding activity and RNA cleavage function. Purification was monitored using a classical radiobinding assay, ultraviolet covalent crosslinking method and denaturing-renaturing affinity blotting assay. The purified enzyme was a 160-kDa dimer that migrated with an apparent molecular mass of 78 kDa and was > 80% pure, as assessed by silver-stained SDS gels. Both a 160-kDa dimer and 78-kDa monomer were found in the cellular extract at a 5:1 ratio. Binding of radiolabeled (2'-5')An to (2'-5')An-dependent RNase either in crude extract or in purified form reached equilibrium by 5 h at 4 degrees C. 2-Mercaptoethanol was required to obtain (2-'5')An-binding activity but, interestingly, in the absence of this reducing agent, (2'-5')An-binding activity was initiated by preincubation with poly(U), a synthetic substrate of the nuclease. This new mechanistic feature indicates that interaction of poly(U) with nuclease induced a conformational modification allowing, in a second step, the binding of (2'-5')An. Furthermore, when activated by low amounts of (2'-5')An, the eluted purified enzyme degraded mRNA but there was still degradation in the absence of (2'-5')An. This suggested a loss of regulatory protein(s) during the purification step. Scatchard analysis showed that the purified enzyme had a Kd of 106 pM for (2'-5')An, similar to estimates obtained using crude spleen extracts (Kd 112 pM), indicating that the purified nuclease had almost identical (2'-5')An-binding properties to those identified in spleen extracts.

Adenine Nucleotides↗
PubMedDate not supplied

Ocular drug delivery of progesterone using nanoparticles.

The objective of this study was to evaluate ocular delivery of a lipid-soluble drug, [3H]progesterone, using nanoparticles. Polybutylcyanoacrylate nanoparticles loaded with [3H]progesterone were prepared by an emulsion polymerization technique using a hydrophilic continuous phase. The resulting nanoparticle suspension contained 2 x 10(-5) M progesterone. It was found that, at equilibrium, 99 per cent of the progesterone resided in the nanoparticles and the remainder in the aqueous phase indicating an excellent encapsulation efficiency. In addition, an appropriate control solution of progesterone was prepared, which did not contain polybutylcyanoacrylate. Concentrations of [3H]progesterone in various ocular tissues of the albino rabbit were monitored at various times following topical administration of either the nanoparticle suspension or the control solutions. Comparison of the concentration-time profiles indicates that tissue concentration of progesterone following topical administration of nanoparticles is generally four to five times less than that obtained with control solutions. This decreased concentration suggests that, due to the high affinity of progesterone for the nanoparticles, the drug is being made less available for absorption during its residence time in the precorneal area. The utility of nanoparticles as an ocular drug delivery system may depend on optimizing lipophilic-hydrophilic properties of the polymer-drug system, in addition to increasing retention efficiency in the precorneal pocket.

Animals↗
PubMed1981

Fluorimetric study of yeast tRNAPheCCF in the complex with phenylalanyl-tRNA synthetase. Evidence for a correlation between the structural adaptation of both macromolecules and the appearance of the acylation activity.

The fluorescence properties of yeast tRNAPheCCF (tRNAPhe in which the 3'-terminal adenosine has been replaced by formycin) and tRNAPheCCFoxi-red (tRNAPheCCF after periodate oxidation followed by borohydride reduction) were studied in the complex with the cognate aminoacyl-tRNA synthetase. In both cases a conformational change affecting the 3' end was observed in a magnesium concentration range close to 1 mM. The modification of formycin fluorescence could be ascribed simultaneously to the existence of a tautomeric equilibrium of the fluorescent probe and to a pH effect raising from a prototropic effect at the active site of phenylalanyl-tRNA synthetase, and to a partial destacking of the 3'-formycin from the adjacent C residue. The observed transconformation, which can be related to the structure modification of the anticodon loop previously reported [Ehrlich, Lefèvre, and Remy (1980) Eur. J. Biochem. 103, 145-153], takes place in the magnesium concentration range allowing the transfer of the activated amino acid from the adenylate to the tRNA. The interconnection between the anticodon loop and the accepting end was further supported by the observation that wybutine excision hinders the specific structure modification of 3'-formycin upon binding to the synthetase. The tRNAPhe transconformations occurring in the complex with the cognate synthetase probably reflect a reciprocal adaptation of both macromolecules which might lead to the optimal aminoacylation velocity and thus contribute to the specificity of aminoacylation, since it was previously established that this specificity relies more strongly on the kinetics of the reaction than on a discrimination of tRNAs according to different affinities.

