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Binding of two mono-acylated lipid monomers by the barley lipid transfer protein, LTP1, as viewed by fluorescence, isothermal titration calorimetry and molecular modelling.

The binding of two mono-acylated lipid monomers by plant lipid transfer proteins (LTP1s) presents an attractive field of research that could help our understanding of the functional role of this protein family. This task has been investigated in the case of barley LTP1 because it is known to exhibit a small cavity in its free state. The titration with lipids could not be followed by fluorescence with the native protein. Indeed, this LTP1 possesses a tyrosine residue on its C-terminus, Tyr91, which is not sensitive to lipid binding but mainly contributes to the fluorescence signal intensity. However, the binding of 1-myristoylglycerophosphatidylcholine (MyrGro-PCho) could be monitored by fluorescence after removal of Tyr91 by a carboxypeptidase. These experiments returned a dissociation constant of about 1 microM and showed that the protein can indeed bind two monomers. This result was corroborated by molecular modelling where the structure of the complex between barley LTP1 and MyrGro-PCho was derived from that determined in the case of wheat [Charvolin, D., Douliez, J.P., Marion, D., Cohen-addad, C. & Pebay-Peyroula, E. (1999) Eur. J. Biochem. 264, 562-568.]. Results from isothermal titration calorimetry experiments indicated non-classic titration behaviour but also suggested that two lipids could be bound by the protein. Finally, barley LTP1 binds two omega-hydroxypalmitic acid, a compound found in the family of cutin monomers. The fact that the binding of two lipids could be related to the physiological role of this protein family is discussed.

Calorimetry↗

Titration of hydrophobic polyelectrolytes using Monte Carlo simulations.

The conformation and titration curves of weak (or annealed) hydrophobic polyelectrolytes have been examined using Monte Carlo simulations with screened Coulomb potentials in the grand canonical ensemble. The influence of the ionic concentration pH and presence of hydrophobic interactions has been systematically investigated. A large number of conformations such as extended, pearl-necklace, cigar-shape, and collapsed structures resulting from the subtle balance of short-range hydrophobic attractive interactions and long-range electrostatic repulsive interactions between the monomers have been observed. Titration curves were calculated by adjusting the pH-pK(0) values (pK(0) represents the intrinsic dissociation constant of an isolated monomer) and then calculating the ionization degree alpha of the polyelectrolyte. Important transitions related to cascades of conformational changes were observed in the titration curves, mainly at low ionic concentration and with the presence of strong hydrophobic interactions. We demonstrated that the presence of hydrophobic interactions plays an important role in the acid-base properties of a polyelectrolyte in promoting the formation of compact conformations and hence decreasing the polyelectrolyte degree of ionization for a given pH-pK(0) value.

Journal Article↗

Determination of sulphite in wine by coulometric titration.

A method for the determination of both free and bound S(IV) in white wine samples by coulometric titration with electrogenerated iodine is described, the S(IV) speciation being performed by extracting SO2 from samples acidified wiith hydrochloric acid. Titrations of samples treated with NaOH led to the total S(IV) concentration, the results being in agreement with the ones obtained by the distillation procedure as well as by using the classical Monier-Williams method. An average recovery of 97% was obtained for samples spiked with S(IV) in the range 0.4-1.2 mM, which demonstrated the validity of the proposed procedure. The repeatability was 1.3% and the limit of detection was estimated as 0.01 mM. Results from direct titrations of wine added to the electrochemical cell are reported, which give indications of the stability of adducts towards the reaction with iodine.

Antioxidants↗

An improved olive oil overall migration test for plastics using Karl Fischer titration.

Karl Fischer titration has been used to eliminate the need to humidity-condition food contact plastics under test for overall migration into olive oil. The water content of the oil is measured before and after the migration test. This direct measure of the loss (or uptake) of water by the test plastic is then used in calculations of the overall migration. The use of Karl Fischer titration gives faster analysis with reduced labour input. The time savings varied from a few days to several weeks depending on the type of plastic. Karl Fischer titration is particularly useful in the high temperature testing of polar plastics where incomplete humidity conditioning can lead to erroneous results. The technique should be equally valuable when employing sunflower oil or the triglyceride HB307 in place of olive oil.

