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At least 775 records · Page 43Linked to original sources
PubMed1997

Determination of cell surface charge by photometric titration.

A colloid titration method has been frequently used to determine the number of charged residues at the cell surface. Here we present a new version of this technique, based on photometric measurements of a metachromatic shift in the maximum absorption of toluidine blue as it binds to the cell surface. The major improvements are: (1) simplified methodology and (2) increased precision of equivalence point determination. The data are analyzed using Gran's theory, which allows measurements to be taken at regular intervals instead of being concentrated around the equivalence titration point. We used this method to characterize the cell surface charge of three populations of rat mast cells: (1) peritoneal mast cells (PMC), (2) bone marrow-derived mast cells (BMMC) and (3) a rat cultured mast cell line (RCMC). Our results indicate that PMC have (4.23 +/- 0.59) x 10(8), while BMMC (8.58 +/- 0.26) x 10(7) negatively charged residues per cell. The results for RCMC were similar to those for BMMC. Taking into account the size differences between PMC and BMMC, the average charge density of PMC was also significantly higher than that of BMMC. The differences in cell surface charge were analyzed in the light of different sensitivities of mast cells to polycationic secretagogues.

Animals↗
PubMed2002

Fast and reliable titration of recombinant adeno-associated virus type-2 using quantitative real-time PCR.

In this study, a quantitative real-time PCR (qPCR) was developed to determine genomic rAAV-2 titers using the Light-Cycler technology. Since the CMV promoter is the most commonly used promoter in gene therapeutic approaches, primers were designed which hybridize with the human CMV promoter sequence. PCR products were detected by the addition of SYBR green. qPCR of a 5 log spanning serial dilution of the vector plasmid containing one CMV promoter per plasmid molecule yielded a high amplification efficiency of 1.99 per cycle. To quantify the copy number of viral genomes, the qPCR curves of adeno-associated virus type 2 (AAV-2) samples were related to a standard curve assessed by the 5 log spanning serial vector plasmid dilution (0.01-100 pg DNA). For validation of the method, rAAV-2 preparations were analyzed by a standard method and qPCR in parallel. As standard method, flow cytometry was used for titration of infectious viral particles on HeLa cells using the Enhanced Green Fluorescent Protein as a marker. A significant correlation was found between the results obtained by flow cytometry and the results from the qPCR over a 5 log range (r=0.85, P<0.0001). The mean ratio between infectious rAAV-2 particles titrated via flow cytometry and genomic copies of rAAV-2 measured by qPCR of the same sample was 1:253. The higher titers found by qPCR might be due to multiple transduction of a single cell or to non-infectious particles generated during rAAV-2 preparation. In conclusion, qPCR is a fast and reliable method for determination of rAAV-2 titers and might be a powerful tool for standardization of rAAV-2 preparations particularly in the context of clinical studies.

Cytomegalovirus↗
PubMed1997

Single-antibody in situ enzyme immunoassay for infectivity titration of hepatitis A virus.

Hepatitis A virus (HAV) establishes a persistent infection in cultured cells, with minimal effect on host cell metabolism. As a result, the virus produces very little, if any, cytopathic effect (CPE), even with cell culture-adapted strains. This feature precludes the use of a plaque or standard endpoint assay (using CPE as an indicator of infection) for the titration of infectious virus. The radioimmunofocus assay (RIFA) is the standard method for HAV titration, though this method is labour intensive and requires the use of radioisotopes. To this end, a single-antibody in situ enzyme immunoassay (EIA) has been developed, using binding of a perioxidase-labelled monoclonal antibody to fixed cell monolayers as an indicator of infection. This novel assay is highly reproducible, can be read by eye, and is suitable for high throughput situations. Furthermore, the assay has been validated against the RIFA making it suitable for use in studies validating the safety of therapeutic biologicals for human use.

Animals↗
PubMed1998

Thermodynamics of ligand (substrate/end product) binding to endoxylanase from Chainia sp. (NCL-82-5-1): isothermal calorimetry and fluorescence titration studies.

The binding of xylo-oligosaccharides to Chainia endoxylanase resulted in a decrease in fluorescence intensity of the enzyme with the formation of 1:1 complex. Equilibrium and thermodynamic parameters of ligand binding were determined by fluorescence titrations and titration calorimetry. The affinity of xylanase for the oligosaccharides increases in the order X2<X3<X4</=X5. Contributions from the enthalpy towards the free energy change decreased with increasing chain length from X2 to X4, whereas an increase in entropy was observed, the change in enthalpy and entropy of binding being compensatory. The entropically driven binding process suggested that hydrophobic interactions as well as hydrogen bonds play a predominant role in ligand binding.