Amino Acyl-tRNA Synthetases↗
PubMed2003

On translocation through a membrane channel via an internal binding site: kinetics and voltage dependence.

Here we present a model for maltodextrin translocation through maltoporin channels. In a first step, our theoretical analysis does consider the case of a single binding site for a given substrate in a structurally unaffected channel with a possibly different entrance barrier on either side. It is shown how by means of conventional electrical conductance measurements (including current noise analysis) the basic equilibrium and rate constants can be determined as functions of the applied voltage. Then also the net translocation rate of the substrate becomes accessible quantitatively. This most simple model mechanism has been extended to include a voltage-dependent fast conformational change of the channel that prevents the binding process. The so developed approach has been tested with experimental data for a single maltoporin trimer being reconstituted in black lipid membranes when studied in the presence of maltohexaose as the substrate. The experimental results turned out to be clearly incompatible with binding alone. They are, however, very satisfactorily fitted by pertinent theoretical curves if also inhibition of binding by a conformational transition is taken into account. Accordingly, quantitative evaluations of the underlying parameters and eventually of the translocation rate have been carried out successfully. Our analysis reveals a set of parameters necessary for an optimal translocation that nicely corresponds to natural conditions.

Bacterial Outer Membrane Proteins↗
PubMed2002

Determination of intrinsic bacterial surface acidity constants using a donnan shell model and a continuous pK(a) distribution method.

Intrinsic acidity constants (pK(a)(int)) for Bacillus subtilis (Gram+) and Escherichia coli (Gram-) cells were calculated from potentiometric titration data at different salt concentrations. Master curves were generated by replotting charge excess data as a function of pH(S) (pH at the location of surface reactive sites) where pH(S) was determined as a function of Donnan potential, Psi(DON). This potential decreased in magnitude with increasing ionic strength, from -48.5+/-0.2 to -3.5+/-0.0 mV for B. subtilis and -47.9+/-0.3 to -3.5+/-0.0 mV for E. coli at 0.01 and 0.5 M K(+), respectively, indicating an efficient surface charge neutralization by counterions. A fully optimized continuous (FOCUS) pK(a) distribution method revealed four binding sites on B. subtilis and E. coli surfaces from the master curves with pK(a)(int) values of 3.59+/-0.38, 4.33+/-0.57, 5.94+/-0.66, and 8.64+/-0.57 for B. subtilis and 3.73+/-0.44, 4.85+/-0.71, 6.56+/-0.64, and 8.79+/-0.62 for E. coli. These were assigned to functional groups according to reported pK(a) ranges of 2.0-6.0 (carboxylic acid), 3.2-3.5 (phosphodiesters), 5.6-7.2 (phosphoric acid), and 9.0-11.0 (amine groups). Average points of zero salt effect (pH(pzse)) for B. subtilis experiments were 6.63+/-0.21 and 6.42+/-0.08 as a function of pH(bulk) and pH(S), respectively. Under the same criteria, E. coli calculations yielded 5.73+/-0.23 and 5.45+/-0.05. An understanding of metal and proton reactivity on bacterial cell surfaces can be addressed quantitatively through the use of electrostatic and chemical equilibrium modeling techniques proposed in this study. The results are consistent with those of electrical force microscopy studies used to document the intrinsic electrochemical heterogeneity of bacterial cell surfaces.

Bacillus subtilis↗
PubMed2000

Characterization of heparin binding of human extracellular superoxide dismutase.