Dietary Fats, Unsaturated↗

The effect of chloride-containing potassium supplements on chloride titrator estimates of dietary sodium intake.

To examine the effect of chloride-containing potassium supplements on chloride titrator estimates of dietary sodium intake, we gave normal subjects diet containing 10, 100, or 200 mEq/d sodium in random order either as such, or supplemented with one of two potassium supplements. One regimen consisted of potassium 45 mEq/d with 12 mEq/d chloride and 33 mEq/d of citrate and gluconate; the other contained 48 mEq/d potassium and 48mEq/d chloride. Increased potassium intake with either supplemented regimen resulted in increased 24-hour potassium excretion, which was manifested in only the diurnal collections. Increased chloride intake resulted in increased urinary chloride excretion both during the day and at night. At all chloride intakes, urinary sodium and chloride excretion were highly correlated. The 48 mEq/d chloride intake generated a relationship with the same slope but with a different intercept from the other two regimens. The highest chloride intake resulted in a greater chloride titrator reading; however, the relationship was sufficiently predictable that adjustments in interpretation could be easily made. We conclude that if daily potassium chloride intake is known, chloride titrators continue to be reliable tools for estimating dietary sodium intake.

Adult↗

[Metal logarithmic scale titration as a tool for complexing ligand distribution determination: an application by DPASV].

A new logarithmic scale titration is proposed for the characterisation of natural organic matter-trace metals interactions in natural systems. The Suwannee River Fulvic Acid complexation with Cd and Pb ions has been analysed by this technique, using Differential Pulse Anodic Stripping Voltammetry (DPASV) for labile metal concentrations measurement. Results of titrations have been modelled by four ligands. Their complexing properties (CLi, Ki(Cd), Ki(Pb)) have been determined by a non-linear optimisation based on the speciation program MINEQL, coupled with a simplex. These ligands were called "very weak, weak, strong and very strong" with respect to their complexing constants (Ki(Cd), Ki(Pb)) ranging from 10(4) to 10(11). The obtained ligands concentrations (CLi) are respectively 1.9 microM, 150 nM, 25.1 nM and 21.1 nM for a 7.9 ppm carbon content. Moreover, this model takes account of the pH dependency and metals competition respectively by experiments at pH 7.8 and 4.6, and by definition of stability constants for each ligand toward each analysed metal. As a conclusion, the authors suggest a systematic use of the logarithmic scale titrations when full characterisation of the metal-organic matter interactions is necessary.

Cadmium↗

Inhibition of influenza virus multiplication by alkyl derivatives of benzimidazole. II. Measurement of inhibitory activity by hemagglutination titrations.

The activity of compounds which inhibit the multiplication of influenza virus can be measured in chorioallantoic membrane cultures in vitro by means of hemagglutination titrations on the medium. Studies on the reproducibility of virus reproduction in membrane cultures have revealed the major variables which affect the results and thus have led to the development of a precise technique. Under strictly controlled experimental conditions, the extent of reproduction of the virus in membrane cultures is predictable within narrow limits of variation. With 10(5.5) EID(50) of influenza B virus, Lee strain, and 5.75 cm.(2) of chorioallantoic membrane per ml., the ratio of infective virus particles to susceptible allantoic cells appears to be approximately 1:28. Under these conditions, the evidence indicates that two cycles of multiplication occur and nearly maximal hemagglutination titers are found with culture medium at 36 hours. The extent of the deviation in the absolute titer in different experiments was only 0.112 log unit. At a concentration of 0.0017 M, 2,5-dimethylbenzimidazole caused inhibition of the multiplication of influenza B virus, Lee strain, which persisted for at least 70 hours as measured by hemagglutination titrations on the culture medium. The degree of inhibition was closely comparable to that demonstrated by infectivity titrations on the membrane at the end of the first cycle of virus reproduction (1).

Benzimidazoles↗

Titration and characterization of two rhesus-derived SIVmac challenge stocks.