Actinomycetales↗
PubMed1998

Titration streptomycin therapy in Meniere's disease: long-term results.

As soon as the use of streptomycin was started for the treatment of tuberculosis, its ototoxic effect was discovered. Initial reports demonstrated that streptomycin was successful in reducing vestibular responses while preserving cochlear activity. Streptomycin sulfate has been used for almost half a century for the treatment of vertigo in patients with Meniere's disease. At the Gruppo Otologico, Piacenza, Italy, between 1987 and 1995 we treated 16 patients with either bilateral Meniere's disease or with Meniere's disease in their only hearing ear, adopting the titration streptomycin therapy protocol of Graham et al. Three patients with follow-up periods of less than 2 years were not included in this study. The ages of the patients ranged from 36 to 64 years with a mean of 49.2 years. The patients were observed for 2 to 8.8 years. The dose of administered streptomycin varied from 14 to 49 gm. Episodic vertigo was totally relieved in all of the patients after initial treatment, whereas hearing remained the same or was improved when compared with the pretreatment levels. Five of the patients experienced recurring vertigo during a period of 4 to 24 months after initial treatment. Oscillopsia persisted in two patients and ataxia in darkness persisted in another two patients. Hearing was unchanged in 14 ears, improved in 2 ears, and worsened in 4 ears at the last follow-up evaluation. The overall outcome indicated that intramuscular titration streptomycin therapy seems to be one of the most important therapeutic options in the treatment of disabling vertigo in patients with either bilateral Meniere's disease or Meniere's disease in an only hearing ear.

Adult↗
PubMed2001

Multiple pathways regulating DnaA function in Escherichia coli: distinct roles for DnaA titration by the datA locus and the regulatory inactivation of DnaA.

Escherichia coli DnaA protein forms a multimeric complex at the chromosomal origin of replication (oriC), where a series of initiation reactions occurs and DNA polymerase III holoenzyme is loaded. The ATP-bound form of DnaA, which is active for initiation, is converted to the inactive ADP-bound form through interaction with the sliding clamp, the beta subunit of DNA polymerase III holoenzyme loaded on DNA. This negative regulation, termed RIDA, is required for preventing untimely initiations. Here, we asked if RIDA is functionally related to another negative regulation, DnaA titration by the datA site. The datA site can harbor hundreds of DnaA molecules, and is also required for preventing untimely initiations. We reveal here that, in growing cells of the datA(+) and datA-deleted strains, the ATP-DnaA levels were both maintained in a limited range of about 20-30% of the total ATP- plus ADP-DnaA molecules. This indicates that RIDA functions in the absence of datA. In synchronized datA-deleted cells, the ATP-DnaA level fluctuated in a manner similar to that observed in datA(+) cells. This suggests that RIDA operates independent from DnaA titration to datA. We suggest that these two mechanisms may play complementary roles during the cell cycle to prevent untimely initiations and thus ensure the scheduled initiation.

Adenosine Triphosphate↗
PubMed1979

Proton N.M.R. study of the conformational dynamics of porcine pancreatic colipase. Titration of aromatic residues.

The low-field portion of the 360 MHz proton N.M.R. spectrum of native porcine pancreatic colipase has been studied as a function of pH over the pH range 2-12. Resonances associated with the 26 protons of the aromatic rings of the two histidines, two phenylalanines and three tyrosines have been identified and tentatively assigned to specific residues. Titrations of pH yielded apparent pKa's of 7.9, 6.9, 10.4, 10.3 and 11.3 for His I (His 30), His II (His 86), Tyr I (Tyr 56 or 57), Tyr II (Tyr 56 or 57) and Tyr III (Tyr 53) respectively (tentative assignments). The high pKa value of His 30 is attributed to the vicinity of Asp 31. The mobility of the aromatic ring of Tyr 53 is hindered and an upper bound of 500 s-1 on the rate of rotation can be estimated. The aromatic rings of the 2 other tyrosine residues and of the 2 phenylalanine residues can rotate freely on the N.M.R. time scale. The study of perturbations in titration profiles and chemical shift values reveals a specific interaction of His 86 with Tyr I and, to a lesser extent, Tyr II. The existence of this interaction indicates that the protein folding brings in close spatial vicinity two distant regions of the covalent structure to form a "hydrophobic-aromatic" site which might be involved in the binding of bile salt micelles to pancreatic colipase.