The C-terminal domain of human extracellular superoxide dismutase (hEC-SOD) plays a crucial role in the protein's interaction with heparin. Here we investigated this interaction in more detail by comparing the heparin-binding characteristics of two variants of hEC-SOD: the two fusion proteins containing the hEC-SOD C-terminal domain and a synthetic peptide homologous to the C-terminal. The interaction studies were performed using a surface plasmon resonance based technique on a BIAcore system. It should be emphasized that this is a model system. However, the kinetic constants, as measured, are valid in a comparative sense. Comparison of affinities for size-fractionated heparins revealed that octa- or decasaccharides are the smallest heparin fragments that can efficiently interact with the C-terminal domain of hEC-SOD. At physiological salt concentration, and pH 7.4, the hEC-SOD/heparin interaction was found to be of a high-affinity type, with an equilibrium dissociation constant, K(d), of 0.12 microM, which is 700 and 10-20 times lower than the K(d) values for the synthetic peptide and the fusion proteins, respectively. However, when an alpha-helical structure was induced in the synthetic peptide, by addition of 10% trifluoroethanol, the K(d) decreased to 0.64 microM. The differences in the K(d) values were mainly governed by differences in the association rate constants (k(ass)). The hEC-SOD/heparin interaction itself was found to have a fairly high dissociation rate constant (0.1 s(-)(1)), and a very high association rate constant (8 x 10(5) M(-)(1) s(-)(1)), suggesting that the interaction is mainly controlled by the association. These results together with circular dichroism spectra of the synthetic peptide suggest that an alpha-helical structure in the C-terminal is essential for optimal binding to heparin and that other parts of hEC-SOD moderate the affinity. Our data also demonstrate that the tetramerization itself does not substantially increase the affinity.

Amino Acid Sequence↗
PubMed1989

Electric dichroism and bending amplitudes of DNA fragments according to a simple orientation function for weakly bent rods.

The linear dichroism is calculated for DNA fragments in their thermal bending equilibrium. These calculations are given for relatively short fragments, where bent molecules can be described by an arc model. Using the measured value of 350 A for the persistence length, the limit dichroism (corresponding to complete alignment) decreases due to thermal bending, e.g., for a fragment with 100 base pairs to 80% of the value expected for straight molecules. Thermal bending should lead to a strong continuous decrease of the dichroism with increasing chain length, which is not observed, however, in electric dichroism experiments due to electric stretching. The influence of the electric field on the bending equilibrium is described by a contribution to the bending energy, which is calculated from the movement of charge equivalents against the potential gradient upon bending. The charge equivalents, which are assigned to the helix ends, are derived from the dipole moments causing the stationary degree of orientation. By this procedure the energy term inducing DNA stretching is given for induced, permanent, and saturating induced dipole models without introduction of any additional parameter. The stationary dichroism at a given electric field strength is then calculated according to an arc model by integration over all angles of orientation of helix axes or chords with respect to the field vector, and at each of these angles the contribution to the dichroism is calculated by integration over all helices with different degrees of bending. Orientation functions obtained by this procedure are fitted to dichroism data measured for various restriction fragments. Optimal fits are found for an induced dipole model with saturation of the polarizability. The difference between orientation functions with and without electric stretching is used to evaluate dichroism bending amplitudes. Both chain length and field strength dependence of bending amplitudes are consistent with experimental amplitudes derived from the dichroism decay in low salt buffers containing multivalent ions like Mg2+, spermine, or [CoNH3)6]3+. Bending amplitudes can be used to evaluate the persistence length from electrooptical data obtained for a single DNA restriction fragment. Bending and stretching effects are considerable already at relatively low chain length, and thus should not be neglected in any quantitative evaluation of experimental data.

DNA↗
PubMed1995

Purification, characterization, and investigation of the mechanism of aminoglycoside 3'-phosphotransferase type Ia.

Aminoglycoside 3'-phosphotransferases [APH(3')s] are the most common cause of bacterial high-level resistance to aminoglycoside antibiotics in clinical isolates. A one-step affinity chromatography was used to purify APH(3') type Ia. The kinetic parameters for turnover of seven aminoglycosides and the corresponding minimum inhibitory concentrations for a strain of Escherichia coli harboring APH-(3')-Ia were determined. The enzyme phosphorylates its substrates with kcat/Km values of 10(6)-10(8) M-1 s-1, including substrates such as amikacin and butirosin A which traditionally have been considered poor substrates for this enzyme. The optimal pH for the phosphotransferase activity was observed to be 7.0-7.5. The purified enzyme was found to be prone to dimerization in the absence of a reducing agent. Treatment of the enzyme with trypsin excised a 4 kDa fragment from the N-terminus which contained the amino acid residue Cys-10. The 27 kDa proteolyzed APH(3')-Ia did not dimerize, suggesting that Cys-10 was involved in dimerization via a disulfide bond. The phosphorylated kanamycin A was isolated, and the phosphorylation was confirmed to occur at the 3'-hydroxyl. Furthermore, both APH(3')-Ia and APH(3')-IIa were shown to phosphorylate water ("ATP hydrolase" activity) at a rate of ca. 10(4)-10(6)-fold slower (effect on kcat/Km) than that for the phosphoryl transfer to a typical aminoglycoside. The results of product-inhibition and alternative substrate diagnostics indicate an equilibrium-random mechanism for phosphorylation of aminoglycosides by APH(3')-Ia.