Simian immunodeficiency virus infection of macaques is a model for human immunodeficiency virus infection of humans. In vivo-titrated stocks of SIV are essential for the utilization of this model for vaccine development. The elicitation of anti-human cell antibodies by some vaccines prepared in human cells and the related protective effects of the vaccine produced in human cells suggest a need for new macaque-derived SIV stocks. Here we describe the titration and characterization of two stocks of SIVmac that were produced in primary rhesus macaque cells. The first virus is SIVmac251, isolated from tissues of macaque 251, and the second is a molecular clone designated as SIVmac239. A 50% rhesus monkey infectious dose (MID50) was titrated for each virus stock by intravenous inoculation. An additional five macaques were inoculated with 10 MID50 of the SIVmac251 stock and were followed for disease outcome. All five monkeys developed antigenemia by 14 days postchallenge. Two of the five monkeys developed strong anti-SIV humoral immunity, whereas three developed little or no humoral immunity. As has been observed previously, the rapidity of disease progression correlated with the lack of a strong antibody response. The three animals with low humoral immunity died within 7 months of challenge, with antigenemia, cachexia, hypoproteinemia, hypoalbuminemia, weight loss, and intractable diarrhea, while maintaining their circulating CD4 numbers. One animal died at 1.5 years of more typical simian AIDS.

Animals↗

DNA minor groove recognition by bis-benzimidazole analogues of Hoechst 33258: insights into structure-DNA affinity relationships assessed by fluorescence titration measurements.

Fluorescence titration measurements have been used to examine the binding interaction of a number of analogues of the bis -benzimidazole DNA minor groove binding agent Hoechst 33258 with the decamer duplex d(GCAAATTTGC)2. The method of continuous variation in ligand concentration (Job plot analysis) reveals a 1:1 binding stoichiometry for all four analogues; binding constants are independent of drug concentration (in the range [ligand] = 0.1-5 microM). The four analogues studied were chosen in order to gain some insight into the relative importance of a number of key structural features for minor groove recognition, namely (i) steric bulk of the N -methylpiperazine ring, (ii) ligand hydrophobicity, (iii) isohelicity with the DNA minor groove and (iv) net ligand charge. This was achieved, first, by replacing the bulky, non-planar N -methylpiperazine ring with a less bulky planar charged imidazole ring permitting binding to a narrower groove, secondly, by linking the N -methylpiperazine ring to the phenyl end of the molecule to give the molecule a more linear, less isohelical conformation and, finally, by introducing a charged imidazole ring in place of the phenolic OH making it dicationic, enabling the contribution of the additional electrostatic interaction and extended conformation to be assessed. Delta G values were measured at 20 degrees C in the range -47.6 to -37.5 kJ mol-1 and at a number of pH values between 5.0 and 7.2. We find a very poor correlation between Delta G values determined by fluorescence titration and effects of ligand binding on DNA melting temperatures, concluding that isothermal titration methods provide the most reliable method of determining binding affinities. Our results indicate that the bulky N -methylpiperazine ring imparts a large favourable binding interaction, despite its apparent requirement for a wider minor groove, which others have suggested arises in a large part from the hydrophobic effect. The binding constant appears to be insensitive to the isohelical arrangement of the constituent rings which in these analogues gives the same register of hydrogen bonding interactions with the floor of the groove.

Base Sequence↗

Escherichia coli strains that allow antibiotic-free plasmid selection and maintenance by repressor titration.

We report the construction of two novel Escherichia coli strains (DH1lacdapD and DH1lacP2dapD) that facilitate the antibiotic-free selection and stable maintenance of recombinant plasmids in complex media. They contain the essential chromosomal gene, dapD, under the control of the lac operator/promoter. Unless supplemented with IPTG (which induces expression of dapD) or DAP, these cells lyse. However, when the strains are transformed with a multicopy plasmid containing the lac operator, the operator competitively titrates the LacI repressor and allows expression of dapD from the lac promoter. Thus transformants can be isolated and propagated simply by their ability to grow on any medium by repressor titration selection. No antibiotic resistance genes or other protein expressing sequences are required on the plasmid, and antibiotics are not necessary for plasmid selection, making these strains a valuable tool for therapeutic DNA and recombinant protein production. We describe the construction of these strains and demonstrate plasmid selection and maintenance by repressor titration, using the new pORT plasmid vectors designed to facilitate recombinant DNA exploitation.