Amino Acid Sequence↗
PubMed2000

Titration calorimetry of surfactant-membrane partitioning and membrane solubilization.

The interaction of surfactants with membranes has been difficult to monitor since most detergents are small organic molecules without spectroscopic markers. The development of high sensitivity isothermal titration calorimetry (ITC) has changed this situation distinctly. The insertion of a detergent into the bilayer membrane is generally accompanied by a consumption or release of heat which can be measured fast and reliably with modern titration calorimeters. It is possible to determine the full set of thermodynamic parameters, i.e., the partitioning enthalpy, the partitioning isotherm, the partition coefficient, the free energy, and the entropy of transfer. The application of ITC to the following problems is described: (i) measurement of the critical micellar concentration (CMC) of pure detergent solutions; (ii) analysis of surfactant-membrane partitioning equilibria, including asymmetric insertion; and (iii) membrane-surfactant phase diagrams. Finally, the thermodynamic parameters derived for non-ionic detergents are discussed and the affinity for micelle formation is compared with membrane incorporation.

Calorimetry↗
PubMed1977

[Detection and titration of antitetanus antibodies using an automated passive hemagglutination method].

An automated reversed passive haemagglutination technique is described, using human erythrocytes sensitized by the chromic chloride method with higly purified tetanus toxoid. Such erythrocytes are agglutinated by the specific antibodies in a Technicon autoanalyzer. Clots are decanted, supernatant is hemolyzed and optical density is measured at 550 millimicron. Assay standards for comparison, ranging from 5 to 25 UI/ml, are prepared from human antitetanus immunoglobulins titrated by toxin neutralisation assay in mice. This method allows to screen donors' sera for presence of high titers of tetanus antibodies (larger than or equal to 5 UI) suitable for preparation of antitetanus immunoglobulins. 4,4% of donors have circulating antitetanus antibody levels corresponding to 5 UI/ml or more, by this method. The specificity of antibodies has been confirmed by the neutralisation assay in mice. The results obtained among 1.000 donors well agree with those of the counter-immuno-electrophoresis technique (C.E.P.). But, when, compared with C.E.P., the haemagglutination assay appears more objective, more quantitative and sensitive, and allows to get not only a rapid screening test but also a precise titration simultaneously.

Antibodies, Bacterial↗
PubMed2001

Microionization constants: novel approach for the determination of the zwitterionic equilibrium of hydroxyphenylalkylamines by photometric titration.

The record of the formation of the phenolate and the zwitterionic form in the course of titration by photometry makes it possible to estimate the tautomeric equilibrium, K(Z), between the zwitterionic and the uncharged form of an ampholyte, provided that (1) the absorptivity of the phenolate and the zwitterionic form are identical and (2) the absorptivities of both forms are distinct from the absorptivities of the protonated and the uncharged form. The relation between the absorbance and K(Z), the degree of titration and the degree of overlapping of the basic and the acid ionization constant is given.

Algorithms↗
PubMed2002

Development and validation of a photometric titration method for the quantitation of sodium chondroitin sulfate (bovine) in Cosequin DS chewable tablet.

A photometric titration method was developed and validated to quantitate sodium chondroitin sulfate in raw materials and Cosequin DS chewable tablet(1). About 0.1% (w/v) cetylpyridinium chloride was used to titrate sodium chondroitin sulfate with photometric indication at wavelength 420 nm. The standard curves for sodium chondroitin sulfate showed linearity (r> or = 0.99) over the selected concentration range from 0.6 to 1.4 mg/ml. The chewable tablet was ground to fine powder and extracted with water and the resulting solutions filtered through a 0.45 microm membrane filter. Recovery between 97 and 103%,. The intra- and inter- day precision as indicated by the relative standard deviation (R.S.D.) were not greater than 0.33 and 0.78%, respectively. The method was found to be specific and with excellent linearity, accuracy and precision and is well suited for the quantitation of sodium chondroitin sulfate in raw material and Cosequin DS chewable tablet.

Animals↗
PubMed2002

Automatic potentiometric flow titration procedure for ascorbic acid determination in pharmaceutical formulations.