Adenosine Diphosphate↗
PubMed1992

Comprehensive model of transport and metabolism of adenosine and S-adenosylhomocysteine in the guinea pig heart.

Regulation of blood flow and mitochondrial respiration in the heart would be clarified by improved knowledge of interstitial concentrations and cellular production rates of adenosine; however, these variables cannot be measured directly. To interpret indexes that are available, a comprehensive mathematical model was developed, based on a large body of experimental data. The model describes most of the important pathways of capillary-tissue transport and cellular metabolism of adenosine in the guinea pig heart. It includes capillary flow, solute transport between tissue regions, nonlinear enzyme kinetics for adenosine kinase and adenosine deaminase, and reversible biunireactant kinetics for S-adenosylhomocysteine hydrolase in cardiomyocytes and endothelial cells, intracellular production of adenosine via AMP hydrolysis and transmethylation, and extracellular production of adenosine. A single set of parameter values for the model was obtained in the first stage of the analysis by taking certain values directly from published sources, other values were subject to specific constraints, and other values were determined by parameter optimization. The effects of flow and endothelial metabolism on the relation between interstitial and venous adenosine concentrations were determined. The relation between myocardial adenosine production rate and S-adenosylhomocysteine accumulation in the presence of excess homocysteine was estimated. In the second stage of the analysis, the model was used to investigate the mechanism of myocardial adenosine production, without changing the parameter values. Cellular adenosine production rates were estimated by fitting measurements of venous adenosine release obtained during altered energetic conditions in experiments by different investigators. The original results showed a dissociation between measurements of cytosolic AMP concentrations and venous adenosine release. It is concluded that 1) it is essential to account for the effect of flow on interstitial and venous adenosine concentrations, since decreased flow may produce effects outwardly resembling inhibition of the enzyme 5'-nucleotidase, 2) adenosine concentrations in epicardial transudate are not in equilibrium with interstitial fluid, and 3) the rate of cellular adenosine production increases monotonically with free cytosolic concentrations of AMP during a variety of alterations in energy balance of the guinea pig heart.

Adenosine↗
PubMed1986

Interaction of the Escherichia coli trp aporepressor with its ligand, L-tryptophan.

We have examined the interaction of the Escherichia coli trp aporepressor with its ligand, L-tryptophan, using both equilibrium dialysis and flow dialysis methods. Results obtained by the two procedures were equivalent and indicate that the trp aporepressor binds L-tryptophan with an equilibrium dissociation constant (Kd) of 40 microM at 25 degrees C under standard binding assay conditions (10 mM potassium phosphate, pH 7.4, 0.2 M potassium chloride, 0.1 mM EDTA, 5% glycerol). Molecular sizing of the purified trp aporepressor shows that in the absence of ligand the regulatory protein exists as a dimeric species with greater than 99% purity and an apparent molecular weight of 30,000. Under the storage and assay conditions used, the dimer appears quite stable, and essentially no monomer or higher multimeric species are detected. Analysis of binding data by Scatchard and direct linear plot methods shows two identical and independent ligand-binding sites/native trp aporepressor dimer. When examined as a function of temperature, L-tryptophan binding by trp aporepressor varied over 7-fold (Kd = 28 microM at 6.5 degrees C to Kd = 217 microM at 40 degrees C). At the optimal growth temperature for E. coli (37 degrees C), the dissociation constant was 160 microM for the ligand, L-tryptophan. From the relationship between temperature and L-tryptophan binding by trp aporepressor, the apparent enthalpy change delta H = -10.6 +/- 0.6 kcal mol-1 and the apparent entropy change delta S = -17 +/- 2 cal degree-1 mol-1 were determined.