Amino Acid Sequence↗

Site-specific 13C-labeling of Trp 62 in hen egg-white lysozyme: preparation and 13C-NMR titration of [delta 1-13C]Trp 62-lysozyme.

The indole C-2(delta 1) carbon of Trp 62 in hen egg-white lysozyme was selectively labeled with 13C through a series of reactions involving N'-formylkynurenine 62-lysozyme with K13CN, NaBH4-reduction, and acid-catalyzed dehydration. [delta 1-13C]Trp 62-lysozyme in which Trp 62 is labeled with 90% 13C has the same chemical and enzymatic properties as the native protein. The reverted lysozyme gave a single 13C-NMR signal at 125 ppm. pH-titration of the 13C signal indicated a transition at pH 3.9 for the free enzyme. In the presence of (GlcNAc)3, the resonance signals were shifted 0.5-1 ppm upfield, and the transitions in the titration curve were observed at pH 3.9 and 6.5. Asp 52 and Glu 35 were assigned to the groups with pKas of 3.9 and 6.5, respectively. In [2-13C]AHT 62-lysozyme, which has 3-(2-amino-3-hydroxy-3H-[2-13C]indol-3-yl)alanine (AHT) at position 62, AHT 62 behaved quite differently from Trp 62 on pH-titration of the 13C-label. These results suggest that a conformational change around Trp 62 is induced upon ionization of the catalytic residue and that the structural flexibility of the side chain of this aromatic residue in the substrate binding site is closely related to the function of lysozyme.

Amino Acids↗

Determination of the pKa values of titratable groups of an antigen-antibody complex, HyHEL-5-hen egg lysozyme.

The titration behavior of the ionizable residues of the HyHEL-5-hen egg lysozyme complex and its individual components has been studied using continuum electrostatic calculations. Several residues of HyHEL-5 had pKa values shifted away from model values for isolated residues by more than three pH units. Shifts away from the model values were smaller for the residues of hen egg lysozyme. A moderate variation in the pKa values of the titratable groups was observed upon increase of the ionic strength from 0 to 100 mM, amounting to 1-2 pH units in most cases. Under physiological conditions, the net charge of HyHEL-5 was opposite that for hen egg lysozyme. Several residues, including those involved in the Arg-Glu salt bridges that have been proposed to be important in antibody-antigen binding, had pKa values that were changed significantly upon binding. The main titration event upon antibody-antigen binding appears to be loss of a proton from residue GluH50 of the Fv molecule. The limitations of our calculation methods and the role they might play in the design of antibodies for use in assays, sensors and separations are discussed.

Amino Acids↗

Effects of a titratable oral appliance on supine airway size in awake non-apneic individuals.

STUDY OBJECTIVES: To define morphological changes in the upper airway and its surrounding structures after the insertion of a titratable mandibular repositioner. DESIGN: Ten non-apneic adult males participated in this study. A set of supine lateral cephalograms was taken for each subject at the end of expiration with a titratable oral appliance in place in four mandibular positions: most retruded (RP), maximum protrusion (MAX), 33% of MAX (MAX33), and 67% of MAX (MAX67). Changes in the anteroposterior width of the upper airway, positions of the hyoid bone and the third cervical vertebra were compared between the four mandibular positions. An ANOVA was used to test for statistical significance. SETTING: N/A. PATIENTS OR PARTICIPANTS: N/A. INTERVENTIONS: N/A. MEASUREMENTS AND RESULTS: The anteroposterior width of the velopharynx significantly increased when the mandible was advanced from RP to MAX67 and MAX. However, there were no significant changes in the anteroposterior width of the oropharynx. Significant forward displacement of the hyoid bone and third cervical vertebra together with the mandible was found in MAX67 and MAX compared to RP. CONCLUSION: Especially in MAX67 and MAX, the titratable oral appliance significantly enlarges upper airway size in the velopharynx and results in a forward displacement of the hyoid bone and the third cervical vertebra.