A flow procedure for the determination of ascorbic acid in pharmaceutical formulations exploiting potentiometric titration is described. The method is based on the reduction of IO3- by ascorbic acid and the detection was carried out employing a flow-through ion selective electrode for iodide. The flow network controlled by a microcomputer was designed to implement multicommutation for ease of operation and robustness. The titration system allowed the determination of ascorbic acid in pharmaceutical formulations with concentrations ranging from 7.5 to 15.0 mmol l(-1). No significant differences at the 95% confidence level were observed in comparison with results obtained by a manual procedure. Merit figures of results such as a relative standard deviation of 1.0% (n=6) and a reagent consumption of 21.4 mg IO3- per determination were obtained.

Ascorbic Acid↗
PubMed1998

Lidocaine and benzalkonium analysis and titration in drugs using new ISFET devices.

Two new ISFETs recently developed by us have now been applied to some pharmaceutical determinations in real matrices; the first device, responsive to cationic surfactants, was employed in the determination of benzalkonium chloride contained in two different disinfectant solutions and in three types of commercial collyrium; the second device, responsive to cocaine hydrochloride, showed an appreciable response also to lidocaine hydrochloride and was used in the determination of lidocaine hydrochloride contained in some injectable antibiotics. The repeatability and accuracy of measurements performed in the analysis of these pharmaceutical matrices using new solid state sensors were evaluated. A further aspect of the research involved the use of two sensors to record complete titration curves for the determination of benzalkonium chloride, cocaine hydrochloride and lidocaine hydrochloride, respectively. Applications to real matrices were also performed by analysing by titration pharmaceutical formulations containing benzalkonium chloride, or lidocaine hydrochloride and an illicit powder containing cocaine hydrochloride and sugars.

Benzalkonium Compounds↗
PubMed1991

Events in the Cardiac Arrhythmia Suppression Trial (CAST): mortality in patients surviving open label titration but not randomized to double-blind therapy.

The patient characteristics and outcomes were studied in the 318 patients who survived open label drug titration in the Cardiac Arrhythmia Suppression Trial (CAST) and who were not randomized to double-blind therapy and in 942 patients, who were randomized to double-blind placebo therapy. The patients randomized to placebo therapy had a lower total mortality or resuscitated cardiac arrest rate (4% vs. 8.5%). However, at baseline, nonrandomized patients were dissimilar from patients randomized to placebo in the following ways: older; lower left ventricular ejection fraction; greater use of digitalis, diuretic drugs and antihypertensive agents; lesser use of beta-adrenoceptor blocking agents and more frequent prior cardiac problems, including runs of ventricular tachycardia and left bundle branch block. A matched comparison that took these inequities into account showed no significant differences in mortality or rate of resuscitation from cardiac arrest between nonrandomized patients and clinically equivalent patients randomized to placebo. Cox regression analysis indicated that two factors significantly increased the hazard ratio for arrhythmic death or resuscitated cardiac arrest in the nonrandomized patients: female gender (4.7, p less than 0.05) and electrocardiographic events (ventricular tachycardia, proarrhythmia, widened QRS complex, heart block, bradycardia) during open label titration (7.0, p less than 0.005). However, some potentially important differences between men and women were not included in the Cox regression model. Of the nonrandomized patients, approximately 70% were not randomized because of lack of suppression of ventricular premature depolarizations or adverse events, or both, and the remaining 30% because of patient or private physician request with no indication of another reason.(ABSTRACT TRUNCATED AT 250 WORDS)

Anilides↗
PubMedDate not supplied

Numeration of DTH-mediating T lymphocytes in mice under optimal titration conditions.

The magnitude of the delayed-type hypersensitivity (DTH) reaction observed in appropriately immunized mice appears to be dependent on an early non-specific inflammatory reaction. The volume of injection and dose of antigen are two major components of this early reaction. The determination of optimal conditions for revealing the DTH reaction enabled definition of a titration method for DTH-mediating T lymphocytes. Injection of a subinflammatory dose of antigen led to conditions of limiting dilution analysis. Thereafter, the initiation of DTH reaction by local injection of a minute number of sensitized cells mixed with an optimal dose of antigen allowed in vivo titration of sensitized T lymphocytes. Single-hit linear curves were obtained when the percentages of negative transfers were plotted against the number of transferred cells in the classical semi-log plot of Poisson's distribution. Initiation of reaction by a quantal mechanism and results giving a linear plot (a single cell type being diluted out) served as an indication that a numeration of sensitized T cells had been performed for the more diluted concentrations of cells assayed.