Chromatography, High Pressure Liquid↗
PubMed1993

Nonequilibrium freezing of one-cell mouse embryos. Membrane integrity and developmental potential.

A thermodynamic model was used to evaluate and optimize a rapid three-step nonequilibrium freezing protocol for one-cell mouse embryos in the absence of cryoprotectants (CPAs) that avoided lethal intracellular ice formation (IIF). Biophysical parameters of one-cell mouse embryos were determined at subzero temperatures using cryomicroscopic investigations (i.e., the water permeability of the plasma membrane, its temperature dependence, and the parameters for heterogeneous IIF). The parameters were then incorporated into the thermodynamic model, which predicted the likelihood of IIF. Model predictions showed that IIF could be prevented at a cooling rate of 120 degrees C/min when a 5-min holding period was inserted at -10 degrees C to assure cellular dehydration. This predicted freezing protocol, which avoided IIF in the absence of CPAs, was two orders of magnitude faster than conventional embryo cryopreservation cooling rates of between 0.5 and 1 degree C/min. At slow cooling rates, embryos predominantly follow the equilibrium phase diagram and do not undergo IIF, but mechanisms other than IIF (e.g., high electrolyte concentrations, mechanical effects, and others) cause cellular damage. We tested the predictions of our thermodynamic model using a programmable freezer and confirmed the theoretical predictions. The membrane integrity of one-cell mouse embryos, as assessed by fluorescein diacetate retention, was approximately 80% after freezing down to -45 degrees C by the rapid nonequilibrium protocol derived from our model. The fact that embryos could be rapidly frozen in the absence of CPAs without damage to the plasma membrane as assessed by fluorescein diacetate retention is a new and exciting finding. Further refinements of this protocol is necessary to retain the developmental competence of the embryos.

Animals↗
PubMed1994

Ionic strength dependence of calcium, adenine nucleotide, magnesium, and caffeine actions on ryanodine receptors in rat brain.

[3H]Ryanodine binding studies of ryanodine receptors in brain membrane preparations typically require the presence of high salt concentrations in assay incubations to yield optimal levels of binding. Here, radioligand binding measurements on rat cerebral cortical tissues were conducted under high (1.0 M KCl) and low (200 mM KCl) salt buffer conditions to determine the effects of ionic strength on receptor binding properties as well as on modulation of ligand binding by Ca2+, Mg2+, beta, gamma-methylene-adenosine 5'-triphosphate (AMP-PCP), and caffeine. In 1.0 M KCl buffer, labeled titration/equilibrium analyses yielded two classes of binding sites with apparent KD (nM) and Bmax (fmol/mg of protein) values of 2.4 and 34, respectively, for the high-affinity site and 19.9 and 157, respectively, for the low-affinity site. Unlabeled titration/equilibrium measurements gave a single high-affinity site with a KD value of 1.9 nM and a Bmax value of 95 fmol/mg of protein. The apparent KD value derived from association and dissociation studies was 20 pM. Equilibrium binding was activated by Ca2+ (KD/Ca2+ = 14 nM), inhibited by Mg2+ (IC50 = 5.0 mM), and unaffected by AMP-PCP or caffeine. In 200 mM KCl buffer conditions, labeled titration analyses gave only a single site with a KD value similar to and a Bmax value 1.8-fold greater than those obtained for the low-affinity site in 1.0 M KCl buffer. In unlabeled titration measurements, the KD value was fivefold lower, whereas the Bmax value was unaffected. The KD value derived from association and dissociation analysis was 2.4-fold greater in 200 mM KCl compared with 1.0 M KCl buffer conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed2000

Cryoprotective agent and temperature effects on human sperm membrane permeabilities: convergence of theoretical and empirical approaches for optimal cryopreservation methods.