Adult↗

Postoperative pain management by intranasal demand-adapted fentanyl titration.

The aim of the present study was to investigate whether intranasal administration of fentanyl allows a demand-adapted postoperative opioid titration. Forty-two patients who had undergone surgery for lumbar intervertebral disk protrusion were included in a prospective randomized double-blind study. When complaining about intense pain, 22 patients received six sprays of fentanyl (0.027 mg) intranasally and 6 ml sodium chloride 0.9% intravenously and 20 patients received six sprays of sodium chloride 0.9% intranasally and 6 ml of a diluted fentanyl solution (0.027 mg) intravenously. In both groups, these doses were repeated every 5 min until the patients were free of pain or refused further analgesic. Before the beginning of opioid titration and then every 10 min for at least 1 h, pain was evaluated with the aid of a 101-point numerical rating scale and a verbal rating scale. Blood pressure, heart rate, arterial hemoglobin oxygen saturation, respiratory rate, and side effects were recorded. All patients were satisfied with the pain reduction achieved. The total fentanyl dose was 0.073 mg (range 0.027-0.162) in the intravenous group and 0.11 mg (range 0.027-0.243) in the intranasal group. The onset of action after intranasal application was nearly as fast as after intravenous titration. The pain reduction achieved was comparable in both groups. Only at the (10-), 20- and 30-min measurement points was the pain intensity significantly lower in the intravenous than in the intranasal group. One patient of the intravenous group showed a decrease in arterial hemoglobin oxygen saturation to less than 90%. Other serious side effects were not observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Intranasal↗

Relationships between measurement of pain using visual analog score and morphine requirements during postoperative intravenous morphine titration.

BACKGROUND: Although intravenous morphine titration is widely used to obtain rapid and complete postoperative pain relief, the relationship between measurement of pain and morphine requirements varies, and the evolution of pain during titration is poorly understood. METHODS: Intravenous morphine titration was administered as a bolus of 2 (body weight < or = 60 kg) or 3 mg (body weight > 60 kg) during the immediate postoperative period in the PACU. The interval between each bolus was 5 min. The visual analog scale (VAS) score threshold required to administer morphine was 30, and pain relief was defined as a VAS score of 30 or less. RESULTS: Data from 3,045 patients were analyzed. The mean initial VAS score was 73 +/- 19 (mean +/- SD), and the mean morphine dose required to obtain pain relief was 0.17 +/- 0.10 mg/kg, i.e., a median of four boluses (range, 1-20). When patients were grouped according to several classes of initial VAS score (31-39, 40-49, 50-59, 60-69, 70-79, 80-89, 90-100), it seemed that the relationship between VAS score and morphine requirements was a sigmoid curve. A VAS score of 70 or greater predicted the need for a high (>0.15 mg/kg) morphine dose (sensitivity, 0.77; specificity, 0.54). During the pain relief process, the relationship between VAS score and time was depicted by a sigmoid curve. CONCLUSION: A VAS score of 70 or greater should be considered indicative of severe pain. The relationship between the initial VAS score and morphine requirements is not linear, and the evolution of the VAS score during the pain relief process is described by a sigmoid curve.

Aged↗

Propofol and sufentanil titration with the bispectral index to provide anesthesia for coronary artery surgery.