Animals↗
PubMed1999

A comparison of the efficacy and duration of action of candesartan cilexetil and losartan as assessed by clinic and ambulatory blood pressure after a missed dose, in truly hypertensive patients: a placebo-controlled, forced titration study. Candesartan/Losartan study investigators.

The purpose of this double-blind, forced titration study was to compare the antihypertensive effect duration of candesartan cilexetil, which has a longlasting binding to the human AT1-receptor, to that of losartan on ambulatory BP (ABP) not only during the 24-h dosing interval but also during the day of a missed dose intake. After a 4-week placebo lead-in period, 268 patients with sitting diastolic BP 95 to 110 mm Hg and mean awake ambulatory DBP > or =85 mm Hg were randomized to receive either 8 mg of candesartan, 50 mg of losartan, or placebo for a 4-week period. Thereafter, the doses were doubled in all patients for an additional 4-week period. Ambulatory BP monitoring was performed for 36 h after dosing and clinic BP measured 48 h after dosing. Candesartan cilexetil (16 mg) reduced ABP to a significantly greater extent than 100 mg of losartan, particularly for systolic ABP during daytime (P<.05), nighttime (P<.05), and 24-h (P<.01) periods, systolic (P<.01) and diastolic (P<.05) ABP between 0 and 36 h, and both systolic (P<.001) and diastolic (P<0.001) ABP during the day of a missed dose. Clinic BP at 48 h after dosing was significantly reduced exclusively with 16 mg of candesartan. The differences in BP reduction between 8 mg of candesartan and 50 mg of losartan were statistically significant for systolic ABP during daytime (P<.01), nighttime (P<.05), 24-h (P<.01), 0 to 36 h (P<.05) and during the day of missed dose (P<.05). Moreover, although losartan did not significantly reduce ambulatory BP in a dose-related manner, ambulatory systolic and diastolic BP reductions with 16 mg of candesartan were significantly greater (P<.01 and <.001) than those seen with 8 mg of candesartan during every period at the ABP supporting a dose-response relationship. In conclusion, this forced titration study in ambulatory hypertensive patients demonstrates that candesartan cilexetil provides significant dose-dependent reduction in both clinic and ambulatory BP in doses ranging from 8 to 16 mg once daily. Furthermore, candesartan cilexetil is superior to losartan in reducing systolic ABP and in controlling both systolic and diastolic ABP on the day of a missed dose. The differences observed between both agents are most likely attributable to a tighter binding to, and a slower dissociation from, the receptor binding site with candesartan cilexetil.

Adult↗
PubMed1994

The efficacy and tolerability of inhaled salmeterol and individually dose-titrated, sustained-release theophylline in patients with reversible airways disease.

In a double-blind, double-dummy, cross-over, multicentre study, 141 patients with moderate reversible airways disease were randomized to receive either 50 micrograms salmeterol via a metered dose inhaler or individually dose-titrated oral theophylline, both twice daily for a 2-week period. Just over half (n = 77) the patients had received theophylline before, but 64 patients were new to theophylline therapy. Efficacy was based on lung function data and symptom scores. Salmeterol produced significantly higher increase in mean morning peak expiratory flow (PEF) of 161 min-1 (P < 0.001; 95% Confidence Interval (CI); 8-241 min-1) and mean evening PEF of 151 min-1 (p < 0.001; 95% CI; 7-221 min-1) compared with theophylline therapy. Further analysis of the data revealed that the increase in PEF with salmeterol compared with theophylline was highest in a sub-group of patients new to theophylline therapy. Patients on salmeterol had significantly less night-time awakenings than those on theophylline (P = 0.011) and significantly less daytime symptoms (P = 0.017). There was also a significant increase in the number of nights (P = 0.013) and days (P < 0.001) on salmeterol when no additional salbutamol was required compared with theophylline. Theophylline produced a higher incidence of adverse events compared with salmeterol. The results of this study show that inhaled salmeterol is more effective and better tolerated than individually dose-titrated oral theophylline over a 2-week study period in patients with moderate asthma.

Administration, Inhalation↗
PubMed1997

Characterization of binding interactions by isothermal titration calorimetry.

Isothermal titration calorimetry is a high-accuracy method for measuring binding affinities. Titration calorimetry is a universal method that has broad impact throughout biotechnology. In recent years, microcalorimeters that are capable of characterizing binding interactions of biological macromolecules have become commercially available. Results from these studies are providing new insight into the molecular nature of macromolecular interactions.

Journal Article↗