Previous reports have left unresolved discrepancies between human sperm cryopreservation methods developed using theoretical optimization approaches and those developed empirically. This study was designed to investigate possible reasons for the discrepancies. Human spermatozoa were exposed to 1 mol/l glycerol, 1 mol/l dimethyl sulphoxide (DMSO), 1 mol/l propylene glycol (PG) or 2 mol/l ethylene glycol (EG) at 22, 11 and 0 degrees C, then returned to isosmotic media while changes in cell volume were monitored. Activation energies (E(a)) of the hydraulic conductivity (L(p)) in the presence of cryoprotective agents (CPA) (L(p)(CPA)) were 22.2 (DMSO), 11.9 (glycerol), 15.8 (PG), and 7.8 (EG) kcal/mol. The E(a) values of the membrane permeability to CPA (P(CPA)) were 12.1 (DMSO), 10.4 (glycerol), 8.6 (PG) and 8.0 (EG) kcal/mol. These data indicated that even at low temperatures, EG permeates fastest. The high L(p)(CPA) in the presence of EG and low associated E(a) would allow spermatozoa to remain closer to equilibrium with the extracellular solution during slow cooling in the presence of ice. Collectively, these data suggest that the increase of the E(a) of L(p) in the presence of CPA at low temperature is the likely reason for the observed discrepancy between theoretical predictions of spermatozoa freezing response and empirical data.

Cell Membrane Permeability↗
PubMed2005

Postural dysfunction in children with cerebral palsy: some implications for therapeutic guidance.

Postural problems play a central role in the motor dysfunction of children with cerebral palsy (CP). Therefore, they spend more time in sitting than in standing to perform vital tasks of daily life. The focus of this article is to describe the pathophysiology of postural control in sitting and outline some implications for management and treatment. In general, children with CP exhibit muscular activity counteracting forces that disturb equilibrium. Only 'non-sitting' children with severe CP lack such 'direction-specific' adjustments, possibly ruling out achievement of independent sitting. Most frequently, the children display dysfunctions in the adaptation of the adjustment. Typical characteristics of this adaptation in children with CP are a top-down recruitment of postural muscles, an excessive degree of antagonistic coactivation, and an incomplete adaptation of the EMG-amplitude to task specific constraints. Despite our knowledge on the pathophysiology underlying the postural problems in children with CP, little 'high-level' evidence (according to Sackett) exists on how different interventions can affect these problems. Therapeutic attention to promote motor performance in sitting focuses on adaptive seating, tilting of the support surface, and ample, variable training in motivating settings. The challenge facing us now is to provide evidence about the efficacy of specific treatment approaches facilitating that children reach an optimal level of functioning in daily life.

Cerebral Palsy↗
PubMed1998

Enhancement of insect antifreeze protein activity by solutes of low molecular mass.

Antifreeze proteins (AFPs) lower the non-equilibrium freezing point of water (in the presence of ice) below the melting point, thereby producing a difference between the freezing and melting points that has been termed thermal hysteresis. In general, the magnitude of the thermal hysteresis depends upon the specific activity and concentration of the AFP. This study describes several low-molecular-mass solutes that enhance the thermal hysteresis activity of an AFP from overwintering larvae of the beetle Dendroides canadensis. The most active of these is citrate, which increases the thermal hysteresis nearly sixfold from 1.2 degrees C in its absence to 6.8 degrees C. Solutes which increase activity approximately fourfold are succinate, malate, aspartate, glutamate and ammonium sulfate. Glycerol, sorbitol, alanine and ammonium bicarbonate increased thermal hysteresis approximately threefold. Interestingly, 0.5 mol l-1 sodium sulfate eliminated activity. Solute concentrations between 0.25 and 1 mol l-1 were generally required to elicit optimal thermal hysteresis activity. Glycerol is the only one of these enhancing solutes that is known to be present at these concentrations in overwintering D. canadensis, and therefore the physiological significance of most of these enhancers is unknown. The mechanism(s) of this enhancement is also unknown. The AFP used in this study (DAFP-4) is nearly identical to previously described D. canadensis AFPs. The mature protein consists of 71 amino acid residues arranged in six 12- or 13-mer repeats with a consensus sequence consisting of Cys-Thr-X3-Ser-X5-X6-Cys-X8-X9-Ala-X11-Thr-X1 3, where X3 and X11 tend to be charged residues, X5 tends to be Thr or Ser, X6 to be Asn or Asp, X9 to be Asn or Lys and X13 to be Ala in the 13-mers. DAFP-4 is shorter by one repeat than previously described D. canadensis AFPs.