BACKGROUND: To provide anesthesia for cardiac surgery, hypnotics and opioids are frequently titrated on variables such as mean arterial pressure and heart rate. In this study conducted in patients scheduled to undergo coronary artery bypass grafting, propofol and sufentanil, both administered by computer-controlled infusion, were titrated on the Bispectral Index (BIS) values using a predefined algorithm. METHODS: After written informed consent, 110 patients, 95 men and 15 women aged 61 (9) yr [mean (SD)], were randomly allocated to receive predicted sufentanil effect site concentrations (Ce) of 0.5, 0.75, 1, 1.25, and 1.5 ng/ml, decreased by a third after sternotomy (groups 1-5). Target induction propofol concentration was 1.5 microg/ml and subsequently adjusted on BIS values. The following parameters were recorded: BIS values, predicted propofol Ce, the number of changes of propofol target, mean arterial pressure, heart rate, the number of bolus injection and doses of vasoconstrictor and vasodilator drugs, time to tracheal extubation, postoperative awareness and satisfaction scores, and cumulative morphine doses for the first postoperative day. RESULTS: One patient randomized to group 1 required 0.75 ng/ml sufentanil Ce instead of 0.5 ng/ml for increased BIS values on tracheal intubation. BIS values were similar in the five groups. The predicted propofol Ce values were different (P < 0.05; analysis of variance) among the five groups: 1.59 (0.47) to 1.23 (0.25) microg/ml in group 1 and group 4, respectively. Significantly fewer changes of propofol target were required in group 4 as compared to group 1. There were no differences among the five groups for mean arterial pressure, heart rate, time to tracheal extubation, awareness, satisfaction scores, and morphine requirements. CONCLUSION: These results suggest the BIS, as part of an algorithm that uses both the absolute BIS value and its increase following tracheal intubation, can be used to effectively titrate both propofol and sufentanil. A predicted sufentanil Ce of 1.25 ng/ml before and 0.8 ng/ml after sternotomy was associated with the lowest predicted propofol Ce and fewer changes of propofol target. Lower sufentanil concentrations required higher propofol concentrations and more frequent changes of the target propofol concentration and were associated with similar hemodynamic tolerance.

Adolescent↗

Misoprostol versus antacid titration for preventing stress ulcers in postoperative surgical ICU patients.

Bleeding from gastroduodenal lesions is a potentially life-threatening complication in patients subjected to overwhelming physiologic stress. Titration of gastric contents with antacid was the first prophylactic treatment regimen proved to decrease the incidence of bleeding and remains the standard by which other methods are compared. We designed a prospective double-blind, double-placebo study comparing the effectiveness of antacid titration with fixed doses of a synthetic prostaglandin E1 analog (misoprostol) for preventing stress gastritis and bleeding. To assess the success of each treatment regimen, we did endoscopic examinations before operation, 72 hours after operation, and after the patient had completed the study. A total of 281 patients entered the study (140 misoprostol, 141 antacid). The two groups were comparable with respect to preoperative parameters and type of operation. We found no statistically significant differences between the two treatment groups concerning upper gastrointestinal tract lesions or serious adverse effects. No clinically evident upper gastrointestinal hemorrhage occurred in either group. Mean gastric pH, measured at two-hour intervals during the initial 72 hours, was maintained at 4.0 or higher in both groups. We conclude that fixed-dose misoprostol is as effective as intensive antacid titration in preventing stress ulcers and bleeding in surgical ICU patients.

Adult↗

Computer-controlled positive end-expiratory pressure titration for effective oxygenation without frequent blood gases.

We have previously designed a computerized system to automatically deliver PEEP to maintain functional residual capacity (FRC) at a desired value. The purpose of this study was to compare the computerized PEEP titration system with a standard clinical PEEP titration algorithm in the animal adult respiratory distress syndrome (ARDS) model. Thirty mongrel dogs were anesthetized, paralyzed, intubated, and ventilated. An acute pulmonary injury was produced using 0.09 ml/kg of oleic acid. The animals were then given PEEP for 5 h. Arterial and venous blood gases, BP, thermodilution cardiac output, heart rate, body temperature, total respiratory system compliance (Ctr), and end-tidal CO2 were measured every 30 min. FRC was measured using an automated sulfur hexafluoride washout system every 15 min. The animals were allocated randomly to three ten-animal groups. The first group had PEEP titrated using a standard clinical protocol; the remaining two groups had PEEP updated at 15-min intervals under computer control to maintain FRC at 1.4 times the postanesthetized, postparalyzed, preinjury value. The second group received fixed 3-cm H2O PEEP steps. The third group had variable size PEEP steps depending on the output of a proportional, integral, and derivative (PID) controller. PaCO2 was maintained at 35.8 +/- 3.4 (SD) torr. There was a significant difference in PEEP delivered between the three groups (p = .0006) and in FRC (p = .005). There was no significant difference in PaO2 (p = .80) or venous admixture (Qva/Qt) (p = .84) between the three groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