Acclimatization↗
PubMed1995

High-performance liquid chromatography method for determination of flunixin in bovine plasma and pharmacokinetics after single and repeated doses of the drug.

A high-performance liquid chromatography method was developed for determination of flunixin in bovine plasma. The extraction procedure was easily performed and made it possible to detect low concentrations of flunixin with high accuracy. The limit of quantitation was 7 ng/ml (relative standard deviation = 18% n = 10). The analytic method permits processing of 60 samples/d. Flunixin, as well as the internal standard (diclofenac sodium), belong to the group of nonsteroidal anti-inflammatory drugs, which are known to have a high degree of binding to plasma proteins. Therefore, an evaluation of several buffer systems was undertaken to optimize analytic conditions. Cattle were given 2.2 mg of flunixin melgumine/kg of body weight. In experiment 1, single injections were administered IV to 1 cow and IV and IM to 1 heifer (7 days apart), and pharmacokinetic variables were calculated. The IV data were best described by a two-compartment model. The half-life after single IV or IM administration was around 4.0 hours. In experiment 2, the decreasing flunixin concentration was determined after the last of either 4 IM injections daily (n = 3 cows) or 2 IM injections daily (n = 3 cows) administered during a 14-day postpartum period. The half-life, determined between 48 and 96 hours after the last dose, was approximately 26 hours in both groups, and flunixin could be detected in plasma up to 8 days, on average. The protein binding of flunixin was studied, using the method of equilibrium dialysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Functional interactions between the zinc fingers of Xenopus transcription factor IIIA during 5S rRNA binding.

We have used a collection of mutant forms of Xenopus transcription factor IIIA (TFIIIA) to study its interaction with 5S rRNA. This collection includes a set of nine mutant proteins, each of which contains a structural disruption in one of the nine zinc fingers of TFIIIA (broken-finger mutants), and a pair of complementary N- and C-terminal truncation mutants. Equilibrium and kinetic binding analyses in conjunction with RNAse protection and interference assays have been used to characterize the RNA-protein interaction in each case. We find that alternative binding modes are available for specific, high-affinity recognition of 5S rRNA by TFIIIA. These binding modes are distinct kinetically and structurally, and the mode of recognition adopted by wild-type TFIIIA when binding to intact 5S rRNA is dependent on the structural integrity of zinc fingers 5 and 6 in TFIIIA and continuity of the sugar-phosphate backbone in loop A of 5S rRNA. Disruption of any of these components allows adoption of one or more alternative modes of binding. In the wild-type TFIIIA-5S rRNA complex, some portions of TFIIIA, most notably the N-terminal three zinc fingers, are prevented from interacting with 5S rRNA in an energetically optimal way, and instead adopt a mode of binding that represents a compromise with the rest of the protein.

Animals↗
PubMed1998

B cell responses to a peptide epitope. V. Kinetic regulation of repertoire discrimination and antibody optimization for epitope.

The influence of imposing various conformational constraints on immune responses to a model epitope within a synthetic peptide immunogen was examined in mice. Although overall immunogenicity was affected, the model epitope (sequence DPAF) remained the predominant recognition site regardless of the conformation in which it was presented. A comparison of anti-DPAF mAbs obtained in response to two analogue peptides, PS1CT3 and CysCT3, in which the DPAF segment was either unconstrained or held within a cyclic loop, respectively, revealed a significant homology in the paratope composition. At one level a subset of anti-PS1CT3 and anti-CysCT3 mAbs was found to share a common heavy chain variable region. In addition, nucleotide sequence homology comparisons of both heavy and light chain variable regions identified the presence of anti-PS1CT3 and anti-CysCT3 mAbs that collectively appeared to derive from a common progenitor, but with nonidentical somatic mutations. Interestingly, however, no bias toward homologous Ag could be discerned on measurement of relative affinities of the mAbs for the two peptides. In contrast, mAb binding on-rates clearly discriminated between peptides representing the homologous vs the heterologous confomer of the DPAF epitope. Thus, it would appear that the kinetics of Ag recognition dominate over equilibrium binding criteria both in epitope-driven repertoire selection and Ab maturation in a humoral response.

Amino Acid Sequence↗